QUANTITATIVE-DETERMINATION OF TOTAL AND SPECIFIC HUMAN IGE WITH THE USE OF MONOCLONAL-ANTIBODIES
QUANTITATIVE-DETERMINATION OF TOTAL AND SPECIFIC HUMAN IGE WITH THE USE OF MONOCLONAL-ANTIBODIES
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DOI：
10.1016/0091-6749(86)90378-7
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发表时间：
1986-06-01
影响因子：
14.2
通讯作者：
STADLER, BM
中科院分区：
文献类型：
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作者：
GRASSI, J;DIDIERLAURENT, A;STADLER, BM
我们使用识别人IgE恒定区两个不同表位的两种单克隆抗体（BS17和Le27）来测定人血清中所含的总IgE和特异性IgE。这两种类型的分析都是基于经典的PRIST和RAST程序，并涉及一个或两个单克隆抗体标记的125I碘。将这两种检测方法的性能与市售多克隆示踪剂获得的结果进行系统比较。我们首先研究了单克隆抗体的125i标记条件。在我们手中，最好的结果是使用比放射性接近10 μ μ ci / μ μ g的示踪剂。我们已经能够证明，由于其有限的亲和力，放射性示踪剂总是不完全结合到固相IgE。尽管如此，该检测的灵敏度与多克隆抗体相当，因为通过PRIST程序可以测量< 0.5 IU/ml。相比之下，用单克隆抗体在RAST中获得的稀释曲线与用多克隆示踪剂观察到的稀释曲线有很大不同。事实上，这些曲线与稀释曲线和PRIST方法得出的标准曲线严格平行，从而表明单克隆抗体对所有IgE的识别都一样好，无论它们以何种方式与固相结合。在此基础上，我们提出了一种以PRIST标准曲线为参考的特异性IgE定量测定方法。我们的结果表明，如果使用高容量，固相以及长孵育时间，这种方法确实是可能的。
We have used two monoclonal antibodies (BS17 and Le27) that recognize two differnet epitopes of the constant region of human IgE in order to determine the total and specific IgE contained in human serum. Both types of assay are based on classic PRIST and RAST procedures and involve one or two monoclonal antibodies labeled with 125I iodine. The performance of these two assays were compared systematically with those obtained with a commercially available polyclonal tracer. We first investigated the 125I-labeling conditions for monoclonal antibodies. In our hands the best results were obtained by use of a tracer of specific radioactivity close to 10 .mu.Ci/.mu.g. We have been able to demonstrate that as a consequence of its limited affinity, the radioactive tracer is always incompletely bound to the solid-phase IgE. Nonetheless, the sensitivity of the assay is comparable to that obtained with the polyclonal antibody, since < 0.5 IU/ml can be measured by the PRIST procedure. In contrast, the dilution curves obtained in the RAST with the monoclonal antibodies are very different from those observed with the polyclonal tracer. In fact, these curves are strictly parallel to the dilution curve and to the standard curve derived from the PRIST method, thus indicating that the monoclonal antibodies recognize all IgE equally well regardless of the way in which they are bound to the solid phase. On the basis of this observation, we propose a quantitative assay of specific IgE with the PRIST standard curve as reference. Our results demonstrate that this approach is indeed possible if high-capacity, solid-phase as well as long incubation times are used.