title: 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.
authors: Immunalysis Corporation, 829 Towne Center Dr., Pomona, CA 91767, USA gwang@immunalysis.com.
authors: Guohong
authors: G
authors: Wang
authors: Immunalysis Corporation, 829 Towne Center Dr., Pomona, CA 91767, USA.
authors: Kim
authors: K
authors: Huynh
authors: Immunalysis Corporation, 829 Towne Center Dr., Pomona, CA 91767, USA.
authors: Rekha
authors: R
authors: Barhate
authors: Immunalysis Corporation, 829 Towne Center Dr., Pomona, CA 91767, USA.
authors: Jialin
authors: J
authors: Liu
authors: Immunalysis Corporation, 829 Towne Center Dr., Pomona, CA 91767, USA.
authors: Phillip
authors: P
authors: Tam
authors: Immunalysis Corporation, 829 Towne Center Dr., Pomona, CA 91767, USA.
authors: Warren
authors: W
authors: Rodrigues
authors: Immunalysis Corporation, 829 Towne Center Dr., Pomona, CA 91767, USA.
authors: Cynthia
authors: C
authors: Coulter
authors: Immunalysis Corporation, 829 Towne Center Dr., Pomona, CA 91767, USA.
authors: Christine
authors: C
authors: Moore
authors: Immunalysis Corporation, 829 Towne Center Dr., Pomona, CA 91767, USA.
authors: James
authors: J
authors: Soares
mesh_terms: D000701:Analgesics, Opioid / Q000652:urine; D002853:Chromatography, Liquid; D006207:Half-Life; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007124:Immunoenzyme Techniques; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D057230:Limit of Detection; D009020:Morphine / Q000652:urine; D009022:Morphine Derivatives / Q000652:urine*; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D015813:Substance Abuse Detection; D053719:Tandem Mass Spectrometry
chemical_list: D000701:Analgesics, Opioid; D007128:Immunoglobulin Fragments; D009022:Morphine Derivatives; D009020:Morphine; C026979:6-O-monoacetylmorphine