MONOCLONAL ANTI-LAMBDA-5 ANTIBODY FS1 IDENTIFIES A 130 KDA PROTEIN ASSOCIATED WITH LAMBDA-5 AND V(PRE-B) ON THE SURFACE OF EARLY PRE-B-CELL LINES
MONOCLONAL ANTI-LAMBDA-5 ANTIBODY FS1 IDENTIFIES A 130 KDA PROTEIN ASSOCIATED WITH LAMBDA-5 AND V(PRE-B) ON THE SURFACE OF EARLY PRE-B-CELL LINES
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DOI：
10.1093/intimm/6.3.393
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发表时间：
1994-03-01
影响因子：
4.4
通讯作者：
JONGSTRA, J
中科院分区：
文献类型：
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作者：
SHINJO, F;HARDY, RR;JONGSTRA, J
用细菌合成的重组lambda5蛋白免疫地鼠的脾细胞与小鼠骨髓瘤细胞系SP2/0-Ag14融合，制备了针对小鼠lambda5蛋白的单抗。在这里，我们报告了FS1杂交瘤产生的抗体的特征。FS1抗体可染多种小鼠前B细胞系，但不能染B细胞系或T细胞系。藻红蛋白(PE)标记的FS1(FS1-PE)对前B细胞系A-1和C-7的染色可被未结合的FS1抗体或亲和纯化的多克隆lambda5特异性Ig预育所阻断，但不能与正常仓鼠或小鼠的Ig或亲和纯化的多克隆抗Mb-1 Ig共同孵育。从这些实验中，我们得出结论，FS1特异性识别lambda5蛋白。我们用FS_1-PE对正常BALB/c小鼠骨髓表面(S)lambda5+细胞进行了探测。当分析总骨髓或FACS分类亚群时，这些细胞是检测不到的。当B220+、CD43+、slambda5-的骨髓细胞在含有IL-7的基质细胞系FLST2上培养4d后，slambda5的表达明显增强。仅在IL7中这些细胞的进一步扩张就将slambda5的表达增强到容易检测到的水平。这种调节可能表明，体内正常骨髓细胞上的slambda5表达是短暂的，在任何给定的时刻，只有一小部分骨髓细胞表面表达低水平的lambda5蛋白。或者，我们的FS1单抗与正常骨髓细胞上的slambda5分子的结合可能会被体内和体外直接与slambda5复合体结合的其他蛋白质所阻断。以前用抗lambda5的多克隆兔抗血清对早期小鼠Pre-B细胞系表面lambda5相关蛋白的分析表明，slambda5蛋白与一种高相对分子质量的蛋白有关。使用我们的FS1单抗对早期Pre-B细胞系的lambda5及其相关蛋白的分析证实了我们之前的发现，并表明早期的lambda5受体至少包含三种蛋白质：lambda5，V(Pre-B)，以及一个尚未鉴定的蛋白质，分子量为130,000，被指定为p130。
mAbs specific for mouse lambda5 protein were prepared by fusion of spleen cells from a hamster immunized with recombinant lambda5 protein synthesized in bacteria and the mouse myeloma cell line SP2/0-Ag14. Here we report the characteristics of the antibodies produced by the FS1 hybridoma. FS1 antibody stains a variety of mouse pre-B cell lines but not B cell lines or T cell lines. The staining of the pre-B cell lines A-1 and C-7 by phycoerythrin (PE)-conjugated FS1 (FS1 - PE) can be blocked by preincubation of these cells with unconjugated FS1 antibody or with affinity purified polyclonal lambda5 specific Ig but not with normal hamster or mouse IgG or with affinity purified polyclonal anti-Mb-1 Ig. From these experiments we concluded that FS1 specifically recognizes lambda5 protein. We used FS1 - PE to probe for surface (s) lambda5+ cells in normal BALB/c mouse bone marrow. Such cells were undetectable when total bone marrow or FACS sorted subpopulations were analyzed. However, when B220+, CD43+, slambda5- bone marrow cells were cultured for 4 days on the stromal cell line FLST2 in the presence of IL-7, slambda5 expression became apparent. Further expansion of these cells in IL7 alone augmented the slambda5 expression to readily detectable levels. This modulation may indicate that slambda5 expression on normal bone marrow cells in vivo is transient and that at any given moment only a small fraction of bone marrow cells expresses low levels of lambda5 protein on the surface. Alternatively the binding of our FS1 mAb to the slambda5 molecules on normal bone marrow cells may be blocked by other proteins binding to the slambda5 complex in vivo and directly ex vivo. Previous analysis of surface lambda5 associated proteins on early mouse pre-B cell lines using a polyclonal anti-lambda5 rabbit antiserum had suggested that slambda5 protein was associated with a high molecular weight protein. Analysis of lambda5 and its associated proteins on early pre-B cell lines using our FS1 mAb confirmed our previous finding and showed that the early lambda5 receptor contains at least three proteins: lambda5, V(pre-B), and an as yet uncharacterized protein with a molecular weight of 130,000 designated p130.