hello, I used ovalbumin as antigen to immunize mouse, I want find out whether the antibody affinity maturation or not, there is a good way? Thank you.
Hi Martin
Thanks for your detalied answer, you are really warm-hearted. I had ever thought about NP-KLH, but for some reason I eventually used OVA protein as immunogen in my study, it really puzzle me now. Any way, thanks very much.
Hi Martin
Thanks for all your suggestions, I will take account of it, thanks again!
Can I use different concentration OVA to coat the plate, for example: 10ug/ml or 0.5ug/ml to detect low or high affinity Ab? Is this can be workable?
What I understand is that you have developed antibody against ovalbumin. Now you want to check that first of all antibody has been developed or not. If developed what is its affiniy?
First of all you have to have anti-mouse antibody conjugated with enzyme (enzyme conjugated secondary antibody) and its substrate. Second thing you require is micro-wells for coating ovalbumin.
First you have to do coating of ovalbumin in different concentration 100 micrograme per millilitre to 10 micrograme per millilitre (Use four dilutions. 100, 50, 25 and 10 micrograme per millilitre; one dilution per eight well). After coating and blocking by inert protein like gelatiin. Add different dilution of primary antibody in vertical fashion; one dilution per two wells (Use four dilutions. 1:500, 1:1000, 1:2000 and 1:4000). After incubation and washing add diluted enzyme conjugated secondary antibody; at that dilution there is no non specific binding. This assay is known as checker board assay. This is to find out optimal dilution of coating antigen and primary antibody.
After finding optimal dilutions of coating antigen (ovalbumin) and primary antibody. Now using these optimal dilution now perform displacement analysis by using ovalbumin as antigen (from pico gram to micro gram concentration) to construct the standard curve. Use standard curve data to calculate affinity of antibody by Scatchard analysis/curve. If you have any query kindly revert me back.
You can go through attached paper about how to set up checkerboard and standard assay.
With best wishes
If you immunize mice using NP-OVA or NP-KLH, then test antibody binding on ELISA using NP(low conjugating ratio)-BSA vs. NP(high-conjugating ratio)-BSA, all NP-specific antibodies bind NPhigh-BSA, but only high affinity antibodies bind NPlow-BSA, therefore by using ratio of NPlo/NPhi and compare among different conditions you have, the higher the ratio the higher the affinity in serum antibody.
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