Double immunocytochemical staining for the in situ study of allotype distribution during an anti-trinitrophenyl immune response in chimeric rabbits.
After incubation of tissue sections with anti-allotype-enzyme conjugates, the localization of immunoglobulin-allotype-bearing cells in the lymphoid tissues of conventional and chimeric rabbits could be established. The use of anti-allotype sera bearing distinct enzyme labels allowed simultaneous recognition of B cells producing immunoglobulin of one or the other parental types in heterozygous rabbits, or of B cells from the donor and recipient in chimeras. After immunization of chimeric rabbits with trinitrophenyl-keyhole limpet hemocyanin, anti-trinitrophenyl antibody-forming cells could be demonstrated through the use of a trinitrophenyl-alkaline phosphatase conjugate. Simultaneous incubation of sections with this reagent and with horseradish peroxidase coupled to (donor or recipient) anti-allotype sera made possible the determination of the origin (donor or recipient) of the antibody-forming cells. In agreement with the results of plaque assays and analyses of serum antibodies, all the anti-TNP producing cells were of donor origin when the chimeras had been created through injection of spleen or lymph node cells from trinitrophenyl primed donors. With this study we introduce a simple, direct method for the simultaneous identification of cells that produce antibody of a given allotype and a given specificity, applicable to appropriate studies in heterozygous or chimeric rabbits. The procedure has various advantages over previously reported methods.
D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D000918:Antibody Specificity; D000921:Antibody-Producing Cells / Q000276:immunology*; D002678:Chimera; D006651:Histocytochemistry; D006735:Horseradish Peroxidase / Q000032:analysis; D007126:Immunoglobulin Allotypes / Q000032:analysis*; D009578:Nitrobenzenes / Q000276:immunology*; D011817:Rabbits; D013194:Staining and Labeling; D014301:Trinitrobenzenes / Q000276:immunology*
D007126:Immunoglobulin Allotypes; D009578:Nitrobenzenes; D014301:Trinitrobenzenes; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase