definition of experiment in chemistry

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Introduction to Experimental Chemistry

Welcome to the world of chemistry!

In the lecture courses [1] , we explain the conceptual framework by which chemists currently describe and explain matter and its interactions. However, ultimately, we are trying to explain what is going on in nature.

In doing so, a quote from Stephen Hawkings’ A Brief History of Time  comes to mind.

No matter how many times the results of experiments agree with some theory, you can never be sure that the next time the result will not contradict the theory. On the other hand, you can disprove a theory by finding even a single observation that disagrees with the predictions of the theory.

Like all other natural sciences, chemistry is fundamentally an experimental subject. While theorists and computational chemists help shape the field, whatever theories and models are developed must be able to explain experimental results. The interplay of theory (used to explain experimental results) and experiment (used to characterize matter and test theories) is critical in the pursuit of science.

Beyond the cutting edge, we rely on experimental results on a day-by-day basis. For example:

• If you look at a packet of food, you will find nutritional facts such as the amount of energy needed. These are typically determined using calorimetry .
• In biochemistry, we often need to determine the concentration of a protein to ensure that our experiment is working. This is typically done with absorption spectroscopy .

You will learn about both of these techniques in this course sequence.

The goals of this course are to:

• Laboratory safety.
• Keeping and using a laboratory notebook.  Proper documentation is critical in all laboratory settings, as well as in healthcare (charting).
• Mastery of techniques used to prepare and conduct experiments, as well as to use chemical instrumentation.
• Design appropriate procedures to perform experiments.
• Illustrate and further explore concepts that are presented in the general chemistry lecture sequence. [2] . We will reference Tro: Chemistry – Structures and Properties , 2nd Ed in our course this year as this is the textbook we will use in the lecture course in 2021-2022; however, any general chemistry textbook will suffice to accompany this course.

The most important part about the chemistry lab is to stay safe and learn about the chemistry. Remember, while you do the experiments, your instructor is the guide. Be sure to ask questions whenever necessary for safety or success! Good luck!

• CHEM-C 105/106, Principles of Chemistry I/II ↵
• CHEM-C 105/106, Principles of Chemistry I/II. ↵

IU East Experimental Chemistry Laboratory Manual Copyright © 2022 by Yu Kay Law is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License , except where otherwise noted.

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Definition of experiment

 (Entry 1 of 2)

Definition of experiment  (Entry 2 of 2)

intransitive verb

• experimentation

Examples of experiment in a Sentence

These examples are programmatically compiled from various online sources to illustrate current usage of the word 'experiment.' Any opinions expressed in the examples do not represent those of Merriam-Webster or its editors. Send us feedback about these examples.

Word History

Middle English, "testing, proof, remedy," borrowed from Anglo-French esperiment, borrowed from Latin experīmentum "testing, experience, proof," from experīrī "to put to the test, attempt, have experience of, undergo" + -mentum -ment — more at experience entry 1

verbal derivative of experiment entry 1

14th century, in the meaning defined at sense 1a

1787, in the meaning defined above

Phrases Containing experiment

• control experiment
• controlled experiment
• experiment station
• pre - experiment
• thought experiment

Articles Related to experiment

This is the Difference Between a...

This is the Difference Between a Hypothesis and a Theory

In scientific reasoning, they're two completely different things

Dictionary Entries Near experiment

experiential time

experimental

Cite this Entry

“Experiment.” Merriam-Webster.com Dictionary , Merriam-Webster, https://www.merriam-webster.com/dictionary/experiment. Accessed 10 Aug. 2024.

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Kids definition of experiment.

Kids Definition of experiment  (Entry 2 of 2)

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Medical Definition of experiment  (Entry 2 of 2)

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68 Best Chemistry Experiments: Learn About Chemical Reactions

Whether you’re a student eager to explore the wonders of chemical reactions or a teacher seeking to inspire and engage your students, we’ve compiled a curated list of the top 68 chemistry experiments so you can learn about chemical reactions.

While the theories and laws governing chemistry can sometimes feel abstract, experiments bridge the gap between these concepts and their tangible manifestations. These experiments provide hands-on experiences illuminating the intricacies of chemical reactions, molecular structures, and elemental properties.

1. Covalent Bonds

By engaging in activities that demonstrate the formation and properties of covalent bonds, students can grasp the significance of these bonds in holding atoms together and shaping the world around us.

Learn more: Covalent Bonds

2. Sulfuric Acid and Sugar Demonstration

Through this experiment, students can develop a deeper understanding of chemical properties, appreciate the power of chemical reactions, and ignite their passion for scientific exploration.

3. Make Hot Ice at Home

Making hot ice at home is a fascinating chemistry experiment that allows students to witness the captivating transformation of a liquid into a solid with a surprising twist.

4. Make a Bouncing Polymer Ball

This hands-on activity not only allows students to explore the fascinating properties of polymers but also encourages experimentation and creativity.

Learn more: Thought Co

5. Diffusion Watercolor Art

This experiment offers a wonderful opportunity for students to explore the properties of pigments, observe how they interact with water, and discover the mesmerizing patterns and textures that emerge.

Learn more: Diffusion Watercolor Art

6. Exploding Baggie

The exploding baggie experiment is a captivating and dynamic demonstration that students should engage in with caution and under the supervision of a qualified instructor.

Learn more: Exploding Baggie

7. Color Changing Chemistry Clock

This experiment not only engages students in the world of chemical kinetics but also introduces them to the concept of a chemical clock, where the color change acts as a timekeeping mechanism.

Learn more: Color Changing Chemistry Clock

8. Pipe Cleaner Crystal Trees

By adjusting the concentration of the Borax solution or experimenting with different pipe cleaner arrangements, students can customize their crystal trees and observe how it affects the growth patterns.

Learn more: Pipe Cleaner Crystal Trees

9. How To Make Ice Sculptures

Through this experiment, students gain a deeper understanding of the physical and chemical changes that occur when water freezes and melts.

Learn more: Ice Sculpture

10. How to Make Paper

Through this hands-on activity, students gain a deeper understanding of the properties of cellulose fibers and the transformative power of chemical reactions.

Learn more: How to Make Paper

11. Color Changing Chemistry

Color changing chemistry is an enchanting experiment that offers a captivating blend of science and art. Students should embark on this colorful journey to witness the mesmerizing transformations of chemicals and explore the principles of chemical reactions.

12. Gassy Banana

The gassy banana experiment is a fun and interactive way for students to explore the principles of chemical reactions and gas production.

Learn more: Gassy Banana

13. Gingerbread Man Chemistry Experiment

This hands-on activity not only introduces students to the concepts of chemical leavening and heat-induced reactions but also allows for creativity in decorating and personalizing their gingerbread creations.

Learn more: Gingerbread Man Chemistry Experiment

14. Make Amortentia Potion

While the love potion is fictional, this activity offers a chance to explore the art of potion-making and the chemistry behind it.

Learn more: How to Make Amortentia Potion

15. Strawberry DNA Extraction

This hands-on experiment offers a unique opportunity to observe DNA, the building blocks of life, up close and learn about its structure and properties.

16. Melting Snowman

The melting snowman experiment is a fun and whimsical activity that allows students to explore the principles of heat transfer and phase changes.

Learn more: Melting Snowman

17. Acid Base Cabbage Juice

The acid-base cabbage juice experiment is an engaging and colorful activity that allows students to explore the pH scale and the properties of acids and bases.

By extracting the purple pigment from red cabbage leaves and creating cabbage juice, students can use this natural indicator to identify and differentiate between acidic and basic substances.

Learn more: Acid Base Cabbage Juice

18. Magic Milk

The magic milk experiment is a mesmerizing and educational activity that allows students to explore the concepts of surface tension and chemical reactions.

By adding drops of different food colors to a dish of milk and then introducing a small amount of dish soap, students can witness a captivating display of swirling colors and patterns.

Learn more: Magic Milk

19. Melting Ice with Salt and Water

Through this hands-on activity, students can gain a deeper understanding of the science behind de-icing and how different substances can influence the physical properties of water.

Learn more: Melting Ice with Salt and Water

20. Barking Dog Chemistry Demonstration

The barking dog chemistry demonstration is an exciting and visually captivating experiment that showcases the principles of combustion and gas production.

21. How to Make Egg Geodes

Making egg geodes is a fascinating and creative chemistry experiment that students should try. By using common materials like eggshells, salt, and food coloring, students can create their own beautiful geode-like crystals.

Learn more: How to Make Egg Geodes

22. Make Sherbet

This experiment not only engages the taste buds but also introduces concepts of acidity, solubility, and the chemical reactions that occur when the sherbet comes into contact with moisture.

Learn more: Make Sherbet

23. Hatch a Baking Soda Dinosaur Egg

As the baking soda dries and hardens around the toy, it forms a “shell” resembling a dinosaur egg. To hatch the egg, students can pour vinegar onto the shell, causing a chemical reaction that produces carbon dioxide gas.

Learn more: Steam Powered Family

24. Chromatography Flowers

By analyzing the resulting patterns, students can gain insights into the different pigments present in flowers and the science behind their colors.

Learn more: Chromatography Flowers

25. Turn Juice Into Solid

Turning juice into a solid through gelification is an engaging and educational chemistry experiment that students should try. By exploring the transformation of a liquid into a solid, students can gain insights of chemical reactions and molecular interactions.

Learn more: Turn Juice into Solid

26. Bouncy Balls

Making bouncy balls allows students to explore the fascinating properties of polymers, such as their ability to stretch and rebound.

 27. Make a Lemon Battery

Creating a lemon battery is a captivating and hands-on experiment that allows students to explore the fundamentals of electricity and chemical reactions.

28. Mentos and Soda Project

The Mentos and soda project is a thrilling and explosive experiment that students should try. By dropping Mentos candies into a bottle of carbonated soda, an exciting eruption occurs.

29. Alkali Metal in Water

The reaction of alkali metals with water is a fascinating and visually captivating chemistry demonstration.

30. Rainbow Flame

The rainbow flame experiment is a captivating and visually stunning chemistry demonstration that students should explore.

31. Sugar Yeast Experiment

This experiment not only introduces students to the concept of fermentation but also allows them to witness the effects of a living organism, yeast, on the sugar substrate.

32. The Thermite Reaction

The thermite reaction is a highly energetic and visually striking chemical reaction that students can explore with caution and under proper supervision.

This experiment showcases the principles of exothermic reactions, oxidation-reduction, and the high temperatures that can be achieved through chemical reactions.

33. Polishing Pennies

Polishing pennies is a simple and enjoyable chemistry experiment that allows students to explore the concepts of oxidation and cleaning methods.

34. Elephant Toothpaste

The elephant toothpaste experiment is a thrilling and visually captivating chemistry demonstration that students should try with caution and under the guidance of a knowledgeable instructor.

35. Magic Potion

Creating a magic potion is an exciting and imaginative activity that allows students to explore their creativity while learning about the principles of chemistry.

36. Color Changing Acid-Base Experiment

Through the color changing acid-base experiment, students can gain a deeper understanding of chemical reactions and the role of pH in our daily lives.

Learn more: Color Changing Acid-Base Experiment

37. Fill up a Balloon

Filling up a balloon is a simple and enjoyable physics experiment that demonstrates the properties of air pressure. By blowing air into a balloon, you can observe how the balloon expands and becomes inflated.

38. Jello and Vinegar

The combination of Jello and vinegar is a fascinating and tasty chemistry experiment that demonstrates the effects of acid on a gelatin-based substance.

Learn more: Jello and Vinegar

39. Vinegar and Steel Wool Reaction

This experiment not only provides a visual demonstration of the oxidation process but also introduces students to the concept of corrosion and the role of acids in accelerating the process.

Learn more: Vinegar and Steel Wool Reaction

40. Dancing Rice

The dancing rice experiment is a captivating and educational demonstration that showcases the principles of density and buoyancy.

By pouring a small amount of uncooked rice into a clear container filled with water, students can witness the rice grains moving and “dancing” in the water.

Learn more: Dancing Rice

41. Soil Testing Garden Science

Soil testing is a valuable and informative experiment that allows students to assess the composition and properties of soil.

By collecting soil samples from different locations and analyzing them, students can gain insights into the nutrient content, pH level, and texture of the soil.

Learn more: Soil Testing Garden Science

42. Heat Sensitive Color Changing Slime

Creating heat-sensitive color-changing slime is a captivating and playful chemistry experiment that students should try.

Learn more: Left Brain Craft Brain

43. Experimenting with Viscosity

Experimenting with viscosity is an engaging and hands-on activity that allows students to explore the flow properties of liquids.

Viscosity refers to a liquid’s resistance to flow, and this experiment enables students to investigate how different factors affect viscosity.

Learn more: Experimenting with Viscosity

44. Rock Candy Science

Rock candy science is a delightful and educational chemistry experiment that students should try. By growing their own rock candy crystals, students can learn about crystal formation and explore the principles of solubility and saturation.

Learn more: Rock Candy Science

45. Baking Soda vs Baking Powder

Baking soda and baking powder have distinct properties that influence the leavening process in different ways.

This hands-on experiment provides a practical understanding of how these ingredients interact with acids and moisture to create carbon dioxide gas.

46. Endothermic and Exothermic Reactions Experiment

The endothermic and exothermic reactions experiment is an exciting and informative chemistry exploration that students should try.

By observing and comparing the heat changes in different reactions, students can gain a deeper understanding of energy transfer and the concepts of endothermic and exothermic processes.

Learn more: Education.com

47. Diaper Chemistry

By dissecting a diaper and examining its components, students can uncover the chemical processes that make diapers so effective at absorbing and retaining liquids.

Learn more: Diaper Chemistry

48. Candle Chemical Reaction

The “Flame out” experiment is an intriguing and educational chemistry demonstration that students should try. By exploring the effects of a chemical reaction on a burning candle, students can witness the captivating moment when the flame is extinguished.

49. Make Curds and Whey

This experiment not only introduces students to the concept of acid-base reactions but also offers an opportunity to explore the science behind cheese-making.

Learn more: Tinkerlab

50. Grow Crystals Overnight

By creating a supersaturated solution using substances like epsom salt, sugar, or borax, students can observe the fascinating process of crystal growth. This experiment allows students to explore the principles of solubility, saturation, and nucleation.

Learn more: Grow Crystals Overnight

51. Measure Electrolytes in Sports Drinks

The “Measure Electrolytes in Sports Drinks” experiment is an informative and practical chemistry activity that students should try.

By using simple tools like a multimeter or conductivity probe, students can measure the electrical conductivity of different sports drinks to determine their electrolyte content.

52. Oxygen and Fire Experiment

The oxygen and fire experiment is a captivating and educational chemistry demonstration that students should try. By observing the effects of oxygen on a controlled fire, students can witness the essential role of oxygen in supporting combustion.

53. Electrolysis Of Water

The electrolysis of water experiment is a captivating and educational chemistry demonstration that students should try.

Learn more: Electrolysis Of Water

54. Expanding Ivory Soap

The expanding Ivory Soap experiment is a fun and interactive chemistry activity that students should try. By placing a bar of Ivory soap in a microwave, students can witness the remarkable expansion of the soap as it heats up.

Learn more: Little Bins Little Hands

55. Glowing Fireworks

This experiment not only introduces students to the principles of pyrotechnics and combustion but also encourages observation, critical thinking, and an appreciation for the physics and chemistry behind.

Learn more: Glowing Fireworks

56. Colorful Polymer Chemistry

Colorful polymer chemistry is an exciting and vibrant experiment that students should try to explore polymers and colorants.

By combining different types of polymers with various colorants, such as food coloring or pigments, students can create a kaleidoscope of colors in their polymer creations.

Learn more: Colorful Polymer Chemistry

57. Sulfur Hexafluoride- Deep Voice Gas

This experiment provides a firsthand experience of how the density and composition of gases can influence sound transmission.

It encourages scientific curiosity, observation, and a sense of wonder as students witness the surprising transformation of their voices.

58. Liquid Nitrogen Ice Cream

Liquid nitrogen ice cream is a thrilling and delicious chemistry experiment that students should try. By combining cream, sugar, and flavorings with liquid nitrogen, students can create ice cream with a unique and creamy texture.

59. White Smoke Chemistry Demonstration

The White Smoke Chemistry Demonstration provides an engaging and visually captivating experience for students to explore chemical reactions and gases. By combining hydrochloric acid and ammonia solutions, students can witness the mesmerizing formation of white smoke.

60. Nitrogen Triiodide Chemistry Demonstration

The nitrogen triiodide chemistry demonstration is a remarkable and attention-grabbing experiment that students should try under the guidance of a knowledgeable instructor.

By reacting iodine crystals with concentrated ammonia, students can precipitate nitrogen triiodide (NI3), a highly sensitive compound.

61. Make a Plastic- Milk And Vinegar Reaction Experiment

Through the “Make a Plastic – Milk and Vinegar Reaction” experiment, students can gain a deeper understanding of the chemistry behind plastics, environmental sustainability, and the potential of biodegradable materials.

Learn more: Rookie Parenting

62. Eno and Water Experiment

This experiment not only introduces students to acid-base reactions but also engages their senses as they witness the visible and audible effects of the reaction.

63. The Eternal Kettle Experiment

By filling a kettle with alcohol and igniting it, students can investigate the behavior of the alcohol flame and its sustainability.

64. Coke and Chlorine Bombs

Engaging in this experiment allows students to experience the wonders of chemistry firsthand, making it an ideal choice to ignite their curiosity and passion for scientific exploration.

65. Set your Hand on Fire

This experiment showcases the fascinating nature of combustion and the science behind fire.

By carefully following proper procedures and safety guidelines, students can witness firsthand how the sanitizer’s high alcohol content interacts with an open flame, resulting in a brief but captivating display of controlled combustion.

66. Instant Ice Experiments

The Instant Ice Experiment offers an engaging and captivating opportunity for students to explore the wonders of chemistry and phase changes.

By using simple household ingredients, students can witness the fascinating phenomenon of rapid ice formation in just a matter of seconds.

67. Coke Cans in Acid and Base

Engaging in this experiment allows students to gain a deeper understanding of the chemical properties of substances and the importance of safety protocols in scientific investigations.

68. Color Changing Invisible Ink

The Color Changing Invisible Ink experiment offers an intriguing and fun opportunity for students to explore chemistry and learn about the concept of chemical reactions.

Learn more: Research Parent

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Validity, Accuracy and Reliability Explained with Examples

This is part of the NSW HSC science curriculum part of the Working Scientifically skills.

Part 1 – Validity

Part 2 – Accuracy

Part 3 – Reliability

Science experiments are an essential part of high school education, helping students understand key concepts and develop critical thinking skills. However, the value of an experiment lies in its validity, accuracy, and reliability. Let's break down these terms and explore how they can be improved and reduced, using simple experiments as examples.

Target Analogy to Understand Accuracy and Reliability

The target analogy is a classic way to understand the concepts of accuracy and reliability in scientific measurements and experiments. 

Accuracy refers to how close a measurement is to the true or accepted value. In the analogy, it's how close the arrows come to hitting the bullseye (represents the true or accepted value).

Reliability  refers to the consistency of a set of measurements. Reliable data can be reproduced under the same conditions. In the analogy, it's represented by how tightly the arrows are grouped together, regardless of whether they hit the bullseye. Therefore, we can have scientific results that are reliable but inaccurate.

• Validity  refers to how well an experiment investigates the aim or tests the underlying hypothesis. While validity is not represented in this target analogy, the validity of an experiment can sometimes be assessed by using the accuracy of results as a proxy. Experiments that produce accurate results are likely to be valid as invalid experiments usually do not yield accurate result.

Validity refers to how well an experiment measures what it is supposed to measure and investigates the aim.

Ask yourself the questions:

• "Is my experimental method and design suitable?"
• "Is my experiment testing or investigating what it's suppose to?"

For example, if you're investigating the effect of the volume of water (independent variable) on plant growth, your experiment would be valid if you measure growth factors like height or leaf size (these would be your dependent variables).

However, validity entails more than just what's being measured. When assessing validity, you should also examine how well the experimental methodology investigates the aim of the experiment.

Assessing Validity

An experiment’s procedure, the subsequent methods of analysis of the data, the data itself, and the conclusion you draw from the data, all have their own associated validities. It is important to understand this division because there are different factors to consider when assessing the validity of any single one of them. The validity of an experiment as a whole , depends on the individual validities of these components.

When assessing the validity of the procedure , consider the following:

• Does the procedure control all necessary variables except for the dependent and independent variables? That is, have you isolated the effect of the independent variable on the dependent variable?
• Does this effect you have isolated actually address the aim and/or hypothesis?
• Does your method include enough repetitions for a reliable result? (Read more about reliability below)

When assessing the validity of the method of analysis of the data , consider the following:

• Does the analysis extrapolate or interpolate the experimental data? Generally, interpolation is valid, but extrapolation is invalid. This because by extrapolating, you are ‘peering out into the darkness’ – just because your data showed a certain trend for a certain range it does not mean that this trend will hold for all.
• Does the analysis use accepted laws and mathematical relationships? That is, do the equations used for analysis have scientific or mathematical base? For example, `F = ma` is an accepted law in physics, but if in the analysis you made up a relationship like `F = ma^2` that has no scientific or mathematical backing, the method of analysis is invalid.
• Is the most appropriate method of analysis used? Consider the differences between using a table and a graph. In a graph, you can use the gradient to minimise the effects of systematic errors and can also reduce the effect of random errors. The visual nature of a graph also allows you to easily identify outliers and potentially exclude them from analysis. This is why graphical analysis is generally more valid than using values from tables.

When assessing the validity of your results , consider the following: 

• Is your primary data (data you collected from your own experiment) BOTH accurate and reliable? If not, it is invalid.
• Are the secondary sources you may have used BOTH reliable and accurate?

When assessing the validity of your conclusion , consider the following:

• Does your conclusion relate directly to the aim or the hypothesis?

How to Improve Validity

Ways of improving validity will differ across experiments. You must first identify what area(s) of the experiment’s validity is lacking (is it the procedure, analysis, results, or conclusion?). Then, you must come up with ways of overcoming the particular weakness. 

Below are some examples of this.

Example – Validity in Chemistry Experiment 

Let's say we want to measure the mass of carbon dioxide in a can of soft drink.

The following steps are followed:

• Weigh an unopened can of soft drink on an electronic balance.
• Open the can.
• Place the can on a hot plate until it begins to boil.
• When cool, re-weigh the can to determine the mass loss.

To ensure this experiment is valid, we must establish controlled variables:

• type of soft drink used
• temperature at which this experiment is conducted
• period of time before soft drink is re-weighed

Despite these controlled variables, this experiment is invalid because it actually doesn't help us measure the mass of carbon dioxide in the soft drink. This is because by heating the soft drink until it boils, we are also losing water due to evaporation. As a result, the mass loss measured is not only due to the loss of carbon dioxide, but also water. A simple way to improve the validity of this experiment is to not heat it; by simply opening the can of soft drink, carbon dioxide in the can will escape without loss of water.

Example – Validity in Physics Experiment

Let's say we want to measure the value of gravitational acceleration `g` using a simple pendulum system, and the following equation:

$$T = 2\pi \sqrt{\frac{l}{g}}$$

• `T` is the period of oscillation
• `l` is the length of string attached to the mass
• `g` is the acceleration due to gravity

• Cut a piece of a string or dental floss so that it is 1.0 m long.
• Attach a 500.0 g mass of high density to the end of the string.
• Attach the other end of the string to the retort stand using a clamp.
• Starting at an angle of less than 10º, allow the pendulum to swing and measure the pendulum’s period for 10 oscillations using a stopwatch.
• Repeat the experiment with 1.2 m, 1.5 m and 1.8 m strings.

The controlled variables we must established in this experiment include:

• mass used in the pendulum
• location at which the experiment is conducted

The validity of this experiment depends on the starting angle of oscillation. The above equation (method of analysis) is only true for small angles (`\theta < 15^{\circ}`) such that `\sin \theta = \theta`. We also want to make sure the pendulum system has a small enough surface area to minimise the effect of air resistance on its oscillation.

In this instance, it would be invalid to use a pair of values (length and period) to calculate the value of gravitational acceleration. A more appropriate method of analysis would be to plot the length and period squared to obtain a linear relationship, then use the value of the gradient of the line of best fit to determine the value of `g`. 

Accuracy refers to how close the experimental measurements are to the true value.

Accuracy depends on

• the validity of the experiment
• the degree of error:
• systematic errors are those that are systemic in your experiment. That is, they effect every single one of your data points consistently, meaning that the cause of the error is always present. For example, it could be a badly calibrated temperature gauge that reports every reading 5 °C above the true value.
• random errors are errors that occur inconsistently. For example, the temperature gauge readings might be affected by random fluctuations in room temperature. Some readings might be above the true value, some might then be below the true value.
• sensitivity of equipment used.

Assessing Accuracy 

The effect of errors and insensitive equipment can both be captured by calculating the percentage error:

$$\text{% error} = \frac{\text{|experimental value – true value|}}{\text{true value}} \times 100%$$

Generally, measurements are considered accurate when the percentage error is less than 5%. You should always take the context of the experimental into account when assessing accuracy. 

While accuracy and validity have different definitions, the two are closely related. Accurate results often suggest that the underlying experiment is valid, as invalid experiments are unlikely to produce accurate results.

In a simple pendulum experiment, if your measurements of the pendulum's period are close to the calculated value, your experiment is accurate. A table showing sample experimental measurements vs accepted values from using the equation above is shown below. 

All experimental values in the table above are within 5% of accepted (theoretical) values, they are therefore considered as accurate. 

How to Improve Accuracy

• Remove systematic errors : for example, if the experiment’s measuring instruments are poorly calibrated, then you should correctly calibrate it before doing the experiment again.
• Reduce the influence of random errors : this can be done by having more repetitions in the experiment and reporting the average values. This is because if you have enough of these random errors – some above the true value and some below the true value – then averaging them will make them cancel each other out This brings your average value closer and closer to the true value.
• Use More Sensitive Equipments: For example, use a recording to measure time by analysing motion of an object frame by frame, instead of using a stopwatch. The sensitivity of an equipment can be measured by the limit of reading . For example, stopwatches may only measure to the nearest millisecond – that is their limit of reading. But recordings can be analysed to the frame. And, depending on the frame rate of the camera, this could mean measuring to the nearest microsecond.
• Obtain More Measurements and Over a Wider Range:  In some cases, the relationship between two variables can be more accurately determined by testing over a wider range. For example, in the pendulum experiment, periods when strings of various lengths are used can be measured. In this instance, repeating the experiment does not relate to reliability because we have changed the value of the independent variable tested.

Reliability

Reliability involves the consistency of your results over multiple trials.

Assessing Reliability

The reliability of an experiment can be broken down into the reliability of the procedure and the reliability of the final results.

The reliability of the procedure refers to how consistently the steps of your experiment produce similar results. For example, if an experiment produces the same values every time it is repeated, then it is highly reliable. This can be assessed quantitatively by looking at the spread of measurements, using statistical tests such as greatest deviation from the mean, standard deviations, or z-scores.

Ask yourself: "Is my result reproducible?"

The reliability of results cannot be assessed if there is only one data point or measurement obtained in the experiment. There must be at least 3. When you're repeating the experiment to assess the reliability of its results, you must follow the  same steps , use the  same value  for the independent variable. Results obtained from methods with different steps cannot be assessed for their reliability.

Obtaining only one measurement in an experiment is not enough because it could be affected by errors and have been produced due to pure chance. Repeating the experiment and obtaining the same or similar results will increase your confidence that the results are reproducible (therefore reliable).

In the soft drink experiment, reliability can be assessed by repeating the steps at least three times:

The mass loss measured in all three trials are fairly consistent, suggesting that the reliability of the underly method is high.

The reliability of the final results refers to how consistently your final data points (e.g. average value of repeated trials) point towards the same trend. That is, how close are they all to the trend line? This can be assessed quantitatively using the `R^2` value. `R^2` value ranges between 0 and 1, a value of 0 suggests there is no correlation between data points, and a value of 1 suggests a perfect correlation with no variance from trend line.

In the pendulum experiment, we can calculate the `R^2` value (done in Excel) by using the final average period values measured for each pendulum length.

Here, a `R^2` value of 0.9758 suggests the four average values are fairly close to the overall linear trend line (low variance from trend line). Thus, the results are fairly reliable. 

How to Improve Reliability

A common misconception is that increasing the number of trials increases the reliability of the procedure . This is not true. The only way to increase the reliability of the procedure is to revise it. This could mean using instruments that are less susceptible to random errors, which cause measurements to be more variable.

Increasing the number of trials actually increases the reliability of the final results . This is because having more repetitions reduces the influence of random errors and brings the average values closer to the true values. Generally, the closer experimental values are to true values, the closer they are to the true trend. That is, accurate data points are generally reliable and all point towards the same trend.

Reliable but Inaccurate / Invalid

It is important to understand that results from an experiment can be reliable (consistent), but inaccurate (deviate greatly from theoretical values) and/or invalid. In this case, your procedure  is reliable, but your final results likely are not.

Examples of Reliability

Using the soft drink example again, if the mass losses measured for three soft drinks (same brand and type of drink) are consistent, then it's reliable. 

Using the pendulum example again, if you get similar period measurements every time you repeat the experiment, it’s reliable.  

However, in both cases, if the underlying methods are invalid, the consistent results would be invalid and inaccurate (despite being reliable).

Do you have trouble understanding validity, accuracy or reliability in your science experiment or depth study?

Consider getting personalised help from our 1-on-1 mentoring program !

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What is chemistry?

Chemistry is the branch of science that deals with the properties, composition, and structure of elements and compounds , how they can change, and the energy that is released or absorbed when they change.

How are chemistry and biology related?

Chemistry is the study of substances—that is, elements and compounds —while biology is the study of living things. However, these two branches of science meet in the discipline of biochemistry , which studies the substances in living things and how they change within an organism.

Recent News

chemistry , the science that deals with the properties, composition , and structure of substances (defined as elements and compounds), the transformations they undergo, and the energy that is released or absorbed during these processes. Every substance, whether naturally occurring or artificially produced, consists of one or more of the hundred-odd species of atoms that have been identified as elements. Although these atoms, in turn, are composed of more elementary particles, they are the basic building blocks of chemical substances; there is no quantity of oxygen , mercury , or gold , for example, smaller than an atom of that substance. Chemistry, therefore, is concerned not with the subatomic domain but with the properties of atoms and the laws governing their combinations and how the knowledge of these properties can be used to achieve specific purposes.

The great challenge in chemistry is the development of a coherent explanation of the complex behaviour of materials, why they appear as they do, what gives them their enduring properties, and how interactions among different substances can bring about the formation of new substances and the destruction of old ones. From the earliest attempts to understand the material world in rational terms, chemists have struggled to develop theories of matter that satisfactorily explain both permanence and change. The ordered assembly of indestructible atoms into small and large molecules , or extended networks of intermingled atoms, is generally accepted as the basis of permanence, while the reorganization of atoms or molecules into different arrangements lies behind theories of change. Thus chemistry involves the study of the atomic composition and structural architecture of substances, as well as the varied interactions among substances that can lead to sudden, often violent reactions.

Chemistry also is concerned with the utilization of natural substances and the creation of artificial ones. Cooking , fermentation , glass making, and metallurgy are all chemical processes that date from the beginnings of civilization. Today, vinyl, Teflon, liquid crystals, semiconductors , and superconductors represent the fruits of chemical technology. The 20th century saw dramatic advances in the comprehension of the marvelous and complex chemistry of living organisms, and a molecular interpretation of health and disease holds great promise. Modern chemistry, aided by increasingly sophisticated instruments, studies materials as small as single atoms and as large and complex as DNA (deoxyribonucleic acid), which contains millions of atoms. New substances can even be designed to bear desired characteristics and then synthesized. The rate at which chemical knowledge continues to accumulate is remarkable. Over time more than 8,000,000 different chemical substances, both natural and artificial, have been characterized and produced. The number was less than 500,000 as recently as 1965.

Intimately interconnected with the intellectual challenges of chemistry are those associated with industry. In the mid-19th century the German chemist Justus von Liebig commented that the wealth of a nation could be gauged by the amount of sulfuric acid it produced. This acid, essential to many manufacturing processes, remains today the leading chemical product of industrialized countries. As Liebig recognized, a country that produces large amounts of sulfuric acid is one with a strong chemical industry and a strong economy as a whole. The production, distribution, and utilization of a wide range of chemical products is common to all highly developed nations. In fact, one can say that the “iron age” of civilization is being replaced by a “polymer age,” for in some countries the total volume of polymers now produced exceeds that of iron .

The scope of chemistry

The days are long past when one person could hope to have a detailed knowledge of all areas of chemistry. Those pursuing their interests into specific areas of chemistry communicate with others who share the same interests. Over time a group of chemists with specialized research interests become the founding members of an area of specialization. The areas of specialization that emerged early in the history of chemistry, such as organic, inorganic, physical , analytical , and industrial chemistry, along with biochemistry , remain of greatest general interest. There has been, however, much growth in the areas of polymer, environmental, and medicinal chemistry during the 20th century. Moreover, new specialities continue to appear, as, for example, pesticide, forensic , and computer chemistry.

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Scientific Theory Definition and Examples

A scientific theory is a well-established explanation of some aspect of the natural world. Theories come from scientific data and multiple experiments. While it is not possible to prove a theory, a single contrary result using the scientific method can disprove it. In other words, a theory is testable and falsifiable.

Examples of Scientific Theories

There are many scientific theory in different disciplines:

• Astronomy : theory of stellar nucleosynthesis , theory of stellar evolution
• Biology : cell theory, theory of evolution, germ theory, dual inheritance theory
• Chemistry : atomic theory, Bronsted Lowry acid-base theory , kinetic molecular theory of gases , Lewis acid-base theory , molecular theory, valence bond theory
• Geology : climate change theory, plate tectonics theory
• Physics : Big Bang theory, perturbation theory, theory of relativity, quantum field theory

Criteria for a Theory

In order for an explanation of the natural world to be a theory, it meets certain criteria:

• A theory is falsifiable. At some point, a theory withstands testing and experimentation using the scientific method.
• A theory is supported by lots of independent evidence.
• A theory explains existing experimental results and predicts outcomes of new experiments at least as well as other theories.

Difference Between a Scientific Theory and Theory

Usually, a scientific theory is just called a theory. However, a theory in science means something different from the way most people use the word. For example, if frogs rain down from the sky, a person might observe the frogs and say, “I have a theory about why that happened.” While that theory might be an explanation, it is not based on multiple observations and experiments. It might not be testable and falsifiable. It’s not a scientific theory (although it could eventually become one).

Value of Disproven Theories

Even though some theories are incorrect, they often retain value.

For example, Arrhenius acid-base theory does not explain the behavior of chemicals lacking hydrogen that behave as acids. The Bronsted Lowry and Lewis theories do a better job of explaining this behavior. Yet, the Arrhenius theory predicts the behavior of most acids and is easier for people to understand.

Another example is the theory of Newtonian mechanics. The theory of relativity is much more inclusive than Newtonian mechanics, which breaks down in certain frames of reference or at speeds close to the speed of light . But, Newtonian mechanics is much simpler to understand and its equations apply to everyday behavior.

Difference Between a Scientific Theory and a Scientific Law

The scientific method leads to the formulation of both scientific theories and laws . Both theories and laws are falsifiable. Both theories and laws help with making predictions about the natural world. However, there is a key difference.

A theory explains why or how something works, while a law describes what happens without explaining it. Often, you see laws written in the form of equations or formulas.

Theories and laws are related, but theories never become laws or vice versa.

Theory vs Hypothesis

A hypothesis is a proposition that is tested via an experiment. A theory results from many, many tested hypotheses.

Theory vs Fact

Theories depend on facts, but the two words mean different things. A fact is an irrefutable piece of evidence or data. Facts never change. A theory, on the other hand, may be modified or disproven.

Difference Between a Theory and a Model

Both theories and models allow a scientist to form a hypothesis and make predictions about future outcomes. However, a theory both describes and explains, while a model only describes. For example, a model of the solar system shows the arrangement of planets and asteroids in a plane around the Sun, but it does not explain how or why they got into their positions.

• Frigg, Roman (2006). “ Scientific Representation and the Semantic View of Theories .”  Theoria . 55 (2): 183–206. 
• Halvorson, Hans (2012). “What Scientific Theories Could Not Be.”  Philosophy of Science . 79 (2): 183–206. doi: 10.1086/664745
• McComas, William F. (December 30, 2013).  The Language of Science Education: An Expanded Glossary of Key Terms and Concepts in Science Teaching and Learning . Springer Science & Business Media. ISBN 978-94-6209-497-0.
• National Academy of Sciences (US) (1999). Science and Creationism: A View from the National Academy of Sciences (2nd ed.). National Academies Press. doi: 10.17226/6024  ISBN 978-0-309-06406-4. 
• Suppe, Frederick (1998). “Understanding Scientific Theories: An Assessment of Developments, 1969–1998.”  Philosophy of Science . 67: S102–S115. doi: 10.1086/392812

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[ noun ik- sper - uh -m uh nt ; verb ek- sper - uh -ment ]

a chemical experiment; a teaching experiment; an experiment in living.

a product that is the result of long experiment.

Synonyms: investigation , research

• Obsolete. experience .

verb (used without object)

to experiment with a new procedure.

• a test or investigation, esp one planned to provide evidence for or against a hypothesis: a scientific experiment
• the act of conducting such an investigation or test; experimentation; research

a poetic experiment

• an obsolete word for experience
• intr to make an experiment or experiments

/ ĭk-spĕr ′ ə-mənt /

• A test or procedure carried out under controlled conditions to determine the validity of a hypothesis or make a discovery.
• See Note at hypothesis

Derived Forms

• exˈperiˌmenter , noun

Other Words From

• ex·peri·menter ex·peri·mentor ex·peri·men·tator noun
• preex·peri·ment noun
• proex·peri·ment adjective
• reex·peri·ment verb (used without object) noun
• unex·peri·mented adjective

Word History and Origins

Origin of experiment 1

Synonym Study

Related words.

• examination
• experimentation
• observation
• undertaking

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• Open access
• Published: 10 August 2024

Covalent penicillin-protein conjugates elicit anti-drug antibodies that are clonally and functionally restricted

• Lachlan P. Deimel   ORCID: orcid.org/0000-0003-3803-871X 1   nAff9 ,
• Lucile Moynié 2 ,
• Guoxuan Sun 2 ,
• Viliyana Lewis   ORCID: orcid.org/0000-0002-4882-336X 2 ,
• Abigail Turner   ORCID: orcid.org/0000-0002-3995-6558 2 ,
• Charles J. Buchanan   ORCID: orcid.org/0000-0002-1840-5706 2 , 3 , 4 ,
• Sean A. Burnap 4 , 5 ,
• Mikhail Kutuzov   ORCID: orcid.org/0000-0003-3386-4350 1 ,
• Carolin M. Kobras 1 ,
• Yana Demyaneko   ORCID: orcid.org/0000-0002-6628-1912 2 , 6 ,
• Shabaz Mohammed   ORCID: orcid.org/0000-0003-2640-9560 2 , 3 , 5 ,
• Mathew Stracy 1 ,
• Weston B. Struwe   ORCID: orcid.org/0000-0003-0594-226X 4 , 5 ,
• Andrew J. Baldwin   ORCID: orcid.org/0000-0001-7579-8844 2 , 3 , 4 ,
• James Naismith   ORCID: orcid.org/0000-0001-6744-5061 2 ,
• Benjamin G. Davis   ORCID: orcid.org/0000-0002-5056-407X 2 , 3 , 6 &
• Quentin J. Sattentau   ORCID: orcid.org/0000-0001-7170-1937 1 , 7 , 8  

Nature Communications volume  15 , Article number:  6851 ( 2024 ) Cite this article

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• Chemical biology
• NMR spectroscopy
• Structural biology

Many archetypal and emerging classes of small-molecule therapeutics form covalent protein adducts. In vivo, both the resulting conjugates and their off-target side-conjugates have the potential to elicit antibodies, with implications for allergy and drug sequestration. Although β-lactam antibiotics are a drug class long associated with these immunological phenomena, the molecular underpinnings of off-target drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, using the classical β-lactam penicillin G (PenG), we probe the B and T cell determinants of drug-specific IgG responses to such conjugates in mice. Deep B cell clonotyping reveals a dominant murine clonal antibody class encompassing phylogenetically-related IGHV1 , IGHV5 and IGHV10 subgroup gene segments. Protein NMR and x-ray structural analyses reveal that these drive structurally convergent binding modes in adduct-specific antibody clones. Their common primary recognition mechanisms of the penicillin side-chain moiety (phenylacetamide in PenG)—regardless of CDRH3 length—limits cross-reactivity against other β-lactam antibiotics. This immunogenetics-guided discovery of the limited binding solutions available to antibodies against side products of an archetypal covalent inhibitor now suggests future potential strategies for the ‘germline-guided reverse engineering’ of such drugs away from unwanted immune responses.

Introduction

In isolation, non-protein, low molecular weight compounds are typically non-immunogenic to the mammalian immune system. As exemplified by classical hapten-carrier biology, antibody responses against small molecules such as 4-hydroxy-3-nitrophenol acetyl (NP) require conjugation to a suitable carrier protein 1 . However, upon attachment to protein, resulting epitopes arise within an antigenic complex that may cross-link cognate B cell receptors (BCRs) and that are associated with peptidic components that may be presented to T helper (Th) cells; together, these can impart combined help to propagate a specific B cell population to a given attached compound 2 , 3 , 4 .

In principle, these mechanisms may extend to small-molecule drugs, particularly those with reactive functional groups that drive covalent conjugation with endogenous proteins under physiological conditions 5 , 6 . Unwanted immune responses to covalent bond-forming drugs, particularly in the form of anti-drug antibody (ADA) responses, include hypersensitivity and allergy reactions, the most severe of which can be life-threatening. Whilst the number of covalent bond-forming drugs (e.g. covalent inhibitors) in clinical use has been traditionally limited 7 , in recent years there has been strong renewed interest 8 , 9 yet notably little analysis of unwanted drug-immune system interactions.

The best-characterised examples of unwanted immunogenicity from drug-protein conjugates are β-lactam antibiotics, such as penicillin G (PenG) 10 . As the electrophilic source of its inhibitory activity, the β-lactam group of PenG may also drive background / side reactivity with off-target biological nucleophiles leading to protein conjugation via primary amine-containing sidechains of lysine (and potentially other nucleophilic residues including arginine, histidine, and cysteine), as has been observed under some buffer conditions 11 , 12 . Such protein-PenG complexes are the antigenic determinants of antibiotic hypersensitivity. The mechanistic underpinnings of the hypersensitivity reaction are immunologically heterologous, with the most common and well characterised being T helper (Th) cell-mediated (type IV) that may be elicited in up to 30% of the population 13 , 14 , 15 , 16 , 17 . However, the most clinically severe forms of drug hypersensitivity are antibody-mediated, particularly IgE-induced anaphylaxis 18 . IgG-mediated hypersensitivity is less severe but relatively common 18 , 19 .

Penicillin is one of the most frequent causes of anaphylaxis and anaphylaxis-related deaths in humans 19 . However, penicillin allergy diagnosis is currently highly inaccurate. Nearly 6% of the general population in the UK are recorded as having a penicillin allergy, yet more than 95% of these patients can ultimately tolerate this class of drug, indicating that most patients are falsely recorded as allergic 20 . Patients with a penicillin allergy record have an increased risk of Clostridioides difficile and Methicillin-resistant Staphylococcus aureus infections and death; this is presumably through increased use of alternatives to β-lactam antibiotics 21 . Furthermore, penicillin allergy diagnosis is associated with higher numbers of total antibiotic prescriptions 22 , undermining antimicrobial stewardship goals and increasing the risk for antimicrobial resistance 23 . A better understanding of the immunological basis of penicillin hypersensitivity is therefore vitally needed to help predict which antibiotic recipients are, or will become, allergic 24 , 25 , and to inform potential future deleterious immune reactions against new generations of covalent bond-forming drugs.

Notably, although the first descriptions of penicilloyl-directed serological responses were made in 1961 12 , key phenomena remain incompletely understood, including (i) the biochemical basis of PenG–protein adduction in vivo and in vitro; (ii) the relative immunogenicity of fully chemically characterised and purified PenG adducts; (iii) the immunophylogenetics of B cells specific to PenG-protein complexes; and (iv) the structure/function characteristics of antibody clones specific to these adducts.

Here, through systematic complementary biochemical, structural and clonotypic analyses of the relationship between the protein-conjugating properties of PenG and its immunogenicity in a mouse model, we now fully map the PenG-specific antibody response. We find that the ADA response is based upon a restricted cluster of highly related B cell germline clonal families that, regardless of CDRH3 length, engage the penicillin sidechain via conserved binding modes. These findings offer a rational basis for understanding ADA responses, and suggest that antibodies have limited binding solutions that in turn may inform drug ‘reverse engineering’ to avoid ADA.

Immunogenicity of ‘pre-complexed’ penicillin-protein conjugate antigen

PenG has constituent β-lactam, thiazolidine and benzylamide sidechain moieties (Fig.  1a ); the β-lactam ring is long known to react with nucleophilic protein sidechains, including the off-target ε-amino groups of lysine residues leading to the formation of subsequent ε-amide-drug adduct forms such as those formed via β-lactam ring opening 3 , 11 , 12 , 26 , 27 , 28 (Fig.  1b ). To probe PenG-protein off-target conjugation, we first titrated adduct formation on the model protein hen egg lysozyme (HEL, ~14 kDa, 129 a.a.), chosen for its stability and relatively evenly distributed (six) lysine (Lys) residues. Various buffer, drug amounts and pH conditions were tested in vitro, and global site-specific drug occupancy was evaluated via mass spectrometry (MS). These revealed pH- and buffer-modulated conjugation levels (Fig  S1 a; Document  S1 ). Mapping of adduct formation through tryptic digest followed by site-specific liquid chromatography tandem MS (LC-MS/MS) analysis confirmed adduct formation at all lysine residues 1, 13, 33, 96/97 and 116 (Fig  S1b ). These reaction data informed ex vivo conjugation of PenG to various recombinant carrier proteins at close-to-physiological pH for subsequent immunisation.

a Chemical structure of PenG. b Proposed target residues of electrophilic β-lactam; primary reported target of lysine with potential targeting of arginine and histidine. c 1 mg/mL HSA and PenG (1:200 per Lys) were mixed in vitro with 0.1 M HCO 3 -, pH = 8.0. This was left at 25 °C for 16 h before dialysis into PBS. d Sex-matched 6-week-old naïve WT C57BL/6 mice were twice immunized (wk 0 and 4) with 10 µg HSA or HSA-PenG in alum. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. e Terminal HEL-specific IgG EPT was evaluated. PenG-specific endpoint titres were evaluated by screening cross-reactivity against HEL-PenG. IgG titres against PenG were evaluated both ( f ) longitudinally and ( g ) at the terminal timepoint. h Competition ELISA was conducted, wherein HSA-PenG antisera binding for HEL-PenG was competed out with soluble PenG. i Longitudinal protein backbone-specific, HSA, and ( j ) terminal HSA-PenG-specific IgG endpoint titres were screened. e – j Dots represent data from a single animal ( n  = 4 per group), and bars/text denotes the median (ND = not detected). Groups were compared via Mann–Whitney test (two-sided). Source data are provided as a Source Data file.

Pilot immunogenicity analysis of HEL-PenG conjugates formulated in aluminium hydroxide (alum) adjuvant was conducted by subcutaneous (s.c.) administration to C57BL/6 mice. Antisera were titrated by ELISA on an unrelated PenG-modified protein (human serum albumin-PenG; HSA-PenG) to determine the titres specifically against the penicilloyl adduct. This revealed modest but significant ( P  < 0.05 Mann–Whitney U) isotype-switched IgG responses raised against the drug adduct (Fig  S1d–g ).

Although a useful model antigen for biochemical characterisation and pilot immunogenicity analysis, HEL is a weak Th cell antigen in mice 29 , 30 . We therefore next evaluated the antibody response generated by PenG pre-complexed to more antigenic HSA using the conditions optimised for HEL (Fig.  1c ). Site-specific occupancy of penicilloyl adducts was evaluated via LC-MS/MS and again diverse lysine occupancy was observed (Fig  S2 ). These occupancy data are concordant with previously published drug modification sites of HSA 28 , 31 , 32 , 33 . Mice were immunised with HSA or HSA-PenG formulated in alum, followed by periodic blood sampling (Fig.  1d ). Anti-penicilloyl serum IgG responses were measured by ELISA against HEL-PenG; no IgG cross-reactivity was detected against unmodified HEL (Fig.  1e ) . However, strikingly, post-prime HSA-PenG antisera displayed considerable IgG reactivity with HEL-PenG, whereas no reactivity was detected in the HSA-alone antiserum (Fig.  1f ). Together, these immediately suggested an anti-PenG-adduct-specific response. Post-boost and at the terminal timepoint, the median HEL-PenG-specific IgG endpoint titre (EPT) was marked at ~2.2 × 10 5 for the HSA-PenG antisera and a near-baseline EPT of ~1.7 × 10 2 for the control HSA antisera ( P  = 0.029, Mann-Whitney test) (Fig.  1g ). Analysis of CD4 + Th responses revealed significant T cell proliferation and IFN-γ production only in the HSA-PenG-restimulated cells, consistent with the adducted protein being most efficiently captured and processed by B cells and presented to Th cells (Fig  S3 ).

The observed antibody responses were generated against a protein-conjugated PenG derivative. Characterisation was consistent with direct β-lactam opening, but we cannot discount a pathway involving intermediates of penicillanic acid (Document  S1 ) 27 , 28 . HSA-PenG antiserum binding to HEL-PenG was out-competed by free PenG, with a median IC 50 of 1.8 mM, while kanamycin (an unrelated non-β-lactam-type antibiotic) failed to detectably compete for antibody binding (Fig.  1h ). These data reveal that antibodies raised against the autologous PenG adduct are cross-reactive with free PenG, suggesting recognition of a common motif that is not the ‘opened’ β-lactam. Administration of HSA-PenG did not affect the antibody responses against the protein unmodified protein backbone, compared with mice immunised with HSA alone ( P  > 0.9999, Mann–Whitney test) (Fig.  1i,j ).

Self-protein carriers elicit penicillin-specific antibodies

Having demonstrated strong B cell immunogenicity of PenG conjugated to foreign proteins (HEL or HSA), we next tested a native and otherwise tolerogenic self-antigen, mouse serum albumin (MSA), as a more physiologically relevant model 34 . First, pure MSA was pre-complexed with PenG in buffer under the optimised conditions, as described previously, and the resultant MSA-PenG was analysed via LC-MS/MS (Fig  S4 ). Animals were immunised three times (wk 0, 4 and 8) with or without alum and terminal (wk 10) IgG antibody titres were evaluated by ELISA against OVA-PenG (Fig.  2a ). 5/6 (83%) animals immunised with MSA-PenG in alum elicited a detectable serum endpoint titre against the adduct (median titre of ~ 5.2 × 10 3 ) and 3/8 (38%) responders in those immunised with MSA-PenG even without adjuvant (Fig.  2b ). Surprisingly, these data show that extrinsic adjuvantation is not required to elicit anti-adduct IgG responses even when presented on a modified self-protein. By contrast, no animal immunised with unmodified MSA alone with or without alum gave a detectable PenG-specific response ( P  = 0.033, Kruskal-Wallis test) (Fig.  2b ). Although the MSA-PenG-elicited PenG-specific antibody titres were lower compared with HSA-PenG (Figs.  1a , 2b ), these data show that a penicillin-specific IgG response can be generated even when using an otherwise highly T-immunorecessive ‘self-derived’ backbone, and that self-proteins can act as ‘non-self’ immunogenic carriers when modified by drug. No reactivity was detected against OVA in any group (Fig.  2c ).

a Sex-matched C57BL/6 mice were immunized three times and the serological response at the terminal timepoint (wk 10) was evaluated. b , c Mice were immunized three times (wk 0, 4 and 8) and the terminal (wk 10) IgG PenG-specific EPTs were evaluated. d Mice were bled and 2 mg/mL PenG was added and mixed end-to-end overnight. Animals were subsequently immunized (wk 0 and 4) intravenously with seum-PenG with or without alum. e , f Terminal IgG EPTs were evaluated. b , c , e , f Dots represent data from a single animal ( n  = 4–8 per group), and bars/text denotes the median (ND = not detected). Data were compared via a Kruskal–Wallis test (two-sided). g PenG-Ben structure. h Mice were given PenG-Ben intramuscularly. i PenG-specific IgG titres were evaluated. Dots represent data from a single animal ( n  = 27). Data were compared to pre-administration, evaluating the ratio of responders via Chi-squared test. a , d , h Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.

Finally, to develop an ex vivo model for self-protein:PenG adduct formation and immunogenicity, we tested the ability of complete mouse serum itself to act as a carrier for PenG. PenG-naïve mice were bled, serum isolated and incubated ex vivo with 2 mg/mL PenG for 16 h. This concentration was chosen to mimic a typical serum concentration in humans 35 . The resulting serum-adduct mixture was then administered intravenously (i.v.), into the autologous mice, with and without alum, to mimic a clinical route of penicillin administration (Fig.  2d ). Terminal IgG endpoint titres were evaluated against OVA-PenG, which revealed 6/8 (75%) of animals primed and boosted with adjuvanted serum-PenG responded with a detectable IgG titre against OVA-PenG and a median titre of ~ 2.5 × 10 3 (Fig.  2e,f ). Interestingly, only a single animal that received unadjuvanted serum-PenG gave a detectable PenG-specific IgG titre, implying that adjuvantation, such as might be generated by bacterial infection during therapeutic penicillin use, is likely required under such conditions to overcome a threshold for immunogenicity.

Immunogenicity of free penicillin via varying administration routes

Having shown that PenG is immunogenic when conjugated to diverse protein carriers including mouse serum, we next evaluated whether free penicillin, as would be administered in the clinic, might be sufficient to induce an antigen-specific antibody response. First, we tested whether PenG delivered i.v. daily to mice in 2 × 1 week-long courses was immunogenic (Fig  S5a ). However, no IgG or IgM drug-specific responses were detected when compared to mice given control PBS (Fig  S5b, c ). Second, the immunogenicity of orally administered antibiotic was evaluated, using the gut-stable oxo-homologue penicillin V (PenV). Unlike PenG, PenV is used for oral administration as it does not degrade under the acidic conditions of the stomach 36 . Mice were given PenV ad libitum for two 3.5-day intervals. Some mice were additionally given an i.p. dose of lipopolysaccharide (LPS) (0.5 mg/kg) to mimic possible systemic increase in endotoxin expected from a bacterial infection, where antibiotics such as PenG/V would be clinically used (Fig  S5d ). Despite this, no specific PenG titres were observed (Fig  S5e ). Notably, administration of LPS increased the background reactivity of serum: mice given PenG and LPS or drug-free water and LPS-only both exhibited modest reactivity against HSA-PenG, which we attribute to induction of B cells producing polyspecific IgG responses 37 .

Free drugs can be rapidly cleared; for instance, mice have an extremely high cardiac output 38 —renal clearance of PenG is efficient 39 , with a previously reported half-life of approximately 15 min. Fast clearance will restrict in vivo PenG adduct formation, ultimately reducing the probability for antigen-B cell encounter and BCR cross-linking. Therefore, to extend the availability of free drug in vivo, penicillin G benzathine (PenG-Ben) was used. This is a formulation of PenG as its dibenzylethylene diammonium salt that renders PenG effective for use in slow-release delivery (Fig.  2g ). When administered intramuscularly, PenG-Ben is solubilised over days to weeks, to release PenG systemically 40 . PenG-Ben was administered intramuscularly (i.m.) to 27 mice (Fig.  2h ). After the antibiotic course, 5/27 (18%) mice generated IgG responses to OVA-PenG, significantly higher than the proportion from pre-immune serum (χ 2  = 5.51, P  = 0.019) (Fig.  2i ). Reactivity against unmodified OVA was not detected (Fig  S5f ).

Cross-reactivity of anti-penicillin-adduct IgG responses are drug side-chain and core focused

PenG, of course, shares common structural homology with the other penicillins as well as other β-lactam antibiotics. We therefore screened the cross-reactivity of the HSA-PenG serological response. Ovalbumin (OVA) was modified with a set of penicillin antibiotics with differing side chains (Fig.  3 ), and with β-lactam antibiotics from other classes (including cephalosporins and carbapenems), using the previously determined conditions (Fig.  1c ). Extent of modification by drug was confirmed by evaluating reduced primary amine availability (Fig  S6 ). Autologous reactivity against OVA-PenG was the greatest of the diverse OVA-X panel tested, with a median IgG EPT of ~2.5 × 10 6 (Fig.  3a ). Interestingly, there was limited reactivity against OVA-ampicillin (median EPT of ~2.4 × 10 3 ), which differs only in a single benzylic amine substituent, and similarly carbenicillin (median EPT of ~ 4.9 × 10 3 ), which differs by its benzylic carboxyl substituent. However, there was considerable cross-reactivity against OVA-oxacillin (median EPT of ~ 8.5 × 10 4 ), despite the greater variation in sidechain compared with ampicillin. These data suggest that the polyclonal response tolerates some change in side-chain but that this may also be blocked by simple alterations at pivotal sites (such as the ampicillin H→NH 2 , or the carbenicillin H→COOH change). A subset of 1 st –4 th generation cephalosporin- and carbapenem-type antibiotics were also screened for cross-reactivity. HSA-PenG antisera displayed limited (albeit above the detection limit) cross-reactivity against these modified OVA antigens (IgG EPTs ~ 10 3 ). Since cephalosporins and carbapenems have differing β-lactam-encompassing cores 41 , these data suggested that the PenG-raised antibody response may be in part dependent on the 6-aminopenicillanic acid-derived core.

a HSA-PenG antisera were screened against a set of β-lactam antibiotic-modified OVA. Data reflect the IgG EPT against the drug adducts, with dots denoting reactivity from a single animal ( n  = 4), with bars/text denoting the median. b Sidechains of penicillins tested. Source data are provided as a Source Data file.

Clonotypic B cell responses to PenG adducts

To evaluate the B cell response at the clonal and molecular levels, PenG-specific B cells were isolated and variable regions cloned using techniques previously described 42 , 43 . Mice were immunised with HSA-PenG in alum and draining inguinal lymph nodes (iLN) were harvested 2 weeks post-prime (Fig.  4a ). To isolate PenG-specific B cells, requisite protein-based probes were synthesised by modifying another carrier protein (HIV-1 gp120) that we have validated as giving low background and high specificity in a other hapten-carrier contexts 29 . Gp120 was modified with PenG and then modified with fluorophore Alexa Fluor 647 using a corresponding NHS ester. We further tetramerised biotinylated gp120 with streptavidin-phycoerythrin. These antigen-displaying probes were then used to sort the PenG-specific B cells on pre-gated non-naïve (DUMP - B220 + IgD - ) B cells (Fig.  4b ; Fig  S7a ). B cells were sorted from four mice and cell clonality was inferred according to the VH sequences (Fig.  4c ).

a Immunisation schedule. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. b Antigen probe sorting strategy on pre-gated non-naïve B cells (DUMP - B220+IgD - ). c Inferred clonal families from the PenG probe-sorted B cells (coloured) and singletons (grey). d Inferred V H gene segment across the sequenced B cells. Bar segment sizes proportional to the number of B cells of the same V H origin. Joining of the segments connote the shared utilisation between mice. Segment colours reflect the IGHV subgroup. e Logo plots of the CDRH1 and CDRH2 amino acid sequences from all sequenced B cells in all animals. f Fab binding specificity assay setup. g ELISA trace for a subset of 28 Fabs and ( h ) their OD1.25 intersection (estimate for EC 50 ) values. BAR-1 (grey) is an unrelated negative control specific to sialyllactose. Dots represent data from a single Fab clone ( n  = 28). Source data are provided as a Source Data file.

Considerable sharing of V H gene segments was observed between the mice, suggesting that similar clonotypes were raised across animals (Fig.  4d ; Fig  S7b ). Notably, the V H gene segments utilised were from four highly phylogenetically related subgroups: IGHV2 , IGHV5 , IGHV10 and IGHV14 . This striking homology is reflected in conservation of the CDRH1 and CDRH2 amino acid sequences across the mice (Fig.  4e ). Together, these data suggest that there are preferred structural and functional motifs encoded in these germline segments that facilitate binding with the drug. Immunogenetic analysis revealed a defined and ordered CDRH3; the length was bimodal, either short (5–6 aa) or long (17 aa) (Fig  S8a ). Short CDRH3s were dominated by an ARG motif for the first three residues, with a diverse C-terminal end, while the long class possesses a negative N-terminus, a neutral centre and a positive C-terminus (Fig  S8b ).

Clonal families were evaluated. The two largest families were isolated from mouse 1 and their germinal centre trees determined (Fig  S9 ). The largest clonal family (Fig  S6a ) exhibits a ‘clonal burst’ following the acquisition of the S11T mutation, a characteristic phenomenon reportedly associated with the acquisition of an affinity-improving mutation that renders the clone more competitive for antigen uptake and T cell help 44 . This mutation was also found in a separate clade on the same tree. The T85S mutation was also identified twice on separate clades.

Finally, to validate the PenG specificity of the antibody response, a subset of V-region pairs from all mice and with diverse gene segment origins were cloned, and corresponding fragment antigen-binding regions (FAbs) were expressed and purified. Binding was validated via ELISA; all FAbs bound the PenG adduct probe, while an unrelated antibody FAb (BAR-1) did not detectably bind (Fig.  4f–h ). These data confirmed that the sorting approach was highly specific.

Structural, biochemical and biophysical characterisation of the antibody response to PenG

We selected a subset of PenG-specific clones with divergent, representative CDRH3 lengths to further dissect the binding characteristics of the clonotypical response to penicilloyl adducts: MIL-1 ( IGHV5-6*01 ; 17 a.a. CDRH3), MIL-2 ( IGHV5-17*01 ; 6 a.a. CDRH3) and MIL-3 ( IGHV10-3*01 ; 9 a.a. CDRH3). We designed a soluble Lys-PenG ligand as a reductionist adduct mimic reflecting β-lactam display on the ε-amine of lysine residues (Fig.  5a ).

a Annotated chemical structure of PenG-lys construct used for uSTA analysis. b Saturation transfer difference spectrum was generated from the difference between the raw off-resonance (gaussian at 37 ppm) and the raw on-resonance (gaussian at 9 ppm) spectra. Data showing differences in para engagement in the benzene ring of MIL-2 fab versus MIL-1. c i. Heatmaps corresponding to saturation transfer efficiency of PenG-lys (1 mM) with MIL-1–3 (5 µM). ii. Histographic saturation transfer efficiencies. Red indicates high transfer efficiency. d SPR chip design. e Biophysical characterization of MIL-3 via SPR. f i. Top view of the x-ray structure of the MIL-3 Fab bound to the PenG-Lys (beige sticks). Both heavy (monochrome plum) and light chain (monochrome blue) CDRs are marked. Key residues within 4.0 Å of ligand aspects ii. phylacetamide, iii. thiazolidine and iv. lysine are shown. Hydrogen bonds are shown as black broken lines. Water is marked in red. Source data are provided as a Source Data file.

First, atomic resolution of the binding pose was dissected by protein NMR using universal saturation transfer analysis (uSTA) 29 , 45 . High transfer efficiencies were observed on the phenyl ring of Lys-PenG with all three FAb fragments, with the p -proton showing the greatest engagement for FAbs MIL-1 and MIL-3 (Fig.  5b,c ), suggestive of an end-on binding mode for the PenG phenylacetamide sidechain. Notably, the Lys residue itself exhibited minimal transfer efficiency in all cases, confirming, as was implied by the immunological data, that binding is dominated by drug adduct rather than peptide binding. Interestingly, the thiazolidine ring showed no significant engagement of the protons that are detected by uSTA. This engagement was further confirmed by drug adduct core binding (although less than drug sidechain) through observed transfer efficiencies of the proton at the stereogenic centre of the opened β -lactam ring (NC H ) as well as those of the benzylic sidechain CH 2 . These data revealed substantial uniformity in the binding poses adopted by the MIL series of antibodies.

Next, we determined the X-ray crystal structure of the PenG-Lys•MIL-3 complex at 2.2 Å (Table  S1 ). Three FAb molecules were present in the asymmetric unit (H (heavy)/L (light), A/B and C/D). In both H/L and A/B molecules, the phenylacetamide PenG sidechain and the thiazolidine moieties are well-defined in the electron density whilst the Cβ, Cδ and Cγ portion of the lysine moiety has weak density, indicating it is less well ordered and consistent with our observations by protein NMR (see above). The electron density is considerably weaker in A/B than in H/L most likely due to crystal contacts present in H/L (Fig  S10 a, Fig  S11 ). Apart from this region, the interactions between the ligand and the protein are conserved in both FAbs. In the third FAb molecule (C/D), CDRH loops are highly disordered, and no ligand was fitted.

Our analysis focuses on the H/L molecules (Fig.  5f ). The PenG benzene group is deeply buried in a narrow hydrophobic pocket sandwiched on one face of the benzene ring by CDR L loops (Tyr68 L , Tyr110 L ) and the β-turn formed by CRDH3, mainly the side chain of Ile120 H with some involvement of the main chain of Phe124 H , on the other face (Fig.  5f ii ., Fig S10b ). The interaction with Tyr68 L has strong π-stacking character whilst the planes of the rings benzene and Tyr110 L are offset 70° and thus hydrophobic in character. The methyl group of Cγ2 Ile120 H sits centred above plane of the ring.

The static X-ray structure does not disclose a simple explanation for the observation from NMR for the interactions of the p -hydrogen yet provides detail on PenG sidechain recognition. The nitrogen of the phenylacetamide forms a hydrogen bond with the carbonyl of Thr121 H whilst the phenylacetamide carbonyl oxygen bridges via a water molecule (W1) to the side chain hydroxyl of Tyr110 L . The β-turn conformation of CDRH3 is stabilised by hydrogen bonds between residues Thr122 H and Arg123 H and Tyr 51 H and Asp75 L .The carboxylate of the thiazolidine moiety makes a bidentate salt bridge to the guanidine group of Arg69 H and potentially a salt bridge with His54 H (Fig.  5f iii .); this binding mode is likely to powerfully contribute to binding enthalpy yet places the protons that are observable by NMR more remotely. The dimethyl group and thiazolidine ring make van der Waal contacts with Tyr115 L and Ile120 H . The nitrogen of the thiazolidine ring interacts with a highly coordinated water molecule (W3) bridging CDRs H1, H3 and the lysine linker. The main-chain mimic of the Lys makes five hydrogen bonds with the 3 10 helix of CDRH2 in H/L (Fig.  5f iv .) but only one in A/B (Fig  S10c ), consistent with differences in ordering and previously noted weaker interactions (see above). Torsion angle modification of the lysine side chain would permit conjugated protein to remain outside the binding pocket without any perturbation of the drug adduct thiazolidine and phenylacetamide interactions.

Finally, surface plasmon resonance (SPR) of FAb MIL-3 with a PenG-adduct chip revealed a K D value = 5.3 µM (90% CI: 3.996–7.37, Fig.  5d,e ).

Antibody clones are unlikely to sequester antibiotic during relevant treatments

The biophysical properties of the antibodies that we determined, the structural biology and the physiological concentrations of antibody in vivo all suggested that anti-PenG-adduct antibodies are unlikely to display any drug-sequestering effects. Nevertheless, this was evaluated by developing a model system to test whether PenG-specific antibodies could reduce the bacterial growth-inhibiting function of PenG. Whole antiserum was added to a culture of attenuated, unencapsulated Streptococcus pneumoniae 46 , chosen because of its high sensitivity to PenG (minimum inhibitory concentration (MIC) = 0.01 µg/mL). We conducted two assays: first, we evaluated the effects of anti-HSA-PenG antiserum on PenG sequestration in culture and, second, contrasted PenG kill zones when drug was pre-incubated with high quantities of purified recombinant control or anti-PenG MIL antibodies (Fig  S12 ). As expected, neither antiserum nor recombinant MIL antibody significantly inhibited antibiotic action.

The elicitation of ADA such as those against PenG may have broad implications, including the mediation of drug hypersensitivity 11 , 18 , 47 , 48 . The complex relationship between chemical reactivity and instability, pharmacokinetics, immunological factors and possible downstream functional effects of the resulting humoral responses are poorly understood. To address this, we have systematically evaluated the way in which the protein conjugation reactivity of the common β-lactam antibiotic, PenG, drives formation of antigenic complexes in vitro, and have established that diverse protein carriers are sufficient to propagate a drug-specific IgG response. Using a murine model, we characterised both the clonal B cell and antibody responses revealing striking clonal restriction and strong conservation of antibody-drug binding features despite diversity in CDRH3 length. Our data demonstrate that the production of PenG-specific antibodies is regulated at two distinct levels: (1) the covalent formation of protein adducts via lysine-amide formation is influenced by reaction conditions and time, and (2) immune engagement, including innate recruitment—such as via adjuvant which in vivo would be mimicked by bacterially-elicited inflammation—and T cell help. These factors ultimately determine the overall probability and magnitude of a downstream B cell response against PenG. Collectively, our data now provide a model for how adduct formation and immune engagement trigger the humoral response, phenomena that may inform both the study of allergy and potentially provide a rational approach to predict and even mitigate anti-penicillin antibody responses.

In a human context, most patients undergoing standard courses of PenG to treat bacterial infection exhibit anti-penicilloyl IgG antibodies thereafter 10 , 49 , 50 . These data appear initially incongruent with our murine data since animals given formulations of free penicillin either intravenously or in drinking water failed to exhibit a specific response. However, it is noteworthy that these selective effects may be attributed to dosing; humans are given as much as 50 mg/kg every 4–6 h of PenG via an intravenous line 51 , 52 . Moreover, higher murine cardiac output and drug clearance rate results in shorter drug half-life 38 , 39 . A critical role for PenG concentration, which will be higher in human circulation, in driving relevant adduct formation is supported by the serological data gathered from mice given both static pre-formed serum-PenG immunisations and intramuscular slow-release PenG-Ben. We did not titrate the downstream differences in murine versus human adaptive immune responses to penicillin, although differences are reported in the literature, particularly T cell-mediated responses to drug adducts 14 .

These experiments were not designed to explicitly evaluate allergic outcomes in mice. Isotype-switching to IgE and the downstream mast cell-mediated and other modes of allergic reactivity are determined by both genetic factors in the form of atopic predisposition to IgE production, and environmental factors 53 , 54 . However, the functional effect of the antibody response can not only be a feature of the Fc effector function or isotype—for example, whether type I versus type II hypersensitivity is imparted—but also the inherent binding mode and biophysical properties of the antibody. Our data now show that the murine B cell repertoire responds to the drug adduct with a dominant clonotypic family that primarily engages the side-chain constituent, phenylacetamide, and the carboxylate group of the thiazolidine, as evidenced by our complementary uSTA 45 and x-ray structural characterisation. These data point to a striking homogeneity of response, implying that B cell receptors and subsequent antibodies may be restricted in the binding solutions that they can adopt to recognise such a small non-protein antigen. This is consistent with previous immunological mapping studies of murine 55 , 56 , rabbit 27 and human 57 antibody responses in which side-chain reactivity appears prominent. Importantly, the biophysical features of these antibodies, with monovalent FAb K D in the low–mid µM range and a relatively fast k off , coupled with relatively low concentrations in vivo, are likely to discount even partial drug inhibitory effects.

Our approach, applied here to an archetypal inhibitor with a covalent bond-forming mode-of-action, now creates a potential roadmap for understanding the chemical, pharmacological and immunological factors governing whether a B cell response can be mounted against other protein-reactive covalent therapeutics, for example, the amide-forming acylating drug aspirin 58 . Having identified a dominant clonotype against the PenG adduct, this excitingly suggests a workflow that could be used as a model to ‘reverse engineer’ a PenG analogue that fails to engage the MIL clonotype, potentially reflecting a low/no allergenic alternative against a murine germline and that now instructs a proof of principle for germline-informed drug design in humans.

Ethics and permissions

All experiments were conducted under approved licenses and protocols, consistent with national (as authorised by the Home Office of the United Kingdom) regulations and University of Oxford guidelines.

Ex vivo modification of protein with β-lactam antibiotics

Carrier proteins (HEL, OVA, HSA, BSA and MSA) were purchased commercially (Merck) and dissolved in 0.1 M NaCO 3 (pH = 8, unless otherwise indicated) and concentration was adjusted to 1 mg/mL. β-lactam was added to a molar ratio of 1:200 per lysine residue of carrier protein, resulting in protein concentrations for HEL and HSA of 6.8 and 60 mg/mL respectively. The mixture was rotated end-to-end at 25 °C overnight and dialysed into PBS.

Denaturing MS

Reversed-phase chromatography was performed in-line prior to mass spectrometry using an Agilent 1100 HPLC system (Agilent Technologies inc.—Palo Alto, CA, USA). Concentrated protein samples were diluted to 0.02 mg/ml in 0.1% formic acid and 50 µl was injected on to a 2.1 mm ×12.5 mm Zorbax 5um 300SB-C3 guard column housed in a column oven set at 40 °C. The solvent system used consisted of 0.1% formic acid in ultra-high purity water (Millipore) (solvent A) and 0.1 % formic acid in methanol (LC-MS grade, Chromasolve) (solvent B). Chromatography was performed as follows: Initial conditions were 90% A and 10% B and a flow rate of 1.0 mL/min. After 15 s at 10% B, a two-stage linear gradient from 10% B to 80% B was applied, over 45 s and then from 80% B to 95% B over 3 s. Elution then proceeded isocratically at 95% B for 1 mins 12 s followed by equilibration at initial conditions for a further 45 s. Protein intact mass was determined using a 1969 MSD-ToF electrospray ionisation orthogonal time-of-flight mass spectrometer (Agilent Technologies Inc.—Palo Alto, CA, USA). The instrument was configured with the standard ESI source and operated in positive ion mode. The ion source was operated with the capillary voltage at 4000 V, nebulizer pressure at 60 psig, drying gas at 350 °C and drying gas flow rate at 12 L/min. The instrument ion optic voltages were as follows: fragmentor 250 V, skimmer 60 V and octopole RF 250 V. Obtained MS spectra were processed and deconvoluted using the Agilent MassHunter Qualitative Analysis (B.07.00) software.

Approximately 5 µg protein was reduced, loaded and run on an SDS-PAGE. Gel bands were excised and washed sequentially with HPLC grade water followed by 1:1 (v/v) MeCN/H 2 O. Gel bands were dried (via vacuum centrifuge), treated with 10 mM dithiothreitol (DTT) in 100 mM NH 4 HCO 3 and incubated for 45 min at 56 °C with shaking. DTT was removed and 55 mM iodoacetamide (in 100 mM NH4HCO3) was added and incubated for 30 min in the dark. All liquid was removed and gels were washed with 100 mM NH 4 HCO 3 /MeCN as above. Gels were dried and 12.5 ng/µL trypsin was added separately and incubated overnight at 37 °C. Samples were then washed and peptides were extracted and pooled with sequential washes with 5% (v/v) formic acid (FA) in H 2 O and MeCN. Dried samples were reconstituted in 2% MeCN 0.05% trifluoroacetic acid and run by LC-MS.

Samples were analysed using an Ultimate 3000 UHPLC coupled to an Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific). Peptides were loaded onto a 75 µm × 2 cm pre-column and separated on a 75 µm × 15 cm Pepmap C18 analytical column (Thermo Fisher Scientific). Buffer A was 0.1% FA in H 2 O and buffer B was 0.1% FA in 80% MeCN with 20% H 2 O. A 40-min linear gradient (0% to 40% buffer B) was used. A universal HCD identification method was used. Data was collected in data-dependent acquisition mode with a mass range 375 to 1500 m/z and at a resolution of 70,000. For MS/MS scans, stepped HCD normalized energy was set to 27, 30 and 33% with orbitrap detection at a resolution of 35,000.

Raw data was first searched using the FragPipe (v19.1) 59 Open Search pipeline to determine the modified mass shift caused by PenG conjugation. Approximately 2.3% of peptide spectral matches (PSM) had an unannotated mass shift of 334.099 Da. Next, to determine site specificity and occupancy, raw data was searched using Proteome Discoverer (3.0.0.757). In-house curated FASTA databases were used. The digestion enzyme was set to trypsin with a maximum of 2 miss cleavages. A 10 ppm precursor mass tolerance and 0.6 Da fragment mass tolerance were allowed. Oxidation (+15.995 Da) of methionine and PenG conjugation (+334.099 Da) of lysines and protein N-termini were set to dynamic modifications. Carbamidomethylation (+57.021 Da) of cysteines was set as a static modification. Target false discovery rate (FDR) for peptide spectrum matches, peptide and protein identification was set to 1%. To approximate site-specific occupancy of PenG conjugation the total number of PSMs of peptides containing a specific lysine site in a PenG modified state were expressed as a percentage compared to the total number of PSMs containing the given lysine in both the modified and unmodified state.

To estimate lysine reactivity in HSA, raw data was searched against a database of known contaminants which contains the canonical HSA sequence using FragPipe (21.1) using a standard closed search parameters with an additional variable modification confined to lysines (+334.099). IonQuant (as implemented in FragPipe) was used to calculate peptide intensities with default parameters. Intensity of each modification-specific peptide was normalised against total intensity of all HSA peptides in treated and control samples. For each position, a ratio of intensity of unmodified peptide (without missed cleavages, as present in control sample) between the treated sample and the control was calculated to estimate the extent of lysine modification at that position after treatment.

LC-MS/MS raw data and search results have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository 60 with the dataset identifier: PXD052026.

Free primary amine ELISA

After the drugs were conjugated to a carrier protein using the method outlined above, the relative abundance of free amines was assessed to determine the extent of lysine modification. Protein samples (5 μg) were dissolved in 10 μL of PBS and mixed with 40 μL of 0.1 M sodium bicarbonate buffer. A 5% solution of 2,4,6-trinitrobenzenesulfonic acid (TNBSA) was diluted at a ratio of 1:500 in the bicarbonate buffer, and 25 μL of this mixture was added to the protein samples. Following a 2 h incubation period at 37 °C, 25 μL of 10% SDS and 12.5 μL of 1 M HCl were added. The absorbance at 335 nm was then measured.

Mice and immunisation formulations

Wild-type, specific pathogen-free, sex-matched, 6–8-week-old C57BL/6 mice were purchased from Charles River. Animals were monitored daily and were provided standard chow and water ad libitum . Immunisation formulations and schedules are outlined in the results. Mice were bled periodically from the tail vein. Animals were sacrificed via a rising CO 2 gradient and subsequent cervical dislocation schedule 1 procedure.

Serum samples were serially diluted and transferred onto an antigen-coated and blocked SpectraPlate-96 (PerkinElmer) plate. Binding was detected with an anti-mouse IgG-HRP (STAR120P, Bio-Rad). ELISAs were developed using 1-Step-Ultra TMB ELISA substrate (Life Technologies), terminating the reaction with 0.5 M H 2 SO 4 . For competition ELISAs, serial dilution of soluble ligands as preincubated with antiserum at the pre-determined EC 50 concentration for 1 h. The antisera and ligand mixtures were subsequently transferred onto the antigen-coated and blocked plates and ELISA conducted, as previously outlined. Cytokine ELISAs were performed using commercially available kits (Life Technologies), screening supernatant from antigen-restimulated splenocytes.

Optical densities were measured at 450 and 570 nm on a Spectramax M5 plate reader (Molecular Devices). After background subtraction, logistic dose-response curves were fitted in GraphPad Prism. Endpoint titres were determined as the point at which the best-fit curve reached an OD 450-570 value of 0.01, a value which was always > 2 standard deviations above background.

B cell sorting

Penicilloyl-specific B cells were isolated using antigen probes. Gp120-PenG was modified with an NHS-esterified AF647 dye, as per the manufacturer’s instructions (Life Technologies). To improve true antigen-specific cell sorting efficiency, a negative backbone-specific probe was synthesised, wherein biotinylated gp120 was tetramerised with PE-conjugated strepdavidin (Biolegend).

Single cell suspensions were stained with LIVE/DEAD Fixable Blue and Fc receptors blocked. Surface staining was performed using anti-mouse F4/80-PE (1:200, BM8, Biolegend), anti-mouse Gr-1 (1:200, RB6-8C5, Biolegend), anti-mouse CD3-PE (1:200, 17A2, Biolegend), anti-mouse CD4-PE (1:200, RM4-5, Biolegend), anti-mouse CD8-PE (1:200, RPA-T8, Biolegend), anti-mouse B220-eFluor450 (1:100, RA3-6B2, BD Biosciences), anti-mouse IgD-AF700 (1:200, 11-26 c.2a, Biolegend), anti-mouse IgM-PE/Cy7 (1:200, R6-60.2, BD Biosciences), anti-mouse IgG1-FITC (1:200, A85-1, BD Biosciences), anti-mouse IgG2a/2b-FITC (1:200, R2-40, BD Bioscience) and antigen probes (10 μg/mL). Cells were stained on ice for 1 h, washed and stored on a BD FACSAriaFusion (BD Biosciences). Single cells were sorted into MicroAmp Optical 96-well PCR plates (Life Technologies), isolating LIVE/DEAD - DUMP - B220 + IgD - gp120 - gp120-PenG + events. Cells were sorted directly into 5 µL if 1X TCK buffer supplemented with 1% 2-ME and stored at −80 °C until use.

Thymidine incorporation

Whole splenocytes were stimulated in vitro with 10 µg/mL antigen in cRMPI for 16 h in a flat-bottom 96-well plate. During the final 6 h stimulation, each well was spiked with 0.037 mBq tritiated thymidine (Perkin Elmer). Cells were transferred and lysed on glass filter mats (Perkin Elmer) using a Micro 96 Harvester (Skatron Instruments). Tritium incorporation was measured using Betaplate Scint and a Microbeta Trilux Scintilation counter (Perkin Elmer).

Intracellular cytokine staining

Whole splenocytes were stimulated in vitro with 10 µg/mL antigen in cRPMI for 16 h. For the final 6 h, 5 µg/mL brefeldin A (Biolegend) was added to suspend ET–Golgi trafficking and block cytokine secretion. Cells were stained with TruStain mouse FcX Plus (Biolegend) and LIVE/DEAD Fixable Blue in PBS with 2 mM EDTA for 30 mins. Surface markers were subsequently stained: PE-conjugated anti-mouse CD3 (dilution: 1:200, clone: 17A2, manufacturer: Biolegend), APC-conjugated anti-mouse CD4 (1:200, RM4-5, Biolegend), AF700-conjugated anti-mouse CD8 (1:200, RPA-T8, Biolegend). Following fixation and permeabilization (Biolegend), cells were stained with PE/DAZZLE-conjugated anti-mouse IFN- γ (1:100, XMG1.2, Biolegend). Cells were washed and data was acquired on the BD Fortessa X-20 (BD Biosciences), collecting 500,000 events per sample.

Variable region cloning and antibody expression

B cell receptor variable regions were recovered, as previously described 29 . Briefly, RNA was captured on RNAClean XP beads (Beckman Coulter) and washed with 70% ethanol. RNA was eluted and cDNA was synthesised using SuperScript III (Life Technologies) with random primers (Life Technologies). VH and VK regions were recovered 43 and Q5 polymerase (New England Bioscience), sequencing the amplicons via Sanger. VH amplicon sequences were used to determine B cell clonality.

To validate that the sequences were specific to the penicilloyl adducts, antibodies were recombinantly expressed. The VH/VK amplicons were incorporated into expression vectors: vector-overlapping adapters were incorporated via PCR 42 , and the V regions were inserted into pre-cut recombinant FAb expression 44 vector via Gibson reaction (New England Bioscience). Vector products were transiently transfected into HEK 293Freestyle cells and FAb was purified from supernatant using Ni-NTA resin.

Immunogenetic analyses

Analyses were performed using VH regions. Sequences were aligned to the murine reference genome using the Immunogenetics Information System (IMGT; https://www.imgt.org/IMGT_vquest/input ), as described previously 29 . Sequence outputs of poor quality or those unproductive were excluded from our analyses. Alignments of CDRs were visualised using WebLogo 61 . Clonal lineages were evaluated using GCTree 62 .

SPR was performed using a Biacore T200 instrument. Details of chip design, synthesis and testing, refer to Document  S1 . FAb binding was evaluated by sequentially injecting serial dilutions at a flow rate of 10 µL/min.

Samples for uSTA were prepared by buffer-exchange of purified FAb fragments with D 2 O PBS using Amicon 30 K MWCO. All NMR experiments were conducted on Bruker Avance Neo 600 MHz spectrometer at 25 o C equipped with QCIF cryoprobe and a SampleJet, running TopSpin 4.2.0. The uSTA experiments were recorded using a pseudo-3D pulse sequence based on stddiffesgp.2 from the standard Bruker library as described previously 45 . The following saturation times were used: 0.1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, 1.7, 1.9, 2, 2.5, 3, 3.5, 4 and 5 s. Low-power gaussian excitation pulse was applied at 9 ppm and 37 ppm for the on- and off-resonance spectra respectively, where the specific choice of excitation at 9 ppm minimised artefacts in a ligand-only spectrum 45 (Fig.  S1 ). All experiments were recorded with 16 scans per transient, 32768 complex points and sweep width of 16.01 ppm for a total acquisition time of 7 h 41 min. These were acquired on a protein-only, a ligand-only and a mixed protein/ligand sample. Data were processed using the nmrPipe module embedded within the uSTA workflow, where the protein-only data is subtracted from the mixture to give a uSTA transfer efficiently, from which values from the ‘ligand only’ sample are then subtracted to account for residual ligand excitation, as previously reported 45 . The ‘heatmaps’ were generated by mapping the uSTA transfer efficiency from the detectable protons onto heteroatoms (carbon, sulphur, oxygen and nitrogen) using a 1/r 6 dependency, before rendering in pymol as described previously 45 .

X-ray crystallography

MIL-3 FAb was loaded onto a gel filtration Superdex 200 column 10/30 (GE Healthcare) in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl. Co-crystals appeared at 20 C after a week from a hanging drop of 0.1 μL of protein solution (15 mg/mL with 2.5 mM PenG-Lys with 0.1 μL of reservoir solution containing 20% (w/v) PEG 6000, 0.1 M MES pH 6, 0.2 M ammonium chloride in vapor diffusion with reservoir. Crystals were frozen with the same solution containing 16% glycerol. Data were collected at the Diamond light source oxfordshire (beamlines I04). Data were processed with XIA2 63 , 64 , 65 , 66 . Structure has been solved by molecular replacement using PHASER and pdb file 7bh8 for VL, CH and CL domains and 7n09 for VH domain. The structure was builded with Autobuild program, refined with REFINE of PHENIX with NCS restraints 67 and adjusted with COOT 68 . Coordinates and topologies of ligands were generated by AceDRG 69 .

Microbiological assays

The attenuated, unencapsulated lab strain Streptococcus pneumoniae D39 (Δ cps2A’ -Δ cps2H’ ) 46 was routinely grown in tryptic soy broth (TSB) (BD Biosciences) at 37 °C (standing incubation) in a 5% CO 2 atmosphere.

Microbroth dilutions of S . pneumoniae D39 revealed a PenG MIC of 0.01 μg/mL (that is, the lowest antibiotic concentration that prevented bacterial growth) (data not shown). For the liquid antibiotic rescue assay, 2 ng of PenG in 10 μL PBS were pre-incubated with 10 μL of antisera for 2 h in a flat-bottom 96-well plate. 180 μL of exponentially growing S. pneumoniae cells (OD 600  = 0.2) were added and incubated overnight (such that the final PenG concentration was 0.01 μg/mL). The following day, bacterial growth was measured using a Spectramax M5 plate reader (Molecular Devices), evaluating the OD 600 nm as a proxy for bacterial density.

For disk diffusion assays, 0.1 μg of PenG and mAb (1:50 molar ratio) were spotted onto paper disks. The disk was placed onto a blood agar plate (Merck), carrying a 5 ml nutrient soft agar overlay with 200 μL exponentially growing S. pneumoniae cells (OD 600  = 0.2). Plates were incubated overnight. The following day, the kill zone diameter was manually measured. PenG-only, PenG-raised antibody clones were tested, as well as an irrelevant HIV-1-specific antibodies were tested.

Data and statistics

Flow cytometry data was evaluated using FlowJo V.10.8.2 for Mac. Statistical analyses were conducted in either GraphPad Prism V.10.0.1 or in RStudio V.4.1. Statistical test details are provided in the results, figures and associated figure legends.

Data availability

Data reported in the manuscript are supplied as separate source data files or deposited as otherwise referred throughout or can alternatively be requested directly from the author. LC-MS/MS raw data and search results have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository 60 with the dataset identifier: PXD052026. The structure of BAR-1 bound to PenG-Lys is deposited in the protein database PDB ( https://www.rcsb.org ), under the accession number 8QXC. No custom code was developed for this manuscript. Reagents are available where applicable through an institutional MTA agreement.  Source data are provided with this paper.

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Acknowledgements

The authors thank The Sir William Dunn School of Pathology flow cytometry facility, SPR facility and animal house staff. We extend our gratitude to the Rosetrees Trust, who have supported this work through the Interdisciplinary Award (ID2020/100023). We are also grateful for the funding provided by the Wellcome Trust (224212/Z/21/Z). Additionally, we thank the Wellcome Trust (grant ref: 095872/Z/10/Z) and the Engineering and Physical Sciences Research Council (grant ref: EP/R029849/1) for the instrumental upgrades of the 600-mHz and 950-MHz NMR spectrometers, as well as support from the University of Oxford Institutional Strategic Support Fund, the John Fell Fund, and the Edward Penley Abraham Cephalosporin Fund. A.J.B. is supported by ERC grant (101002859). For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. The Chemistry theme at the Rosalind Franklin Institute is sustained by the EPSRC (V011359/1 (P)). We would like to thank the Membrane Protein Laboratory at Diamond Light Source (funded by Wellcome Trust grant 223727/Z/21/Z) for help and support. C.M.K. is supported by an EPA Cephalosporin Junior Research Fellowship from Linacre College Oxford. L.P.D. is supported by the Clarendon Fund, and Q.J.S. is a Jenner Vaccine Institute Investigator and James Martin School Senior Fellow. We are grateful for the technical advice of Anton van der Merwe (The Sir William Dunn School of Pathology, University of Oxford) in the biophysical analyses.

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Lachlan P. Deimel

Present address: Laboratory of Molecular Immunology, The Rockefeller University, New York, NY, 10065, USA

Authors and Affiliations

Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK

Lachlan P. Deimel, Mikhail Kutuzov, Carolin M. Kobras, Mathew Stracy & Quentin J. Sattentau

Rosalind Franklin Institute, Harwell Science and Innovation Campus, Oxford, OX11 0FA, UK

Lucile Moynié, Guoxuan Sun, Viliyana Lewis, Abigail Turner, Charles J. Buchanan, Yana Demyaneko, Shabaz Mohammed, Andrew J. Baldwin, James Naismith & Benjamin G. Davis

Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK

Charles J. Buchanan, Shabaz Mohammed, Andrew J. Baldwin & Benjamin G. Davis

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Charles J. Buchanan, Sean A. Burnap, Weston B. Struwe & Andrew J. Baldwin

Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK

Sean A. Burnap, Shabaz Mohammed & Weston B. Struwe

Department of Pharmacology, University of Oxford, Oxford, OX1 3QT, UK

Yana Demyaneko & Benjamin G. Davis

The Max Delbrück Centre for Molecular Medicine, Campus Berlin-Buch, 13125, Berlin, Germany

Quentin J. Sattentau

Experimental and Clinical Research Center (ECRC), Charité Universitätsmedizin Berlin and Max-Delbrück-Center for Molecular Medicine, Lindenberger Weg 80, 13125, Berlin, Germany

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Conceptualization of project: L.P.D., B.G.D., Q.J.S.; Methodology: L.P.D., L.M., G.S., V.L., A.T., C.J.B., S.A.B., M.K., C.M.K., M.S., W.B.S., A.J.B., J.N., B.G.D., Q.J.S.; Investigation: L.P.D., L.M., G.S., V.L., A.T., C.J.B., S.A.B., M.K., Y.D., C.M.K., W.B.S.; Funding acquisition: M.S., W.B.S., A.J.B., J.N., B.G.D., Q.J.S.; Project administration: M.S., W.B.S., A.J.B., J.N., B.G.D., Q.J.S.; Supervision: S.M., M.S., W.B.S., A.J.B., J.N., B.G.D., Q.J.S.; Writing—original draft: LPD.; Writing—review & editing: L.P.D., B.G.D., Q.J.S.

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Deimel, L.P., Moynié, L., Sun, G. et al. Covalent penicillin-protein conjugates elicit anti-drug antibodies that are clonally and functionally restricted. Nat Commun 15 , 6851 (2024). https://doi.org/10.1038/s41467-024-51138-7

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