Validation of a New Recombinant Antibody Fragment (rFab)-Based Homogeneous Enzyme Immunoassay for the Highly Specific Detection of 6-Acetylmorphine in Urine

Abstract

The detection of 6-acetylmorphine (6-AM) in urine by immunoassay methods is challenging due to its short half-life and its similarity in structure to many commonly abused opiates that are often present at very high concentrations in urine. Current 6-AM homogeneous enzyme immunoassays use lyophilized reagents because of the instability of 6-AM in water or lack of the required specificity due to high cross-reactivity with morphine. A new 6-AM rFab-based homogeneous enzyme immunoassay (HEIA) has been developed with highly improved specificity. Using a cutoff concentration of 10 ng/mL, morphine or morphine glucuronides did not produce a positive signal up to 300,000 or 1,000,000 ng/mL, respectively. Assay imprecision (n = 80) was less than 1.5% using four replicates per day for 20 days over the range 0-20 ng/mL. Cross-reactivity with structurally related or non-related compounds was assessed at concentrations up to 1,000,000 ng/mL. Interferences from endogenous compounds at ±25% cutoff were also performed at the concentrations ranging from 100,000 to 500,000 ng/mL. The effect of varied pH values on assay performance at ±25% cutoff was investigated; no false-positive or false-negative results were observed between pH 4 and -11. Based on the analysis of 149 authentic urine samples, the accuracy of the 6-AM HEIA compared with LC-MS-MS was 100%. These results demonstrated that rFab can be suitable for traditional HEIA with desired detection sensitivity and stability.

Evaluation of an on‐site test device for the heroin metabolite 6‐acetylmorphine in urine

“…3 One disadvantage is the low levels of this metabolite in urine and a resulting shorter detection time. [7][8][9] Because of the low concentrations and the structure similarities to other morphine alkaloids and metabolites many of the 6-AM immunoassays suffer from unwanted crossreactivity. 5 Modern liquid chromatography-mass spectrometry instruments have made it possible to easily include 6-AM as a parameter in routine confirmation methods.…”

“…3,4 It has been demonstrated, however, that the focus on 6-AM in the confirmation method may not lead to any lower detection rate in applied drug testing. [7][8][9] On-site drug tests are used when immediate results are needed. 5,6 In recent time, 6-AM has become a target analyte already in the screening assay.…”

“…5,6 In recent time, 6-AM has become a target analyte already in the screening assay. [7][8][9] Because of the low concentrations and the structure similarities to other morphine alkaloids and metabolites many of the 6-AM immunoassays suffer from unwanted crossreactivity. [7][8][9] On-site drug tests are used when immediate results are needed.…”

“…[7][8][9] Because of the low concentrations and the structure similarities to other morphine alkaloids and metabolites many of the 6-AM immunoassays suffer from unwanted crossreactivity. [7][8][9] On-site drug tests are used when immediate results are needed. [10][11][12] These tests are based on immunochemistry and share the limited specificity that is typical of immunoassay drug screening tests.…”

Fabricating cholyglycine–glucose‐6‐phosphate dehydrogenase conjugates for cholyglycine detection

“…As a result, the reporter enzyme activity is repressed. EMIT has many advantages, such as rapid assay time , simple assay protocols , and low assay cost . Perhaps the most outstanding advantage compared with ELISA is that EMIT‐based assays can be conducted conveniently in homogeneous solution without washing and separation steps .…”