Preparation of a monoclonal antibody against hyaluronic acid and its application in immunoassay

This study was to develop a quantitative immunoassay for hyaluronic acid(HA). We firstly prepared a monoclonal antibody(MAb)against it. HA, activated by 1-cyano-4-dimethyl-aminopyridinium tetrafluoroborate(CDAP), was coupled to bovine serum albumin(BSA). BALB/c mice were immunized with purified complete antigen BSA-HA and the monoclonal antibody was prepared by hybridoma technique. To characterize the specificity of the antibody, inhibition enzyme-linked immunosorbent assay(ELISA)was conducted and the cross reactivity along with affinity constant was determined. The results showed that the substitution degree of BSA-HA(m_BSA/w_HA)was about 9. Also, a hybridoma cell line named 8B6 was obtained by hybridoma technology. The indirect ELISA titer of the ascetic fluid was 1 ∶256 000; the isotype of MAb 8B6 was IgG1 and the affinity constant was K_a=6. 71×10⁹ mol^-1. To establish the indirect competitive ELISA method, polystyrene microwell plates were coated with 10. 0 ng/mL HA and blocked with 1. 0% BSA. The linear range of indirect competitive ELISA was between 10. 0 ng/mL to 10. 0 μg/mL which confirmed that the method was specific and sensitive.