PCR to identify Ig heavy chains (as a diagnostic for immune response) - (Sep/18/2007 )

Hello immuno experts,

I am new to immunology and have a question.

1) we immunize rabbits/pigs with antigen (3 immunizations + 3 booster doses; all together lasting about 3-4 months).

2) we then use the same antigen intraperitoneally to immunize the same animals.

3) we collect peripheral blood 3 days after step 2, "assuming" that it will have a high titre of B cells that have come up in response to the intraperitoneal injection.

4) can I use PCR, with primers for the conserved regions of the Heavy chain, as a tool to screen if my peripheral blood has cells that encode for antibodies??

let me know if this makes sense?

thanks.

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Straightforward PCR will just amplify your genomic DNA. If you use RT-PCR you can look for expression of heavy chain, again you'd have to has some controls to determine what corresponds to high level antibody production. Also using the constant region isn't going to allow you to identify antibodies specific to your target antigen just the antibody isotype e.g. IgG1, IgM etc. That's why diagnostic kits use immunoassay to look at antibody titres with specific antibodies coated to a plate to capture antigen specific antigens and then anti-antibodies for detection.

Ceri

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Yes, RT-PCR will help. Wouldn't a positive PCR reaction atleast tell that there are circulating peripheral B cells in the blood I collected. And since I am collecting them after intraperitoneal injection, it is also very likely that this B cell population will have cells that code for antibodies to my antigen.

I am just wondering what would be a positive control?

Is this true?

thanks.
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Hi brami.

The B cells circulates all the time and you must remember that there're natural antibodies which may recognise your antigen and these are usually selected from recombinant "naive" antibody libraries.

Therefore in the absence of your antigen of interest, one will always get a positive PCR results as eluded above.

The only approach which normally indicates the presence of antibodies of interest is to use the serum collected prior to immunisation as a negative control and collected blood after infection/immunisation can indicate the increase in presence of your antibodies of interest.

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Hello chick gene,

thanks for your expert comments. I am a biophysicist and very new to immunology. I have a couple of more questions:

1) wouldn't using primers to amplify the heavy chain constant regions always give a positive pcr reaction, since these must be there?

2) could you elaborate a little mor eon the 'naive' antibody library is? and how can it bind to the antigen without having ever seen it??

3) the aim of my experiment is to just see if the peripheral blood that I collect after intra-peritoneal injection with the antigen (after completing the normal course of immunization; 3 immunizations + 3 booster doses) has cells that code for antibodies or not. If these cells are there, then the probability that cells coding for antibodies against my antigen is also high. Does this make sense?

please let me know.

thanks.
Rami
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Hi brami.

The B cells circulates all the time and you must remember that there're natural antibodies which may recognise your antigen and these are usually selected from recombinant "naive" antibody libraries.

Therefore in the absence of your antigen of interest, one will always get a positive PCR results as eluded above.

The only approach which normally indicates the presence of antibodies of interest is to use the serum collected prior to immunisation as a negative control and collected blood after infection/immunisation can indicate the increase in presence of your antibodies of interest.
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