So, we tried both tests but then in the end focused primarily on the immunofluorescence because my wife, Dagmar, had a lot of experience with this test and so she participated and Vera Hauptfeld who joined me from Croatia, born originally in Czechoslovakia, joined me in the laboratory and we together tried to find whether we can detect any antibodies there in the serum that we were producing. We were focusing primarily on the combination of the recombinants that I produced during my earlier studies because they gave, in combination with Jack Stimpfling's recombinant, what we needed... the identity of the K and D loci. Well, eventually... for a long time we couldn't find anything and in the retrospective it seems like... to an outsider it might seem like, how come? I mean, why? Later everybody was able to find out these antibodies... find these antibodies and why did it take so long for you to find them and other people as well? Well the problem was again really technical. In both assays we could see killing of cells but it was never 100%. We never... in all the other assays, especially in agglutination, all the cells become clumped together if you have a good antibody and with the cytotoxicity test with other antibodies, that was the same way. If you had a good antibody you got 100% killing, so that's what we were looking for.
It took a while for us to realize that perhaps there is in the lymphocytes only a small population of cells that bear this antigen and so we should not expect that all the cells will be killed and only some cells will be killed or stained in the immunofluorescence. But then still the problem was that if you used them for cytotoxicity or immunofluorescence you often had the so-called background. You often... you always have a background. Some cells are killed non-specifically, no matter... they are not killed by the antibodies that you are looking at... for, but by some other factors present in the serum, so you always have some cells killed or some cells stained, and the problem was to differentiate what was really the positive and differentiate it from the background. In the case of the complement components, not all the complements were of the same quality. Complement you got as a source of complement usually was used the rabbit, a normal rabbit serum, but you had to test... eventually we realized that you had to test 50 rabbits, sera from 50 rabbits, to find one which was suitable for this, because many of them killed unacceptably high number of the cells.
In the immunofluorescence the test was similar problem but of a different nature. The cells often stained other cells because again you were using a third component which was reacting with these cells non-specifically. So, the problem was to differentiate the background from specific reactivity. Well, eventually... it took us a while to convince ourselves that we have such a difference and that it was reproducible and that you can really distinguish certain... if you had, especially if you a low non-specific background... some specific staining or killing. So, there were antibodies present and further testing revealed that they are indeed specific for antigen that is controlled by the middle portion of the H2 complex and that it was mapping with the help of other recombinants, it could be mapped to what then became to be called the IR region, or IR region of the H2 complex.
