Establishment of ELISA for detection of hepatoma specific γ-glutamyltransferase and its clinical application / 中华检验医学杂志

ABSTRACT

Objective To prepare monoclonal antibody (McAb) against γ-glutamyhransferase(GGT) firmly bound to datura stramonim (DSA) leetin from primary hepatic carcinoma (PHC) tissue and establish an avidin-biotin enzyme-linked immunosorbent assay (ELISA) for evaluating the diagnostic value of serum DSA-GGT for PHC. Methods Anti-DSA-GGT monoclonal antibodies were obtained by McAb technology and purified by protein G-sepharose affinity chromatography. The McAb was labeled with biotin and avidin-biotin ELISA for measurement of serum DSA-GGT was established. Using the avidin-biotin ELISA, serum DSA-GGT levels was detected in 39 patients with PHC, and 122 patients with non-PHC diseases. The distribution of serum DSA-GGT values of 119 healthy subjects were determined by P-P plots. Optimal cut-off value for the diagnosis of PHC was determined by receiver operating characterstic (ROC) curve. Results The protein levels of McAb in the ascites derived from 5 McAb hybridoma cell strains ranged from 2. 12 to 6. 70 mg, The biotin-labeled rate varied from 48. 6% to 72. 2% respectively. The minimum detection limit of serum DSA-GGT in avidin-biotin ELISA was 2 μg/L. Intra-assay and inter-assay coefficients of variation were 8.9% and 11.5% respectively. The distribution of DSA-GGT values of 119 healthy subjects showed Gaussian distribution and its Mean ± SD was ( 1.50±0. 51 ) μg/L. Optimal cut-off value (3.25 μg/L) in the diagnosis of PHC was determined by ROC curve. DSA-GGT was positive in 26 out of 39 patients with PHC and 10 out of 122 patients with non-PHC diseases were positive. The sensitivity and specificity of this assay for the diagnosis of PHC were 66. 7% and 91.8% respectively. Conclusions The convenient avidin-biotin ELISA method was successfully established in our laboratory and it showed a good reproducibility and reliability. It may be a potential tool in the diagnosis of PHC to achieve higher sensitivity and specificity.