Production and Characterisation of Monoclonal Antibodies against 19-Nortestosterone

ObjectiveTo produce anti-19-Nortestosterone (NT) monoclonal antibodies and identify their immunological characteristics.MethodsHybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mice. Noncompetitive and competitive indirect ELISA were employed to screen positive cell clones. A caprylic acid ammonium sulphate (CAAP) method was used to purify NT mAb, and the Batty saturation method was used to determine the affinity constant (Kaff).ResultsFive hybridoma cell lines, named NT-1, NT-2, NT-3, NT-4, and NT-5, were identified and their corresponding mAbs were of the IgG1 isotype with a k light chain. The Kaffs of all mAbs were between 2.6 and 4.7×109 L/mol. The titers and IC50 values of purified ascite fluids were in the range of (0.64-2.56)×105 and (0.55-1.0) ng/mL, respectively. Of all the cross-reacting steroids, (-NT was the most reactive with the mAbs at 62% with NT-1 mAb and 64% with NT-2 mAb. Negligible cross-reactivity (<0.01%) with other steroids was observed.ConclusionThe establishment of these hybridomas allows the potential development of a rapid test kit, and may provide an alternative method for the detection of NT residues in food producing animals.