A SENSITIVE METHOD FOR THE DETECTION OF SPECIFIC ANTIBODY-PRODUCTION IN DIFFERENT ISOTYPES FROM SINGLE LAMINA PROPRIA PLASMA-CELLS
A SENSITIVE METHOD FOR THE DETECTION OF SPECIFIC ANTIBODY-PRODUCTION IN DIFFERENT ISOTYPES FROM SINGLE LAMINA PROPRIA PLASMA-CELLS
复制标题
DOI：
10.1111/j.1365-3083.1986.tb02127.x
复制
发表时间：
1986-10-01
影响因子：
3.7
通讯作者：
LYCKE, N
中科院分区：
文献类型：
--
作者：
LYCKE, N
开发了一种灵敏且可重复的方法，用于在单细胞水平上检测分离的固有层淋巴细胞的特异性抗体产生（或总免疫球蛋白分泌）。通过胶原酶酶提取从小鼠肠粘膜制备细胞，并用固相酶联免疫斑点（ELISPOT）测定证明抗体分泌。口服霍乱毒素或匙孔血蓝蛋白免疫小鼠分离的固有层淋巴细胞之间的高度抗原特异性斑点形成细胞（SFC）的数量很高。斑点是由免疫球蛋白在体外的主动合成引起的。在给定的口服免疫方案后，发现个体动物之间SFC数量的变化为25%，在不同情况下分析的相同组之间为12%。霍乱毒素口服免疫后初级和次级免疫反应的动力学很容易监测。单剂量霍乱毒素产生230抗毒素SFC/107分离的固有层淋巴细胞。在五次免疫后，每增加一个剂量刺激特异性SFC的增加，大约7000抗毒素SFC/107细胞。对口服加强免疫后逐日应答的监测表明，在抗原施用后第8天达到SFC数量峰值。免疫球蛋白分泌（IG）细胞的总数和特异性SFC的同种型分布也可以确定。在峰值抗毒素反应中，8%的分离的总Ig分泌固有层细胞对霍乱毒素有活性，其中80%产生伊加。该方法也已成功用于人和兔，以证明单个固有层浆细胞产生特异性抗体。
A sensitive and reproducible method for the detection of specific antibody production (or total immunoglobulin secretion) at the single cell level from isolated lamina propria lymphocytes was developed. The cells were prepared from mouse intestinal mucosa by enzyme extraction with collagenase, and antibody secretion was demonstrated with a solid phase enzyme-linked immunospot (ELISPOT) assay. Oral immunizations with cholera toxin or keyhole limpet haemocyanin to mice gave high numbers of highly antigen-specific spot-forming cells (SFC) among isolated lamina propria lymphocytes. Spots were shown to result from active synthesis of immunoglobulin in vitro. The variation in SFC numbers between individual animals after a given protocol of oral immunizations was found to be 25% and between equal groups analysed on different occasions, 12%. Kinetics of primary as well as secondary immune responses after oral immunizations with cholera toxin were easily monitored. A single dose of cholera toxin gave rise to 230 antitoxin SFC/107 isolated lamina propria lymphocytes. Each additional dose stimulated to increasing numbres of specific SFC with roughly 7000 antitoxin SFC/107 cells after five immunizations. Monitoring to day-by-day responses after oral booster immunizations demonstrated peak SFC numbers on day 8 after antigen administration. The total number of immunoglobulin-secreting (Ig) cells and the isotype distribution of specific SFC could also be determined. In the peak antitoxin response, 8% of the isolated total Ig-secreting lamina propria cells were active against cholera toxin, and of these 80% were producing IgA. This method has also been successfully used in humans and rabbits to demonstrate specific antibody production by single lamina propria plasma cells.