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 navel sensitive method termed immuno-polymerase chai n reaction (PCR) for the detection of the gastric carcinoma-associated anti gen MG7-Ag in the gastric carcinoma cell line KATO III was reported. Compar ed with the enzyme-linked immunoadsorbent assay, the single determinant imm uno-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 t he 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 bi otinylated monoclonal antibody against gastric carcinoma was added to bind the antigen immobilized on the wells. After the biotinylated antibody was b ound to the antigen, free avidin was used to attach a biotinylated nonoclon al antibody and biotinylated DNA molecule. The biotinylated DNA complexed w ith. antigen-antibody-avidin was amplified by PCR and the PCR products were analyzed by agarose gel electrophoresis. in the current study this method tvas used to detect circulating MG7-Ag in the sera of patients with gastric carcinoma and other various malignancies. For comparison, carcinoembryonic , CA.50, CA 19-9, and TAG-72 were quantitated by radioimmunoassay and immu noradiometric assay using the relevant commercial kits in the same sera sam ples from 86 patients with pathologically confirmed gastric carcinoma and 8 3;patients with relevant benign diseases of the stomach. In addition, the s emiquantitative analysis of PCR products among gastric carcinoma patients w ith or without metastasis was performed to compare the intensity of DNA ban d amplification. RESULTS. Using the immuno-PCR assay, positive results were obtainer 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 carcfnoma (40 of 90 patients), 0% for liver carcinoma (no ne of 84 patients), 2.2% for ovarian carcinoma (1 of 45 patients), 0% for u terine carcinoma(none of 21 patients), and 6.1% for lung carcinoma (4 of 66 patients). The positive results obtained from those patients with benign d iseases were 7.7% for peptic ulcer (6 of 78 patients), 5.9% for chronic gas tritis (7 of 118 patients);, 3.3% for chronic colitis (2 of 60 patients), a nd 0.8% for healthy blood donors (2 of 236 patients). In addition, the semi quantitative analysis of PCR products showed that the intensity of DNA band amplified from the PCR products of those: patients with metastasis tvas mu ch higher than that of patients without metastasis or those with early stag e tumors (1.94 +/- 0.03 vs. 1.28 +/- 0.02). In comparative studies of immun o-PCR and commercial assays for tumor-associated antigens the sensitivity o f immuno-PCR was 81.4% and pseudopositivity was lower (8.4% vs. 7.2-12.0% w ith 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 seru m can improve the sensitivity of detection greatly. This novel assay also m ight be used to monitor the circulating amount of tumor-associated antigen after gastrectomy and provide information regarding recurrence or metastasi s, as well as for screening elderly patients who have no indications for en doscopy and those with precancerous conditions. The application of immuno-P CR in the serologic diagnosis of carcinoma has significant advantages inclu ding ready application in the clinical setting as well as use as a potentia l screening tool in mass surveys of high risk populations with gastric carc inoma. (C) 2000 American Cancer Society.