Balkian G K, Flores K M, Llull R, Ko H S, Walker K W, Black K S, Hewitt C W
Department of Surgery, Robert Wood Johnson Medical School, Cooper Hospital/University Medical Center, Camden, NJ 08103, USA.
Cell Transplant. 1997 Jul-Aug;6(4):431-7. doi: 10.1177/096368979700600411.
A cellular enzyme-linked immunosorbent assay (CELISA) was developed for the detection and quantification of antibodies elicited against allogeneic cell surface determinants. The technique uses a solid-phase cell matrix created by fixing cells with a mild formalin solution onto the bottom of a 96-well microtiter plate. A primary layer of alloantisera is first reacted against rat target cells. The secondary antibody, peroxidase conjugated antirat IgG, is then added to each well and serves as the second sandwich layer. Optimal reagent concentrations were determined by serial dilution analysis of various cell concentrations and secondary antibody dilutions. It was found that 200,000 cells per well was the optimal target cell concentration. However, 100,000 cells per well was also sufficient to run the assay with acceptable performance characteristics. Even lower cell concentrations of 10,000 and 20,000 cells/well, although not optimal, also produced acceptable results. Secondary antibody concentration with respect to the optimal cell concentration was determined to be 1:500. At 200,000 cells per well and a 1:500 secondary antibody dilution, the assay presented excellent coefficients of determination and high positive to negative ratios. The reaction was found to be very sensitive in yielding high antibody titers with low background levels and could be defined mathematically as a linear-log function. Titers of multiple unknown alloantibody samples were easily and accurately predicted in an automated manner by regression analysis form known standards. This immunoassay will be useful in studies of cell surface determinant expression and quantitation of antibodies reactive to such markers.
开发了一种细胞酶联免疫吸附测定法（CELISA），用于检测和定量针对同种异体细胞表面决定簇产生的抗体。该技术使用一种固相细胞基质，通过用温和的福尔马林溶液将细胞固定在96孔微量滴定板底部来创建。首先使一层同种异体抗血清与大鼠靶细胞反应。然后将二级抗体，即过氧化物酶偶联的抗大鼠IgG，添加到每个孔中，并作为第二个夹心层。通过对各种细胞浓度和二级抗体稀释度进行系列稀释分析来确定最佳试剂浓度。发现每孔200,000个细胞是最佳靶细胞浓度。然而，每孔100,000个细胞也足以进行该测定并具有可接受的性能特征。即使是每孔10,000和20,000个细胞的更低细胞浓度，虽然不是最佳的，但也产生了可接受的结果。相对于最佳细胞浓度的二级抗体浓度确定为1:500。在每孔200,000个细胞和1:500的二级抗体稀释度下，该测定呈现出优异的测定系数和高阳性与阴性比率。发现该反应在产生高抗体滴度且背景水平低方面非常灵敏，并且可以数学定义为线性对数函数。通过已知标准的回归分析，可以轻松、准确地自动预测多个未知同种异体抗体样品的滴度。这种免疫测定法将有助于细胞表面决定簇表达的研究以及对与此类标记反应的抗体进行定量。
