Detection of anti-TNP antibody-forming cells (AFC) with TNP-enzyme and TNP-Fab anti-enzyme conjugates.
Peroxidase (PO), alkaline phosphatase, and glucose oxidase, as well as Fab anti-PO, were coupled with varying molar ratios of trinitrophenyl (TNP) hapten. These reagents were evaluated for their ability to detect anti-TNP antibodies in the lymph node cells of Balb/c mice immunized with heavily-substituted TNP45 alkaline phosphate, which gave rise only to anti-hapten antibody-forming cells (AFC). The best results were obtained with lightly-substituted TNP-Fab anti-PO plus PO, TNP-alkaline phosphatase, and TNP-glucose oxidase. These reagents gave strong, specific staining of AFC, and negative background staining. Anti-hapten and anti-carrier AFC could be stained in contrasting colors on the same slide, when immunization was performed with lightly-substituted TNP5.9 alkaline phosphatase. Anti-hapten AFC were detected with TNP-Fab anti-PO or TNP-glucose oxidase, and unsubstituted alkaline phosphatase was used to reveal anti-carrier AFC. The number of AFC detected with these reagents was compared with the number of direct and indirect anti-TNP plaque-forming cells (PFC). At three and five weeks after primary immunization, 40 and 70% more AFC than PFC were detected. These methods can be employed alone, to enumerate anti-TNP AFC and, if desired, anti-carrier AFC; they can also be used in parallel with anti-TNP PFC assays, to determine the fractions of AFC that are not actively involved in antibody secretion.
D000469:Alkaline Phosphatase / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000032:analysis*; D000918:Antibody Specificity; D000921:Antibody-Producing Cells / Q000276:immunology*; D002352:Carrier Proteins / Q000276:immunology; D005189:False Positive Reactions; D005949:Glucose Oxidase / Q000276:immunology; D006241:Haptens; D006462:Hemolytic Plaque Technique; D006735:Horseradish Peroxidase / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009578:Nitrobenzenes / Q000276:immunology*; D010544:Peroxidases / Q000276:immunology*
D000906:Antibodies; D002352:Carrier Proteins; D006241:Haptens; D007140:Immunoglobulin Fab Fragments; D009578:Nitrobenzenes; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D010544:Peroxidases; D000469:Alkaline Phosphatase