Idiotypic Antibodies

Science

#1

@Agauger
Some researchers have used an anti-idiotypic method for finding antibodies with beta-lactamase activity. They reason that the binding region of the antibody is a mirror image of the antigen structure it binds to. So, the first step is to immunize with beta-lactamase and isolate the antibodies to beta-lactamase. This gives you the mirror image of the beta-lactamase. They then immunize with the anti-lactamase antibody. The antibodies raised against the anti-lactamase antibody should have the positive image of the beta-lactamase, and possibly the functional fold itself. It’s analogous to the process of casting negative molds and making sculptures, if you are familiar with that.

It is worth noting that they are simply amplifying pre-existing B-cells. The immune system is not creating completely new antibodies in response to new antigens. V(D)J recombination occurs during embryonic development so that each individual is born with all of the antibodies it will ever have. However, there is somatic hypermutation in B-cell lineages that show initial specificity for a given antigen so there can be an increase in avidity and specificity during B-cell proliferation through positive selection.

This is why the other experiments using naive populations of B-cells is of importance. These populations had never seen beta-lactamase or antibodies raised against beta-lactamase, and yet they were still able to find antibodies that demonstrated beta-lactamase activity.


How would you define "science"?
(Ann Gauger) #2

@T_aquaticus
That says to me that there is something fundamentally different about the vertebrate system than the bacterial system. If unimmunized animals have antibodies that have catalytic activity for beta lactamase function my design intuition tells me there is a reason. that’s a pretty high initial frequency of binding for the unimmunized catalytic antibody isn’t it? Or am I wrong?

Do you find beta lactamase activity in other Ecoli proteins? No need to reply right away. I will be busy.


(Ann Gauger) #3

My response was in direct response to T_aquaticus. If I am off topic so is he and anyone else who is discussing my views on catalytic antibodies on this thread. Unflag me, or flag every one. Or move us all and explain what you have done. This is ridiculous and discriminatory.


(S. Joshua Swamidass) #4

Everyone needs to stay on topic. Flag his post for being off topic if you like.


(Mikkel R.) #5

I don’t understand why this was moved out of the previous discussion. Seems directly relevant to the topic to me. What am I missing?


(Curtis Henderson) #6

Yes, that reason is that the randomness of the B cell maturation process (primarily through VDJ joining) occasionally yields something very novel, like enzymatic activity.


#7

My curiosity is eager to hear what that reason could be. :wink:

Any explanation will need to be couched in terms of V(D)J recombination since this is the process that produces the binding sites on antibodies.

I’m not sure what the context is for that question. I will need a bit more detail to figure out what is being asked.

For clarity, there are around 100 million different B-cell lineages, if memory serves. I believe each has its own unique combination of gene fragments for its variable region, but there may be some redundancy. These are essentially a library of randomly assembled DNA fragments which is why they can serve as a model for finding specific functions in sequence space.


(Ann Gauger) #8

I am trying to establish the reasonableness of the frequency of 1 in 10^10 antibodies match the beta lactamase active site. How many possible protein epitopes are there in the protein universe? How many distinct epitomes does an unimmunized naive animal have? Just by chance, how like is it that a given animal’s sera should contain antibody capable carrying out the beta lactamase reaction?

100 million per animal? According to Mercer, they screened 4 animals (2 immunized and 2 not)… Immunization should increase the proportion of specific antibody in the serum. (By how much probably varies with the antigen and immunization). Their combined library was about 2.7 x 10^9, with pretty good representation among the clones the kinds of antibodies expected

So if we went and looked, would we find trypsin antibody enzymes? Would we find abzymes for just about any protein if we looked? Some will be removed self non-self screening, but still…

If the Abzyme was present at the same frequency in immunized and immunized animals, that is also a concern: they had 5 that bound that they followed up. 2 Abzymes were from the immunized library and 3 were from non immunized. Very small sample size though, and not a direct screen so we can’t tell from this what proportion of the clones from each library were catalytic.

their target they Identified, 2 were from immunized 3 from non-immuniIt should be amplified by immunization. By how much is unknown; it varies according to immunization and antigen. But it definitely should be amplified. They recovered 5 clones with activity after panning.


(S. Joshua Swamidass) #9

@mercer, these are good questions. What are your thoughts?


(S. Joshua Swamidass) #10

@Agauger, from another thread, and relevant here:

Let us say that they were immunized. How much of antibody sequence space do you think these small animals were able to explore? if not 10^10, what exactly? 10^15? 10^20?


(John Mercer) #11

The size of the mammalian immune repertoire is only 10^8.

They don’t “match” the bacterial beta-lactamase. They are structurally similar, but they are constrained to fit within the immunoglobulin fold. That blows the “functional fold” criterion out of the water, correct?


(Ann Gauger) #12

@Mercer

You miss my point. Is the universe of protein epitopes 10^8? Or more? Or less. I am willing to bet much mores since as you point out, an epitope is not a fold, it is only a small portion of a protein with a particular shape and set of amino acid side chains. So we have 4 libraries and we get actually 100 distinct clones that bind their target, showing som affinity. 100 clones! Out of 10^9 And that’s only the 100 they chose to sequence. They follow up 5 and they have measurable beta galactosidase activity. (Mind, though, they didn’t do the best negative control.)

EVEN IF, Doug were wrong, these numbers do not seem reasonable to me. A bacterium has a mutation rate of about 1 in 10^8 per nucleotide per cell. The immune system has only a ten-fold difference in its naive repetoire for a mimicking particular protein’s active site??? If the protein universe has a repetoire of 10^9 epitopes, a number of which recognize beta lactamase, then we had better start rhinking about proteins as being like complicated snap-on toys, on which there are only 10^9 shapes. We should be looking for and finding abzymes for just about every enzyme. Why aren’t we all dead?


(S. Joshua Swamidass) #13

Isn’t that we are thinking already, but with just 20 shapes?


(Ann Gauger) #14

@swamidass

No. Epitopes are bundles of structure, part of the surface of a protein that the antibody binds to. Not a single amino acid.


(John Mercer) #15

I get your point. Yes, 10^8.

Good! How much?

I wouldn’t point out anything of the sort. Epitopes are not only proteins. Have you never heard of a hapten?

Yup.

That’s because you don’t bother to read the literature.

Doug’s single study was laughably sloppy. If Doug believed that he is right, he would have repeated it with many other enzymes. That he hasn’t shows his complete lack of faith in the result.

For binding. Most of catalysis is simple binding.

This makes no sense. The protein universe has many more than that. There’s not a 1-to-1 correspondence between Abs and epitopes.

Are you not aware that proteins are sticky?

That’s the way most biologists do think about them. It works pretty well.

That’s why there are >5000 papers in the field that you and Doug have ignored.


(S. Joshua Swamidass) #16

@Agauger, in terms of scientific standards, this is very true. He is making an audacious claim. It is absurd to do this based on one study. Any scientist wanting to overturn the consensus with such a stunning discovery would repeat this over and over again with protein after protein, resolving every methodological objection that arose.

Axe’s response has been to argue with words rather than demonstrate with experiments. From what I understand, he had resources to do these experiments at Biologics, and did not.

Yeah, that is how I think of them.


#17

There’s a lot to unpack here, so I will do my best.

The number of epitopes is probably as large as the number of proteins and macromolecules that exist. What you need to keep in mind is that antigens can have many epitopes that antibodies can bind to. As I mentioned in another thread, antibodies recognize a very small portion of a protein, sometimes as little as 10 amino acids and perhaps even fewer. If the antibody recognizes a shape in the folded protein it may recognize bits and pieces from separate regions of the amino acid sequence that are near each other in the final tertiary structure of the protein.

Initially, the immune system has about 10^8 unique antibodies. Some may bind to the same epitope, so the number of epitopes the initial adaptive immune system can recognize is less than the total number of unique antibody sequences. Subsequent mutation of immunogenic antibodies may increase the library of antibody sequences, so the initial library will differ from the adapted library. As for antibodies in circulation, you won’t see many of the possible antibodies in serum because the B-cell carrying that antibody has not bound to anything and has not been signalled to proliferate and release anbitody into the serum. This is why these types of antibodies harvest B-cells instead of looking at antibodies in circulation.

That is certainly a good question. There are quite a few auto-immune diseases out there, and I think it is entirely possible for some antibodies to have trypsin activity or other protease activity. From my limited memory of papers I have read, protease activity is usually easy to find in sequence space.I don’t know if we could find abzymes for any old activity we wanted.

We do know that it is much less than the 10^44 discussed by Axe.