RESUMO
A monoclonal antibody (MAb) directed against human trypsin-1 has been produced by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by ultramicro enzymelinked immunosorbent assay (UMELISA). MAb was purified by affinity chromatography on protein A-Sepharose, and MAb had a high affinity for trypsin-1 with the affinity constant equal 1.79 x 10(9) L/mol. Specificity was studied by UMELISA using cross-reactant proteins; MAb gave a positive reaction with native trypsinogen-1 and with the same protein after reduction. Antibody appeared to be directed against sequential epitope. One-step purification is described. The method evolved the adsorption of the enzyme onto a Sepharose-MAb(3H9) affinity column. The collected fraction was characterized and is available for immunization of BALB/c mice and for the preparation of a standard for immunoenzymatic assay.
Assuntos
Anticorpos Monoclonais , Tripsina/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Epitopos , Humanos , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
A monoclonal antibody (MAb) directed against human immunoaffinity purified trypsinogen has been produced by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by ultramicro-enzyme-linked immunosorbent assay (UMELISA). The MAb was purified by affinity chromatography on protein A-sepharose, and MAb had a high affinity for trypsinogen with the affinity constant equal 2.06 x 10(9) L/mol. Specificity was studied by UMELISA using cross-reactant proteins; MAb gave a positive reaction with native trypsinogen-1 but did not react with the same protein after reduction. The antibody seem to be directed against conformational epitope. The MAb obtained was characterized immunologically and used to develop UMELISA for detection Trypsin. This monoclonal assay enabled the detection of 2.8 ng/mL.
