ANTIGENIC PROPERTIES OF KERATAN SULFATE - INFLUENCE OF ANTIGEN STRUCTURE, MONOCLONAL-ANTIBODIES, AND ANTIBODY VALENCY
ANTIGENIC PROPERTIES OF KERATAN SULFATE - INFLUENCE OF ANTIGEN STRUCTURE, MONOCLONAL-ANTIBODIES, AND ANTIBODY VALENCY
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DOI：
10.1016/0003-9861(92)90591-j
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发表时间：
1992-08-01
影响因子：
3.9
通讯作者：
RATCLIFFE, A
中科院分区：
文献类型：
--
作者：
SEIBEL, MJ;MACAULAY, W;RATCLIFFE, A
研究了抗原结构、单克隆抗体类型和抗体二价性对聚集蛋白聚糖中硫酸角质素（KS）免疫化学检测和定量的影响。通过免疫球蛋白G（IgG）-酶联免疫吸附试验（ELISA）测定表观KS表位水平的人聚集蛋白聚糖的制剂，并在一个定义的系列的低分子量蛋白聚糖制剂产生的蛋白水解和碱处理聚集蛋白聚糖。凝胶过滤层析显示KS表位优先在制剂的较高分子量片段中检测到。在单个KS链中，在较高Mr的链中检测到表位。蛋白聚糖在IgG-ELISA中的抑制能力随着蛋白聚糖片段大小的减小而降低，范围在6- 260倍之间，这取决于所使用的抗体。这被认为是一种合作约束力。对于大多数抗体，IgG-ELISA的灵敏度（由抑制斜率的陡度表示）也随着较小的抑制剂尺寸而降低。最低检测限（产生20%抑制所需的KS量）根据所用抗体的不同变化高达60倍。在ELISA中使用单价Fab片段代替完整IgG抗KS抗体表明抗体的二价性也影响测定的定量。在Fab-ELISA中，发现该测定具有增加的可检测性（用聚集蛋白聚糖作为抑制剂增加9.5倍），并且蛋白聚糖片段和聚集蛋白聚糖都产生平行的抑制曲线。虽然Fab-ELISA在一定程度上受到KS结构呈现的影响，但这对于小片段和单链并不明显。因此，合作的结合和抗体效价的影响，可以克服和定量数据，可以获得所有样品，使用木瓜蛋白酶消化的样品和Fab-ELISA。应用该测定法分析体液显示，滑液、血清和尿液中含有KS的片段大小不同，并且可以定量。
The influence of (a) antigen structure, (b) type of monoclonal antibody, and (c) antibody bivalency on the immunochemical detection and quantification of keratan sulfate (KS) from aggrecan has been studied. Apparent KS epitope levels were determined by immunoglobulin G (IgG)-enzyme-linked immunosorbent assay (ELISA) in preparations of human aggrecan and in a defined series of lower molecular weight proteoglycan preparations generated by proteolytic and alkali treatment of aggrecan. Gel filtration chromatography showed KS epitope to be preferentially detected in the higher molecular weight fragments of the preparations. In single KS chains the epitope was detected in the chains of higherMr. The ability of the proteoglycan to inhibit in the IgG-ELISA decreased with a reduction in proteoglycan fragment size, ranging between 6- and 260-fold, depending on the antibody used. This was considered to be a cooperative binding, effect. With most antibodies, the sensitivity of the IgG-ELISA (represented by the steepness of the inhibition slope) was also reduced with smaller inhibitor sizes. The lowest limit of detectability (the amount of KS required to generate 20% inhibition) varied by up to 60-fold depending on the antibody used. The use of monovalent Fab fragments instead of the whole IgG anti-KS antibody in the ELISA showed that the bivalency of the antibody also affected the quantitation of the assay. In the Fab-ELISA the assay was found to have an increased detectability (by 9.5-fold with aggrecan as the inhibitor), and the proteoglycan fragments and aggrecan all generated parallel inhibition curves. Although the Fab-ELISA was somewhat influenced by the structural presentation of the KS, this was not apparent for small fragments and single chains. Thus the effects of cooperative binding and antibody valency could be overcome and quantitative data could be obtained for all samples, using papain-digested samples and the Fab-ELISA. Application of this assay to analysis of body fluids showed the KS-containing fragments in synovial fluid, serum, and urine were of different sizes and could be quantified.