Bioassay

Preparation of genistein monoclonal antibody and establishment of indirect competitive ELISA*

  • LI Hua-yuan ,
  • GUO Yu ,
  • XIE Wei ,
  • XIANG Qian ,
  • ZHANG Wen ,
  • CAO Ya-jing ,
  • WANG Jing ,
  • LU Yan-yun ,
  • DUAN Si-qi ,
  • SUN Ying
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  • 1. Cloud-Clone Corp, Wuhan 430056, China;
    2. Wuhan Cloud-Clone Diagnostic Reagents Institute, Wuhan 430056, China;
    3. USCN Life Science Inc., Wuhan 430056, China

Online published: 2025-10-13

Abstract

Objective: To prepare monoclonal antibodies (MAbs) that can specifically recognize genistein (Gen), and to establish a specific and rapid quantitative method for Gen using indirect competitive enzyme-linked immunosorbent assay (ELISA). Methods: Gen was coupled with carrier protein by the Mannich method to synthesize artificial antigens. Femal Balb/c mice aged 6-8 weeks old were immunized by Gen-BSA. After the booster immunization, splenocytes of the mice were collected and fused with SP2/0 myeloma cells under the action of 50% polyethylene glycol (PEG). Subclones were screened by the limiting dilution method to obtain specific MAbs and an indirect competitive ELISA method was established. Results: Two hybridoma cell lines (C1 and C3) were isolated successfully with the titers of 1 ∶ 32 000, 1 ∶ 40 000, respectively. Under the optimized conditions, the indirect competitive ELISA based on C3 for Gen showed a half maximum inhibition concentration (IC50) of 16.15 ng · mL-1 and detection ranges of 3.78-55.85 ng · mL-1 with cross-reactivity for biochanin A and irisolidone of 4.96% and 2.40%, respectively, negligible cross-reactivity with other Gen analogs including apigenin and 4-oxo-4H-1-benzopyran-2-carboxylic acid. The recovery test showed that the recovery rate was between 85% and 110%, precision tests showed RSD of inter-assay and inter-assay was less than 10%. Conclusion: In this study, Gen-specific MAbs are prepared successfully. An sensitive and accurate indirect competitive ELISA based on MAb for Gen is developed. The developed method can meet the requirements for immunoassay.

Cite this article

LI Hua-yuan , GUO Yu , XIE Wei , XIANG Qian , ZHANG Wen , CAO Ya-jing , WANG Jing , LU Yan-yun , DUAN Si-qi , SUN Ying . Preparation of genistein monoclonal antibody and establishment of indirect competitive ELISA*[J]. Chinese Journal of Pharmaceutical Analysis, 2025 , 45(5) : 859 -866 . DOI: 10.16155/j.0254-1793.2024-0492

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