The relationship between the binding of primary antibody to solid-phase antigen in microtiter plates and its detection by the ELISA.
Radioiodinated monomeric and dimeric M315 (mM315 and dM315) prepared from BALB/c ascites fluid by gel filtration and affinity chromatography were used to study the relationship between primary antibody binding to solid-phase dinitrophenylated gelatin in microtiter ELISAs and its indirect detection by enzyme-antibody conjugates and complexes. The relationship between the amount of mM315 or dM315 which binds to dinitrophenylated gelatin to the amount added is linear over a nearly 3-log range with a slope of 1; the amount of M315 which binds in this linear range after 24 h represents all of the active antibody in the system. On a binding site basis, mM315 was inhibited by a significantly lower amount of dinitrophenyl-(DNP)glycine than was dM315. The indirect detection of bound M315 over the same 3-log range using ELISA yielded a sigmoidal titration curve which encompassed a short linear region that had a slope of 0.9 or less. Plateauing of the titration plots at high input of both mM315 and dM315 was shown to be progressively exaggerated in direct relationship to the size of the enzyme-antibody conjugate used for their detection. The data show that the upper region of the sigmoidal ELISA titration plot is the result of steric hindrance of the detection system. Through the combined use of an 131I-enzyme-antibody immune complex (EIC) detection system and 125I-M315, it was shown that the deviation from the linear binding of 125I-M315 observed during its indirect detection in the so-called linear region of the ELISA titration curve was the result of changing ratios of bound EIC: bound primary antibody, not altered enzymic activity.
D000469:Alkaline Phosphatase; D000818:Animals; D004136:Dinitrobenzenes / Q000276:immunology; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D046911:Macromolecular Substances; D051379:Mice; D011863:Radioimmunoassay; D012995:Solubility
D004136:Dinitrobenzenes; D046911:Macromolecular Substances; D000469:Alkaline Phosphatase