Science Buddies: "Ask an Expert"

My experiment is basically testing a prodrug and seeing if it will bind to the outside of a bacterium and kill it.

I have several questions... (keep in mind I am working on this with no mentor. None are available to help me)

- I have access to two types of labs: a biosaftey level 2 at my local college and the biosaftey level 1 at my school. I can't access the one at the college for a while so do you think I can do assembly, assays, and testing at my high school lab?

- Do you think in-vitro tests and assays will be sufficient data to be used for this project? In-vivo is impossible right now.

- I am unsure of what type of assay to use to measure binding affinity. Will saturation binding or indirect immunofluoresence work? Is there a better option?

- Would a kirby-bauer test suffice to measure how potent the antimicrobial is (antimicrobial tested alone). Or does the assembled prodrug (receptor & antimicrobial) need to be tested instead of the components separately?

- Can I do assembly/bioconjugation of the prodrug at my high school lab?

- Since this project requires assembly of a prodrug, do I need a mass-spectrometer or NMR machine? Some professors say I would need to find out functional groups that way. However, do I really need the machines if I have already researched the functional groups of the binding site of the prodrug? Isn't there a better alternative to determine purity?

Thank you for your help!

Hi,

If I understand your project correctly, you are combining a drug with an antibody (Ab) to a bacterial surface marker to target the drug to a specific bacterium. Is that correct? Are you going to chemically attach the drug to the Ab or put the two together into a nanoparticle that targets the bacteria? Chitosan is a cheap polymer that can be used to make nanoparticles that can be loaded with drugs and modified with Ab's and other proteins for targeting. There are a number of sources for chitosan online. It is a polysaccharide that is harmless to bacteria and other cells.

Yes, you could make the targeted drug at your high school but testing it will still require using bacteria. How do you plan to attach the antibody to the drug or the nanoparticle in the correct orientation--with the FAb domain free to bind to the bacterial antigen? If you have the antigen you could set up a microtiter plate assay for the drug-antibody but you will need a plate reader to analyze the results.

The drug has to be available in an active form when it reaches its target and you need to have a growing bacterial culture to test it on. You will have to test free drug alone, Ab alone and the combination. I don't think a Kirby-Bauer test will give accurate enough results to prove your hypothesis. You should test the compounds on a bacterial culture, make serial dilutions, plate them on agar and count the colonies. This will give you a quantitative result that would be acceptable to other researchers.

Physical measurements such as mass-spec or particle sizing only tell you the mass or dimensions of your pro-drug. This information may be useful but the only test that matters is whether the particles will target and kill the specific bacterium better than free drug. You need that BSL2 lab ASAP!

If you have more questions, please let us know.

Good luck!

Sybee

Good morning sir,

I intend to join the antibody and the drug together, but the problem is i do not know what method is best, or how to for that matter, so you will have to excuse my lack of information for now. I am unsure of the advantages of (bioconjugating?) the prodrug by itself or by loading it up in a nanoparticle. Does the nanoparticle make increase bioavailability or something?

The antibody in question is a dependent on the spacing of cilia on the surface of the bacterial membrane. This spacing must be kept in order to correctly recognize the specific bacteria in question, so I don't want antibodies too close together to mess up overall binding.

For example, if the antibody recognizes bacteria that expresses, spatially (letters represent spacing of receptor, P) "POP" on the outside, and an endothelial cell express "OPP", I don't want to put two antibodies close enough (POPPOP) so it has the potential to be toxic to human cells (notice the OPP in POPPOP? That's the very rough gist with my analogy).

I think I will have the Fab domain free to bind with the antigen, for then binding and recognition will be most effective that way.

Regarding testing the compound on the bacterial culture, do you mean the entire unit as a whole? And then separate tests for different components (the microtiter plate assay for checking binding affinity?)

I would also like to inquire about the necessity of a BSL2 laboratory. The professors don't want to let me work in their lab because I am a minor and be under them and the institution's accountability, which is something not explicitly stated in their rulebook. I am conducting independent research. I am confused because from what I read from other teenage researchers their request usually gets accepted; mentorship and access to a lab. Is this a common problem when asking for access to a university lab? Is there paperwork I have to fill out in order to get their consent? Tbh I never heard of this type of red tape before...

I was wondering if I can do the above experiment (testing the compound on the bacterial culture and counting the colonies) if the strain I am using is a proxy for a more dangerous strain. I am using Ecoli k12 as a proxy because the structure spacing is common with that of a more lethal strain.

Lastly, do you have any resources/links you can send me so I can learn more about the procedures for testing a new drug? Do you know of any other important resources that could increase my understanding of the above material? I need a sharper sense of direction from here on out.

You have no idea how much I needed this, and many thanks!

*Correction: Do the above experiment (testing the complete compound on the bacterial culture and counting the colonies and the microtiter test) in my BS1 high school lab, if I am using a nationally accepted strain (Ecoli k12) as a proxy because the proxy and lethal strains have very similar/same structures.

Wow! I think your post takes the prize for the most questions. Let me see what I can do and hopefully other experts will help out with this one.

1. Working with a mentor in a university lab
All the researchers that I know will take high school students for summer science projects. I don't know exactly what the policies are with regard to minors working in a lab under supervision although it is permitted in our department. Other labs have different rules. The best thing you can do is be polite, persistent, and enthusiastic. Your project has merit and eventually you will find someone to help you and make the way clear.

2. Using E coli K12 as the model organism
This strain of E coli is acceptable as a model for preliminary drug studies. You will have to make sure that your Ab will bind to and inactivate this strain. Where did you get the Ab? How was it made? What animal [rabbit, sheep, mouse, etc] was it made in? You said that the Ab requires a certain spacing of cilia on the bacteria. Where did you get this idea? Can you give a reference to a published use of an Ab this way? Does your Ab specifically bind to a bacterial molecule? If you know what part of the bacterial molecule that the Ab is specific for, called the epitope, you can compare it with the same molecule of E coli K12 and that should tell you if the Ab will recognize K12.

3. Use of nanoparticles for drug delivery
You can wait to test the individual components of your drug delivery system until you find out if it will work. Most researchers use a delivery agent like chitosan or other polymers to support and contain the drug and any targeting molecules like Ab's. Chemically attaching the Ab to the drug can alter it in such a way that it is no longer toxic so it is better to use nanoparticles made from a polymer like chitosan (http://www.jfda-online.com/article/S102 ... 0/abstract) to hold the drug during delivery to bacteria.

4. Testing the drug by microtiter plate assay vs bacterial assay
There are a many ways to test drug delivery systems such as you envision. The plate method allows you to confirm that a targeting Ab will bind to the protein or other molecule that it is supposed to recognize. Simply, all you do is put the target molecule in the wells of the microtiter plate so that it adheres to the plastic. When you add your Ab it should stick to the target. Then you wash the plate and add an Ab against the Ab (I will explain this later.) with a colored molecule attached. Wherever you see color you know your Ab is working. This experiment you can do in a high school lab. If your school permits the use of E coli K12 then you could do the functional assay as well. By function I mean the drug binding to and killing the bacteria. I can explain the details of how to do this later but here's a video on how to do serial dilutions and count bacteria which are the basic steps of the procedure: https://www.youtube.com/watch?v=pmRUBYlPMBM

I'm sure you will have more questions but hopefully I have answered some of them. Keep trying to find someone to help you and let you work in their lab. Prepare a 3 minute 'elevator presentation' of your project in which you explain VERY clearly what it is you want to test and how you will do it. It would also help if you could mention a couple of published references where a similar method has been used successfully.

You have to show your prospective mentor that you are serious, knowledgeable and have a project worth doing. This is going to be difficult but keep working at it and you will succeed. The main thing is to get into a lab where you can learn the techniques of research. Volunteer to help with existing research even if it is not exactly what you want to do. You will learn a lot and make some contacts. Later you can work on your own.

Good luck!

Sybee

Good Morning sir,

So I've been reading and I believe the "microtiter plate test" is ELISA? When you have the antigen stuck to a substrate, have antibodies go into it (and bind if possible), wash them out, add a secondary antibody (bioconjugated with a fluorescent marker) that seeks out the first antibody, and then determine binding affinity through the luminous signal? Is that the gist of it?

Are there any other, less expensive alternatives? I heard about this one test in which a teenager bioconjugated an antibody to a carbon nanotube substrate, and when sufficient binding occured, a voltage difference was detected. He won Intel a couple years back. Would something that be acceptable to use to check binding affinity by a scientific committee? Or is there another alternative?

Yes, you have described the method of doing an ELISA perfectly. There is one modification that you can use if you don't have access to a fluorescence plate reader. There are conjugates to the 2nd Ab that produce a colored substance and you can just look at the plate and know that the Ab is working. This does not give you a quantitative reading but all you really need is to prove that the Ab-drug combo will bind to the antigen. If you don't see any color then you know it did not bind.

The alternative method you describe is possible but you would need a special device to measure the electrical changes.

Here's a company that sells a variety of substrates for colorimetric ELISAs:

https://www.thermofisher.com/us/en/home ... guide.html

You can choose a 2nd Ab that is conjugated to alkaline phosphatase, horseradish peroxidase or other enzyme that reacts with one of the substrates to give a colored product that you can record by eye. You do not need a plate reader unless you want to make quantitative measurements of antigen amounts.

Sybee