Improved sensitivity of enzyme immunoassay for cocaine and benzoylecgonine using heterologous hapten-enzyme conjugates

Antibodies for cocaine and benzoylecgonine were prepared by established methods using diazotized 4-aminococaine or 4-aminobenzoylecgonine conjugated to bovine serum albumin as immunogens. Enzyme immunoassay was first developed using diazotized 4-aminococaine or 4-aminobenzoylecogonine conjugated to horseradish peroxidase as the enzyme labels. The IC₅₀s of cocaine and benzoylecgonine were 4 ng/ml and 2 ng/ml for their respective antibodies. However, the IC₅₀s of cocaine and benzoylecgonine decreased to 0.4 ng/ml and 0.1 ng/ml respectively when two heterologous haptens, 4-formylcocaine and 4-formyl benzoylecgonine, were synthesized and used for enzyme labeling. The sensitivity of the assays was considerably improved using the heterologous bridge strategy.