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

Document Type: Research Paper


1 Food and Drug Control Laboratories, Department of Biology, Tehran, Iran.

2 Department of Biochemistry, Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, Iran.

3 Department of Clinical Biochemistry, Birjand University of Medical Sciences, Birjand, Iran.

4 4Department of Pilot, Pasture Institute of Iran, Tehran, I.R. Iran.


A rapid, sensitive, specific and high through-put enzyme-linked immunosorbant assay (ELISA) method
for determination of morphine in urine samples using penicillinase as label enzyme has been developed. No
extraction or chromatography was included in this assay procedure. Immunoglobulin (Ig) purified polyclonal
anti-bodies against a C6-hemisuccinate derivative of morphine (M-C6-HS) conjugated to bovine
serum 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 conjugate
prepared for M-C3-HS-P showed better properties in term of sensitivity, reproducibility and slope of standard
curve. 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 assay
was 3.2 ×1012 l/mol. The assay was completed within 4 h. The homologous assays performed under different
conditions 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 with
immunochromatography (IC) and 90% percent correlation with latex agglutination inhibition (LAI) test in the
results obtain with urine samples declared positive by authorities. ELISA also showed approximately 90%
correlation with LAI-negative urine samples.