title: Monoclonal antibody-based ELISA and colloidal gold immunoassay for detecting 19-nortestosterone residue in animal tissues.
abstract: This article presents the generation of monoclonal antibodies (mAbs) with high specificity against 19-nortestosterone (NT) through cell fusion techniques and the development of a mAb-based indirect competitive ELISA (icELISA) method and colloidal gold-based immuno-chromatographic assay to detect NT residues in beef and pork samples. A modified carbodiimide method was employed to synthesize the artificial antigen, and BALB/c mice were used to produce anti-NT mAbs. On the basis of the checkerboard titration, an indirect competitive ELISA standard curve was established. This assay was sensitive and had a linear range from 0.03 to 38 ng/mL in phosphate buffered saline (PBS), with IC(50) and LOD values of 0.52 ng/mL and 0.01 ng/mL, respectively. Of all the competitive analogues, the produced mAb exhibited a high cross-reactivity to 17α-nortestosterone (83.6%), the main metabolite of NT in animal tissues. Except for moderate cross-reactivities with trenbolone (22.6%) and β-boldenone (13.8%), the other interference to the assay was negligible (<0.05%). In contrast, the strip test had a visual detection limit of 1 ng/mL in PBS, 2 μg/kg in beef, and 2 μg/kg in pork, respectively, and the results can be judged within 10 min. The ELISA and GC-MS results showed close correlation in beef (R2=0.9945) and in pork (R2=0.9977). Therefore, the combination of two immunoassays provides a useful screening method for quantitative or qualitative detection of NT residues in animal-origin products.
authors.affiliation: College of Animal Science, Henan Institute of Science and Technology, Xinxiang, Henan 453003, China. jjq5678@126.com
authors.forename: Jinqing
authors.initials: J
authors.lastname: Jiang
authors.forename: Ziliang
authors.initials: Z
authors.lastname: Wang
authors.forename: Haitang
authors.initials: H
authors.lastname: Zhang
authors.forename: Xiaojian
authors.initials: X
authors.lastname: Zhang
authors.forename: Xingyou
authors.initials: X
authors.lastname: Liu
authors.forename: Sanhu
authors.initials: S
authors.lastname: Wang
mesh_terms: D045930:Anabolic Agents / Q000032:analysis*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D000918:Antibody Specificity; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D017979:Gold Colloid; D007118:Immunoassay / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D009132:Muscles / Q000737:chemistry; D009277:Nandrolone / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D015203:Reproducibility of Results; D013552:Swine
chemical_list: D045930:Anabolic Agents; D000911:Antibodies, Monoclonal; D017979:Gold Colloid; D009277:Nandrolone