Development of an antigen-biotin conjugate with a protein bridge and its application for the development of an enzyme immunoassay

Antigens with aromatic amino groups can be conjugated to proteins by a diazo bond. This property was used to synthesise an antigen-biotin conjugate using bovine serum albumin (BSA) as a bridge, and tested this conjugate in an enzymoimmunoassay for clenbuterol detection. The assay included a highly specific antibody and the biotin-avidin amplification system. Its coefficient of variation was lower than 20% in the range of 100–8000 pg per well and the limit of detection was 6.4 pg. The synthesis of clenbuterol–BSA–biotin conjugate was reproducible, the reaction product easily purified and the amplification capacity could be set by adjusting the clenbuterol:biotin ratio.