Preparation of monoclonal antibody based indirect competitive ELISA for detecting 19-nortestosterone residue

Abstract

19-Nortestosterone (NT) has been illegally used in horse racing to boost physical performance, and in animal husbandry to accelerate weight gain. To monitor the abuse of NT, our goal was to develop a commercial enzyme linked immunosorbent assay (ELISA) kit. For this purpose, hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mouse. Noncompetitive and competitive indirect ELISA were used to screen positive cell clones. To optimize the indirect competitive ELISA (icELISA) method, various methanol concentrations in assay buffer were evaluated. Matrix effects in urine and spiking test were also investigated. Finally, five hybridoma cell lines named NT-1, NT-2, NT-3, NT-4 and NT-5 were screened out. The corresponding monoclonal antibodies (mAbs) were of the IgG 1 isotype with a k light chain, and the antibody affinity of all mAbs were between 2. 6×10 9 and 4. 7×10 9 L/mol. The titer and IC 50 values of purified ascites were in the range of 0. 64×10 5-2. 56×10 5 and 0. 55-1. 0 ng/mL, respectively. Based on the NT-1 hybridoma, a heterologous icELISA method was developed for the quantitative detection of NT in cattle urine. The dynamic range was from 0. 004 to 85. 8 ng/mL, with a detection limit for the assay and IC 50 values of 0. 002 and 0. 55 ng/mL, respectively. Except for a high cross-reactivity (62%) to α-NT, negligible cross-reactivity to other compounds was observed. After optimization, 10% of methanol was used in the assay buffer, and a 20-fold dilution in cattle urine gave an inhibition curve almost the same as that in phosphate buffered saline. The correlation coefficient between the established icELISA and LC-MS/MS method was 0. 9871. The results showed that the established heterologous icELISA method provides an excellent alternative for the detection of NT residues in food producing animals. © 2011 Science China Press and Springer-Verlag Berlin Heidelberg.