Immunoassay for the determination of morphine-3-glucuronide using a surface plasmon resonance-based biosensor.
Polyclonal antibodies were produced for the development of competitive ELISA's and surface plasmon resonance (SPR)-based BIAcore inhibition assays for the detection of morphine-3-glucuronide (M3G, the main metabolite of heroin and morphine). A conjugate consisting of M3G and ovalbumin was produced and used for the generation of antibodies, for the coating of immunoplates and for immobilisation onto BIAcore chips. Competition ELISA's were developed in PBS and urine to characterise the antibodies ability to recognise free M3G. SPR-based inhibition immunoassays on BIAcore were developed. The regeneration of the surface of a chip immobilised with conjugate following antibody binding, essential for the development of inhibition assays was investigated. Regeneration of the conjugate-coated surface was optimised for both polyclonal antibodies resulting in binding-regeneration capacities of approximately 60 cycles for one antibody and 50 cycles for the second antibody. The inhibition assays developed in urine had ranges of detection of 762-24,400 (antibody 1) and 976-62,500 pg ml(-1) (antibody 2). The inter-day coefficients of variation for the assays ranged from 1.48 to 11.24%.
Dillon
Paul P
PP
School of Biotechnology, Dublin City University, 9, Dublin, Ireland.
Daly
Stephen J
SJ
Manning
Bernadette M
BM
O'Kennedy
Richard
R
D000911:Antibodies, Monoclonal; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D020099:Coated Materials, Biocompatible / Q000138:chemical synthesis; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D009022:Morphine Derivatives / Q000032:analysis / Q000652:urine*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015813:Substance Abuse Detection / Q000295:instrumentation / Q000379:methods; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*
D000911:Antibodies, Monoclonal; D020099:Coated Materials, Biocompatible; D009022:Morphine Derivatives; C018108:morphine-3-glucuronide