Iwagaki Naofumi, Mizuno Motowo, Nasu Jyunichirou, Mizuno Masakatsu, Okazaki Hiroaki, Hori Shinichirou, Yamamoto Kazuhide, Okada Hiroyuki, Tsuji Takao, Fujita Teizo, Shiratori Yasushi
Department of Medicine and Medical Science (Medinen 1). Okayama University Graduate School of Medicine and Dentistry, 2-5-1, Shikata-cho, Okayama 700-8558, Japan.
J Immunoassay Immunochem. 2002;23(4):497-507. doi: 10.1081/ias-120015480.
We have previously shown that stool concentrations of decay-accelerating factor (DAF; CD55), a membrane-bound complement-regulatory protein, are significantly elevated in patients with colorectal cancer and that the measurement of stool DAF may be a valuable test for the detection of colorectal cancer. Accordingly, we are working to develop a clinically useful immunoassay for fecal DAF. A requirement for such assay is a plentiful and reliable supply of anti-DAF antibodies. We developed a sandwich enzyme-linked immunosorbent assay (ELISA) for DAF in stool specimens, using two monoclonal anti-DAF antibodies recognizing different epitopes on the DAF molecule. When we first used a biotin-labeled antibody and enzyme-linked streptavidin method, we often observed stool interference, probably due to the presence of a substance(s) with biotin activity which non-specifically bound to the Fc portion of IgG of the first anti-DAF antibody on the ELISA wells. By the use of inorganic salts in the sample-dilution buffer and HRP-labeled anti-DAF as second antibody, we circumvented the stool interference and established that the new ELISA system could reliably measure DAF at low concentrations in stool specimens. Because the new assay system uses only monoclonal antibodies, we can now consistently supply ample amounts of antibodies for routine measurement of stool DAF.
我们之前已经表明，衰变加速因子（DAF；CD55）是一种膜结合补体调节蛋白，结直肠癌患者粪便中该蛋白的浓度显著升高，并且粪便DAF的检测可能是检测结直肠癌的一项有价值的检测方法。因此，我们正在致力于开发一种临床上有用的粪便DAF免疫测定法。这种测定法的一个要求是要有大量且可靠的抗DAF抗体供应。我们使用两种识别DAF分子上不同表位的单克隆抗DAF抗体，开发了一种用于粪便标本中DAF的夹心酶联免疫吸附测定法（ELISA）。当我们最初使用生物素标记抗体和酶联链霉亲和素方法时，我们经常观察到粪便干扰，这可能是由于存在具有生物素活性的物质，该物质非特异性地结合到ELISA孔中第一种抗DAF抗体IgG的Fc部分。通过在样品稀释缓冲液中使用无机盐以及使用辣根过氧化物酶（HRP）标记的抗DAF作为二抗，我们规避了粪便干扰，并确定新的ELISA系统能够可靠地测量粪便标本中低浓度的DAF。由于新的测定系统仅使用单克隆抗体，我们现在能够持续供应大量抗体用于粪便DAF的常规检测。
