Detection of circulating gastric carcinoma-associated antigen MG7-Ag in human sera using an established single determinant immuno-polymerase chain reaction technique.

BACKGROUND: In 1994, a novel sensitive method termed immuno-polymerase chain reaction (PCR) for the detection of the gastric carcinoma-associated antigen MG7-Ag in the gastric carcinoma cell line KATO III was reported. Compared with the enzyme-linked immunoadsorbent assay, the single determinant immuno-PCR technique could allow for as few as 20 cells to be detected and was found to show an approximately 10,000-fold enhancement in sensitivity of the detection limit. The current study clinically evaluated the significance of serum MG7-Ag detection in gastric carcinoma patients.
METHODS: The sera of patients were immobilized on wells and a specific DNA molecule, which could be amplified by PCR, was employed as a marker. The biotinylated monoclonal antibody against gastric carcinoma was added to bind the antigen immobilized on the wells. After the biotinylated antibody was bound to the antigen, free avidin was used to attach a biotinylated monoclonal antibody and biotinylated DNA molecule. The biotinylated DNA complexed with antigen-antibody-avidin was amplified by PCR and the PCR products were analyzed by agarose gel electrophoresis. In the current study this method was used to detect circulating MG7-Ag in the sera of patients with gastric carcinoma and other various malignancies. For comparison, carcinoembryonic antigen, CA 50, CA 19-9, and TAG-72 were quantitated by radioimmunoassay and immunoradiometric assay using the relevant commercial kits in the same sera samples from 86 patients with pathologically confirmed gastric carcinoma and 83 patients with relevant benign diseases of the stomach. In addition, the semiquantitative analysis of PCR products among gastric carcinoma patients with or without metastasis was performed to compare the intensity of DNA band amplification.
RESULTS: Using the immuno-PCR assay, positive results were obtained in 164 of 198 patients with gastric carcinoma (82.8%). The rates of positivity in other malignancies were 17.4% for esophageal carcinoma (15 of 86 patients), 44.4% for colonic carcinoma (40 of 90 patients), 0% for liver carcinoma (none of 84 patients), 2.2% for ovarian carcinoma (1 of 45 patients), 0% for uterine carcinoma (none of 27 patients), and 6.1% for lung carcinoma (4 of 66 patients). The positive results obtained from those patients with benign diseases were: 7.7% for peptic ulcer (6 of 78 patients), 5.9% for chronic gastritis (7 of 118 patients), 3.3% for chronic colitis (2 of 60 patients), and 0.8% for healthy blood donors (2 of 236 patients). In addition, the semiquantitative analysis of PCR products showed that the intensity of DNA band amplified from the PCR products of those patients with metastasis was much higher than that of patients without metastasis or those with early stage tumors (1.94 +/- 0.03 vs. 1.28 +/- 0.02). In comparative studies of immuno-PCR and commercial assays for tumor-associated antigens the sensitivity of immuno-PCR was 81.4% and pseudopositivity was lower (8.4% vs. 7.2-12.0% with radioimmunoassay or immunoradiometric assay).
CONCLUSIONS: The results of the current study demonstrate that introducing PCR into the indirect determination of tumor-associated antigen in the serum can improve the sensitivity of detection greatly. This novel assay also might be used to monitor the circulating amount of tumor-associated antigen after gastrectomy and provide information regarding recurrence or metastasis, as well as for screening elderly patients who have no indications for endoscopy and those with precancerous conditions. The application of immuno-PCR in the serologic diagnosis of carcinoma has significant advantages including ready application in the clinical setting as well as use as a potential screening tool in mass surveys of high risk populations with gastric carcinoma. (c) 2000 American Cancer Society.