Development of methods detecting HLA-D region genes and their products regulating disease susceptibility

疾病易感性调控HLA-D区基因及其产物的检测方法开发

基本信息

批准号：
60870017
负责人：
KATAGIRI Makoto
金额：
$ 4.61万
依托单位：
Asahikawa Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1986
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-60870017/
关键词：
HLA antigen class <II> antigen class <II> gene HLA-D region disease susceptibility 酵素免疫測定法酵素抗体法

项目摘要

Gene products of HLA-D region (class <II> antigens) are involved in the development of a certain category of autoimmune diseases as an etiologic factor. DNA analysis suggested the presence of extra class <II> antigens in addition to four types of class <II> antigen system (DR, DRw52.53, DQ and DP). For the detection of these class <II> molecules, radioimmunoassay (RIA) instead of cytotoxic assay has been widely used. Since the use of radioisotopes, however, is rather restricted, we have been trying to establish non-radioisotopic assay systems for class <II> molecules in the hope that they can eventually be used in the field of clinical pathology.We have developed three kinds of enzyme immunoassay (EIA) using peroxidase-labeled anti-mouse Ig antibody or biotinylated anti-mouse Ig antibody and avidin-peroxidase.1. Solid phase EIA method in which solubilized class <II> molecules are attached to the well as target antigens: Various detergents and buffer systems for the attachment of target molecules have been tested. Deoxycholic acid as detergent and tris-HCl as buffer have yielded most reproducible results. The plates thus prepared can be used for three months if stored at -40゜C. It has also been confirmed that this method can be applied for immunochemical analysis of class <II> molecules.2. Cellular EIA method in which cells themselves are used as targets: Two versions of this method have been established using either Millititer SV plates (Millipore Corp.) or Immulon 600 flat-bottomed plates (Greiner). The former is convenient in that cells can be washed by aspiration from the bottom of the well, and the latter is advantageous because the plates can be stored for three months at 4゜C once cells are attached to the well. Furthermore, biotin-avidin system has been shown to be more sensitive than RIA in detecting class <II> antigens. We hope that these methods will be used in various fields of medical science.

HLA-D区域（<ii>抗原）的基因产物参与了特定类别的自身免疫性疾病作为病因学因素的发展。 DNA分析表明，除了四种类型的类抗原系统外，还存在额外的类抗原（DR，DRW52.53，DQ和DP）。为了检测这些类<ii>分子，已广泛使用了放射免疫测定（RIA）而不是细胞毒性测定。 Since the use of radioisotopes, however, is rather restricted, we have been trying to establish non-radioisotopic assay systems for class <II> molecules in the hope that they can sometimes be used in the field of clinical pathology.We have developed three kinds of enzyme immunoassay (EIA) using peroxidase-labeled anti-mouse Ig antibody or biotinylated anti-mouse Ig antibody and Avidin-过氧酶1。固相EIA方法，其中溶解的类<ii>分子与靶抗原相连：已测试了用于附着目标分子的各种确定剂和缓冲系统。确定的脱氧胆酸和TRIS-HCL作为缓冲液产生了最可重现的结果。这样准备的板如果存储在-40°C下，则可以使用三个月。还可以证实，该方法可以用于类分子的免疫化学分析2。细胞本身用作靶标的细胞EIA方法：已使用Millitter SV板（Millipore Corp.）或Immulon 600平底板（Greiner）建立了该方法的两个版本。前者很方便，因为可以通过从井的底部抽吸来洗涤细胞，而后者是有利的，因为一旦将细胞连接到井上，板就可以在4°C下在4°C下储存三个月。此外，在检测<ii>抗原中，已经证明生物素 - 阿维丁系统比RIA更敏感。我们希望这些方法将用于医学的各个领域。