Determinación de la constante de afinidad para anticuerpos monoclonales mediante un método inmunoenzimático empleando la tecnología SUMA

Affinity constant of six monoclonal antibodies was determined: anti β-hCG (4G1-C5), anti T4 (F8-B9), anti IgE (IgE1and IgE2), anti HBsAg ( Hep-1 and 3F1-F3) using an immunoenzimatic method adapted to ultramicroanalytical system (SUMA). UltramicroELISA plates sensitized with several concentrations of each antigen, were incubated with the corresponding monoclonal antibody. Conjugate anti-IgG murine/ Alkaline Phosphatase was added and the reaction was revealed with a fluorigenic substrate (4-methylumbelliferylphosphate). The concentration that corresponds to the 50 % of flurescence in each of the sigmoid curves obtained for each antibody, was used to calculated Kaf, according to the formula: Kaf= (n-1)/2 (n[Ab']t [Ab]t) where: n = [Ag]t/[Ag']t. Results showed that the six monoclonal antibodies assayed have present high affinity because their ligands oscilated between 108 y 109 L/mol. This method is quick, reproducible and it is easy to automate and develop.