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

Abstract

A rapid, sensitive, specific and high through-put enzyme-linked immunosorbant assay (ELISA) method<br />for determination of morphine in urine samples using penicillinase as label enzyme has been developed. No<br />extraction or chromatography was included in this assay procedure. Immunoglobulin (Ig) purified polyclonal<br />anti-bodies against a C6-hemisuccinate derivative of morphine (M-C6-HS) conjugated to bovine<br />serum albumin (BSA) was coated onto the wells of microtiter plate. A morphine-C3-hemisuccinate (M-C3-<br />HS) was also prepared and the two derivatives were conjugated to penicillinase (M-C6-HS-P and M-C3-HSP).<br />The heterologous combination of antibody prepared against M-C6-HS-BSA and enzyme conjugate<br />prepared for M-C3-HS-P showed better properties in term of sensitivity, reproducibility and slope of standard<br />curve. The assay was sensitive from 20 pg/ml and detected up to 100 ng/ml of morphine in urine samples.<br />The affinity of antibody in homologous assay was found to be 6.6×1010 l/mol and for heterologous assay<br />was 3.2 ×1012 l/mol. The assay was completed within 4 h. The homologous assays performed under different<br />conditions of coating, concentrations, duration, pH, etc. did not end up with a suitable standard curve.<br />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 with<br />immunochromatography (IC) and 90% percent correlation with latex agglutination inhibition (LAI) test in the<br />results obtain with urine samples declared positive by authorities. ELISA also showed approximately 90%<br />correlation with LAI-negative urine samples.<br /><br />