[Preparation of specific antibodies against recombinant Eml8 antigen and detection of the circulating antigen].

OBJECTIVE

To prepare specific antibodies against the recombinant Eml8 antigen (ReEml8), establish a sandwich ELISA and detect the Eml8 circulating antigen in patients' sera.

METHODS

Rabbits and BALB/c mice were immunized with the ReEml8 antigen, which was purified by affinity method for preparation of the specific poly-clonal and monoclonal antibodies. A sandwich ELISA was established by the specific antibodies.

RESULTS

By immunizing with the ReEml8 antigen, high antibody level was reached with a serum dilution of 1: 204 800 and above in the immunized rabbits. After double selection by ELISA using the ReEml8 antigen and block ELISA using both AE-positive and negative control sera, 14 positive cell clones were obtained with an inhibition rate of more than 50%. Those mono-and poly-clonal antibodies were matched freely in sandwich ELISA tests for detecting the ReEml8 antigen. A combination of monoclonal antibody No.9 and polyclonal antibody showed the best result. The sensitivity to detect ReEml8 antigen was at 3 ng/ml. Six of 11 AE sera were positive when tested with the sandwich ELISA system.

CONCLUSION

Highly specific polyclonal and monoclonal antibodies have been prepared, and a sensitive sandwich ELISA established. Preliminary result is suggested that a detectable level of Eml8 circulating antigen is present in AE patients' sera.