[Study of the affinity characteristics of monoclonal antibodies by solid-phase immunoenzyme analysis].
An approach is proposed for measuring the binding constant (Kb) for monoclonal antibodies (MA) interacting with an immobilized antigen in indirect ELISA. This approach allows the measurement of optical density (A405) in the peroxidase reaction initiated by the conjugate at different concentrations (C0) of antibodies. Using the Scatchard plots, the dependence of A405/C0 = f (A405) for the whole range of MA concentrations was examined, and the tangential of the slope (tg alpha = Kb) of the linear portion of the antigen molecule was calculated. Analysis of MA affinity parameters by using this approach may find wide use in immunodiagnostic studies aimed at measuring antigen and antibody concentrations in biological fluids as well as for estimating the efficiency of vector drugs in which the diagnostic or therapeutic component is conjugated with the vector (MA or F(ab) fragment) responsible for the drug transport to target cells. The method proposed was used for testing mouse (BALB/C) monoclonal antibodies (IgG1) to pig insulin produced by various hybridomas as well as for estimating the effect on MA of pH, temperature and hydrophobization. The minimal detectable concentration (method sensitivity) was found to depend on Kb.
Ermakov
N V
NV
Ishkov
A G
AG
Miroshnichenko
E V
EV
Pliushch
O P
OP
Remizova
E N
EN
Sveshnikov
P G
PG
Chenchikova
E Iu
EIu
Cherchenko
N G
NG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D013552:Swine
D000911:Antibodies, Monoclonal; D007328:Insulin
Issledovanie affinnykh kharakteristik monoklonal'nykh antitel v metode tverdofaznogo immunofermentnogo analiza.