A Heterologous Enzyme Linked Immunosorbant Assay of Morphine Using Penicillinase as Label

A rapid, sensitive, specific and high through-put enzyme-linked immunosorbant assay (ELISA) methodfor determination of morphine in urine samples using penicillinase as label enzyme has been developed. Noextraction or chromatography was included in this assay procedure. Immunoglobulin (Ig) purified polyclonalanti-bodies against a C6-hemisuccinate derivative of morphine (M-C6-HS) conjugated to bovineserum albumin (BSA) was coated onto the wells of microtiter plate. A morphine-C3-hemisuccinate (M-C3-HS) was also prepared and the two derivatives were conjugated to penicillinase (M-C6-HS-P and M-C3-HSP).The heterologous combination of antibody prepared against M-C6-HS-BSA and enzyme conjugateprepared for M-C3-HS-P showed better properties in term of sensitivity, reproducibility and slope of standardcurve. The assay was sensitive from 20 pg/ml and detected up to 100 ng/ml of morphine in urine samples.The affinity of antibody in homologous assay was found to be 6.6×1010 l/mol and for heterologous assaywas 3.2 ×1012 l/mol. The assay was completed within 4 h. The homologous assays performed under differentconditions of coating, concentrations, duration, pH, etc. did not end up with a suitable standard curve.Hence it seems that the ability of morphine to displace the hapten enzyme conjugate dependds on the position of the enzyme coupled to the hapten molecule.This ELISA techniqu showed 100% correlation withimmunochromatography (IC) and 90% percent correlation with latex agglutination inhibition (LAI) test in theresults obtain with urine samples declared positive by authorities. ELISA also showed approximately 90%correlation with LAI-negative urine samples.