Group-selective immunoassay for the detection of morphine in urine.
A new competitive inhibition immunoassay (group-selective immunoassay; GSI) has been developed to detect free morphine in urine with the Fab' fragments of monoclonal antibodies (MAbs) (1B(12)F(9)B(4), IgG(1), kappa, K(aff) = 9.66 x 10(10)M(-1)). At the first assay step, microtiter plates were coated with morphine-ovalbumin (M-6-S-OVA), in which free amino acids were protected by a glutaraldehyde cross-linking modification. The modification did not essentially influence the antibody-binding capacity of the immunosorbent. At the second assay step, anti-morphine MAbs' Fab' fragments, in which free amino groups were biotinylated by N-hydrosuccinimide-biotin ester, were bound to chemically modified immunosorbent. The biotin residues were then detected by the streptavidin-peroxide conjugate. This method has a sensitivity of 3.50 x 10(-15) mol/L using very little volume of sample, covering up to almost 1.20 x 10(-11) mol/L of standard concentration of morphine with good reproducibility. Standard curve prepared in urine indicated a good correlation between the concentration of morphine and the value of OD (y = 1/ax + b; r = 0.99939257, S = 0.01138127). Coefficients of variation for this immunoassay were 1.41 approximately 6.61% within-a-day assay and 2.31 approximately 8.99% between days assay. The recoveries were 94 approximately 101.4% from negative urine and 95.2 approximately 107.5% from positive urine samples, respectively. This method has application as a specific screen for morphine in drug abusers, to study the metabolism of the drug in the body, or to screen the monoclonal antibodies (MAbs) against morphine.
Institute of Neurosciences, Fourth Military Medical University, Xi'an, Shaanxi, People's Republic of China. y_t_b@hotmail.com
Tang-Bin
TB
Yang
Yan-Hong
YH
Yuan
Ping
P
Zhong
Li-Na
LN
Qu
Bin
B
Yang
Ying-Hui
YH
Li
Gong
G
Ju
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D009020:Morphine / Q000652:urine*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D009020:Morphine