A sensitive assay for measuring alpha-Gal epitope expression on cells by a monoclonal anti-Gal antibody.
A sensitive assay for measuring alpha-Gal epitope expression on cells by a monoclonal anti-Gal antibody.
复制标题
通过单克隆抗 Gal 抗体测量细胞上 α-Gal 表位表达的灵敏测定法。
作者：
Uri Galili;D. LaTemple;Marko Z. Radic
背景
评估各种细胞和组织上的α-gal表位（Galalpha 1 - 3Galbeta 1 -4GlcNAc-R）表达对于预测抗Gal介导的异种移植物免疫排斥是重要的。本研究描述了一种酶联免疫吸附试验（ELISA抑制试验）为此目的，它使用单克隆抗半乳糖胺抗体M86。
方法
将不同浓度的细胞与1/100稀释的M86孵育过夜。除去细胞和结合的抗体，用α-gal-牛血清白蛋白作为固相抗原在ELISA测定中测量上清液中的残留抗体。细胞上α-gal表位表达的程度与ELISA中M86结合的后续抑制相关。将不同细胞浓度下的抑制结合曲线与每个细胞具有已知数量表位的标准细胞系的抑制结合曲线进行比较。
结果和结论
小鼠IgM M86单克隆抗体对α-gal表位具有高度特异性。在ELISA抑制测定中使用该抗体，细胞在宽范围的浓度下，能够检测每个细胞至少5 × 10（4）和高达超过5 × 10（7）的α-gal表位。该测定还可用于检测来自组织匀浆的膜上的α-gal表位，因此其能够确定经遗传操作以改变其碳水化合物组成的动物中α-gal表位表达的降低程度。
BACKGROUND
The assessment of a-gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) expression on various cells and tissues is important for the prediction of anti-Gal-mediated immune rejection of xenografts. This study describes an enzyme-linked immunosorbent assay (ELISA inhibition assay) developed for this purpose, which uses the monoclonal anti-Gal antibody M86.
METHODS
Cells at various concentrations were incubated overnight with M86 at 1/100 dilution. The cells and bound antibody were removed, and the residual antibody in the supernatant was measured in an ELISA assay with a-gal-bovine serum albumin as a solid phase antigen. The extent of a-gal epitope expression on cells correlates with the subsequent inhibition of M86 binding in ELISA. The inhibition binding curves at various cell concentrations were compared with those of a standard cell line with a known number of epitopes per cell.
RESULTS AND CONCLUSIONS
The mouse IgM M86 monoclonal antibody was highly specific for a-gal epitopes. Using this antibody in an ELISA inhibition assay with cells at a wide range of concentrations enables the detection of at least 5 x 10(4) and up to more than 5 x 10(7) a-gal epitopes per cell. This assay can be used also for the detection of a-gal epitopes on membranes from tissue homogenates, and thus it enables the determination of the extent of decrease in a-gal epitope expression in animals that are genetically manipulated to alter their carbohydrate make-up.
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DOI：
10.4049/jimmunol.1202204
发表时间：
2013-02-15
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Olaru F;Wang XP;Luo W;Ge L;Miner JH;Kleinau S;Geiger XJ;Wasiluk A;Heidet L;Kitching AR;Borza DB
通讯作者：
Borza DB
灵长类动物之间的心脏异种移植物为α-半乳糖基决定簇在超急性排斥反应中的重要性提供了证据。
DOI：
--
发表时间：
1995
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Collins,BH;Cotterell,AH;McCurry,KR;Alvarado,CG;Magee,JC;Parker,W;Platt,JL
通讯作者：
Platt,JL
鼠α1,3-半乳糖基转移酶。
DOI：
--
发表时间：
1992
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Joziasse,DH;Shaper,NL;Kim,D;VandenEijnden,DH;Shaper,JH
通讯作者：
Shaper,JH
通过α1,3半乳糖基转移酶基因转染改变细胞表面碳水化合物并抑制小鼠黑色素瘤的转移特性。
DOI：
--
发表时间：
1995
期刊：
Cancer research.
影响因子：
--
作者：
Gorelik,E;Duty,L;Anaraki,F;Galili,U
通讯作者：
Galili,U