An immunoglobulin light chain from a lupus-prone mouse induces autoantibodies in normal mice.
An immunoglobulin light chain from a lupus-prone mouse induces autoantibodies in normal mice.
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来自易患狼疮的小鼠的免疫球蛋白轻链会在正常小鼠中诱导自身抗体。
DOI：
10.1084/jem.171.6.1919
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Schwartz,RS
中科院分区：
文献类型：
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作者：
Puccetti,A;Koizumi,T;Migliorini,P;André-Schwartz,J;Barrett,KJ;Schwartz,RS
材料与方法兔抗Id-28/12抗体的制备用CFA中的150 μ g亲和纯化的mAb 28/12皮下免疫兔，然后用IFA中的100 μ g抗体每两周加强三次。将含有最高抗28/12活性的血清合并，并通过在偶联到合并的小鼠Ig（TEPC 183、MOPC 104 E、UPC 10、FLOPC和MOPC 141，均购自利顿Bionetics，Inc.，查尔斯顿，南卡罗来纳州）。纯化的兔抗-28/12以1：2，000的稀释度使用。在固相上稀释后，200 JAI的生物素标记的mAb 28/12在20 min内产生0.600的OD 40 s。纯化的抗-28/12不与轻链F、G和105结合（见下文）。聚苯乙烯板（Immulon II; Dynatech Laboratories，Inc.，Alexandria，VA）用50 μ l兔抗28/12抗独特型抗体包被，该抗体在0.05 M硼酸盐缓冲液（pH 8.6）中以1：2，000稀释，并用3%BSA的PBS溶液封闭。使用含有1%BSA和0.05%Tween的PBS稀释标记和未标记的抗体。将251 μ l生物素标记的mAb 28/12（0.11 μ g/ml）（9），以对应于50%最大结合抗独特型试剂的浓度，与试验抗体一起在平板上于37 ℃温育1小时。在PBSIBSA/Tween中洗涤三次后，加入抗生物素蛋白-碱性磷酸酶（Boehringer曼海姆Biochemicals，Indianapolis，IN）并测量结合的酶活性。抗原结合。通过ELISA、蛋白质印迹和液相竞争测定法进行DNA和SmRNP结合，如别处所详述（9）。在mAb 28/12-Sepharose 4 B柱上从兔胸腺提取物中亲和纯化snRNP抗原（9）。轻链G的DNA序列。用异硫氰酸胍从杂交瘤细胞中分离总RNA，并通过在oligo（dt）-纤维素上传代制备聚腺苷酸化mRNA。将如前所述（10）用17个核苷酸的5 ′ Cu区引物（TGGATGGTGGGAAGATG）产生的cDNA连接到M13-mp 18中，并用从S.刘易斯和D.巴尔的摩（11）。通过双脱氧方法对阳性克隆进行测序（12）。
Materials and Methods Preparation ofRabbitAnti-Id-28/12. A rabbit was immunized subcutaneously with 150 ug of affinity-purified mAb 28/12 in CFA and then boosted biweekly three times with 100 hg of the antibody in IFA. Sera that contained the highest anti 28/12 activity were pooled and made specific for the immunizing idiotype by repeated adsorptions on Sepharose 4B coupled to pooled mouse Igs (TEPC 183, MOPC 104E, UPC 10, FLOPC, and MOPC 141, all purchased from Litton Bionetics, Inc., Charleston, SC). The purified rabbit anti-28/12 was used in a dilution of 1: 2,000. With that dilution on the solid phase, 200 JAI of biotin-labeled mAb 28/12 gave an OD4os of 0.600 within 20 min. The purified anti-28/12 did not bind to light chains F, G, and 105 (see below).Detection of Id-28/12. Polystyrene plates (Immulon II; Dynatech Laboratories, Inc., Alexandria, VA) were coated with 50 pl rabbit anti-28/12 antiidiotype diluted 1: 2,000 in 0.05 M borate buffer, pH 8.6, and blocked with 3% BSA in PBS. PBS containing 1% BSA and 0.05% Tween was used to dilute labeled and unlabeled antibodies. 251A1 ofbiotin-labeled mAb 28/12 (0.1 lAg/ml)(9), at a concentration corresponding to 50% maximal binding to the antiidiotype reagent, was incubated on the plate together with the test antibody for 1 h at 37 C. After three washes in PBSIBSA/Tween, avidin-alkaline phosphatase (Boehringer Mannheim Biochemicals, Indianapolis, IN) was added and the bound enzymatic activity measured. Antigen Binding. DNA and SmRNP binding by ELISA, Western blots, and liquid phase competition assays were conducted as described in detail elsewhere (9). The snRNP antigens were affinity purified from rabbit thymus extract on a mAb 28/12-Sepharose 4B column (9). DNA Sequence ofLight Chain G. Total RNA was isolated from hybridoma cells with guanidine isothiocyanate, and polyadenylated mRNA was prepared by passage over oligo (dt)-cellulose. cDNA, produced as previously described (10) with a 17-nucleotide 5'Cu region primer (TGGATGGTGGGAAGATG) was ligated into M13-mp18 and plaques were screened with a 32P-labeled 3 kb probe obtained from S. Lewis and D. Baltimore (11). Positive clones were sequenced by the dideoxy method (12).