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

Abstract

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. 19-nortestosterone

Biological Activity of the Carrier as a Factor in Immunogen Design for Haptens

“…AF-specific immune response in mice sera was confirmed by testing the interaction of antibodies with soluble AF with IC-ELISA [ 40 ]. We also used IC-ELISA to observe the carrier-specific antibodies in mice sera.…”

Development and Characterization of Anti-Estradiol Polyclonal Antibody

“…The EDC method was employed to synthesize the artificial antigen of E 2 -BSA, according to the literature described by Jiang et al [4] . Briefly, to a solution of 10 mL anhydrous pyridine containing 80 mg of E 2 , 150 mg of succinic anhydride was added and stirred in dark chamber, kept at 50 ℃ for 24 h. Stramineous grease material was acquired after pyridine removed with nitrogen evaporator.…”

Preparation of Artificial Antigen and Immunological Trait for Estradiol

“…Sensitivity was evaluated according to the inhibition rate, and the data were calculated using the IC 50 values, which represented the concentration of E 2 that produced 50% inhibition of antiserum binding to the hapten conjugate. The detection of limit (LOD) was defined as the lowest concentration that exhibits a signal of 15% inhibition [6] . The dynamic range for the icELISA was calculated as the concentration of the analyte providing a 20-80% inhibition rate (IC 20 -IC 80 values) of the maximum signal.…”