Generation of Monoclonal Antibodies to Alpha‐Fetoprotein and Application in Solid‐Phase Enzyme Immunoassay

Abstract

By using an improved hybridoma technique, monoclonal antibodies specific to alpha‐fetoprotein (AFP) were generated. After three subcutaneous immunizations and three intravenous boosters, cell fusion experiments were performed. The hybrid cells were initially cultured in a semisolid medium containing methylcellulose and later transferred to a liquid medium for subculture. Out of 800 colonies recovered after two cell fusion experiments, 16 were shown to exhibit affinity to AFP by radioimmunoassay. Six hybrid cell lines which showed high affinity and specificity were selected for further evaluation. From the results of a cross‐matching procedure, two pairs of antibodies (AFP 3 and AFP 05; AFP 3 and AFP 013) reacting with discrete antigenic determinants were identified for preparing solid‐phase sandwich enzyme immunoassay (EIA) kits. The association constants between AFP and these three antibodies (AFP 3, AFP 05, and AFP 013) were 2.0, 3.7, and 3.8 × 10(9) M‐1, respectively. The immunoglobulin subclass of them was determined to be IgG1. The EIA procedure designed could be performed within 40 min in a one‐stage incubation and 70 min in a two‐stage incubation. The incubation time was shown to be equal to or shorter than that of any other known commercial kits and the sensitivity was less than 1 IU/ml. In order to avoid the high‐dose hook effect which occurred in the one‐stage incubation procedure, a two‐stage incubation protocol was advised.