Loirat M J, Czerwinski M, Duk M, Blanchard D
Atlantique/Vendée, Nantes, France.
Transfus Med. 1999 Mar;9(1):69-79. doi: 10.1046/j.1365-3148.1999.009001069.x.
The murine monoclonal antibody NaM26-4C6 (IgM class), obtained from the splenocytes of a BALB/c mouse immunized with human umbilical cord red blood cells, was characterized by agglutination test and immunoblotting analysis. The structure of the NaM26-4C6 epitope was further elucidated by using a series of peptides synthesized on pins. The antibody agglutinated untreated and chymotrypsin-treated but not trypsin- or neuraminidase-treated human erythrocytes. Agglutination-inhibition test demonstrated that the antibody recognizes an epitope located on the N-terminal trypsin-sensitive portion of glycophorin C. The antibody bound on immunoblots to glycophorin C, and also to the band 3 protein and its 69-kDa N-terminal fragment but did not bind to desialylated and de-O-glycosylated glycophorin C. Peptide mapping allowed localization of the binding site on the 23-kDa N-terminal intracellular peptide of band 3. The antibody binds to the amino-acid sequences 22EDPDIP27 of band 3 protein and 15SLEPDPGM22 of glycophorin C, and residues D and P were found to be essential. The new epitope identified by NaM26-4C6 corresponds to a linear amino acid sequence located on the N-terminal intracellular portion of band 3 and to a more complex structure involving oligosaccharide chains on the N-terminal extracellular domain of GPC.
从用人脐带红细胞免疫的BALB/c小鼠脾细胞中获得的鼠单克隆抗体NaM26-4C6（IgM类），通过凝集试验和免疫印迹分析进行了表征。使用在针上合成的一系列肽进一步阐明了NaM26-4C6表位的结构。该抗体能凝集未处理的和经胰凝乳蛋白酶处理的人红细胞，但不能凝集经胰蛋白酶或神经氨酸酶处理的人红细胞。凝集抑制试验表明，该抗体识别位于血型糖蛋白C N端对胰蛋白酶敏感部分的一个表位。该抗体在免疫印迹上与血型糖蛋白C结合，也与带3蛋白及其69 kDa的N端片段结合，但不与去唾液酸化和去O-糖基化的血型糖蛋白C结合。肽图谱分析确定了带3蛋白23 kDa N端细胞内肽上的结合位点。该抗体与带3蛋白的氨基酸序列22EDPDIP27和血型糖蛋白C的15SLEPDPGM22结合，发现残基D和P是必需的。NaM26-4C6鉴定的新表位对应于位于带3蛋白N端细胞内部分的线性氨基酸序列，以及涉及血型糖蛋白C N端细胞外结构域上寡糖链的更复杂结构。
