Walker K W, Llull R, Balkian G K, Ko H S, Flores K M, Ramsamooj R, Black K S, Hewitt C W, Martin D C
Transplantation Laboratory, University of California, Irvine 92717.
J Immunol Methods. 1992 Sep 18;154(1):121-30. doi: 10.1016/0022-1759(92)90219-j.
A solid phase cellular ELISA was designed and evaluated for the detection of antibodies specific for cell surface determinants. It was hypothesized that certain fixation and freezing procedures would result in stabilization of cell structures for prevention of antigen diffusion and extraction during washing procedures. This would assure assay accuracy and convenient sample management. It was hypothesized that fixation with certain reagents prior to analysis would not alter antigenicity of antibody targeted epitopes. In order to improve the preservation of the cells following cell binding to the solid phase matrix while still retaining antigenicity and morphology, a series of fixatives and storage procedures were screened to determine which were best suited for CELISA. Methanol, washing buffer (WB), Hanks' balanced salt solution (HBSS), and 0.5% formalin in HBSS were examined by comparing their relative cell binding capacity and the subsequent cell morphology. In consideration of all variables, fixation in 0.5% formalin provided the best maintenance of cell antigenicity, morphology, binding, and was associated with consistent results. Cells used immediately after fixation and fixed cells used after storage at -80 degrees C for up to 12 months were compared to determine if long term storage affected antigenicity. Since frozen cells and fresh cells demonstrated statistically identical positive to negative ratios and consistency of antibody binding, it was determined that long term frozen storage of formalin-fixed cells did not adversely affect antibody binding capacity to cell surface determinants.
设计并评估了一种固相细胞酶联免疫吸附测定法（ELISA），用于检测针对细胞表面决定簇的特异性抗体。据推测，某些固定和冷冻程序将导致细胞结构稳定，以防止在洗涤过程中抗原扩散和提取。这将确保检测准确性和方便的样品管理。据推测，在分析前用某些试剂固定不会改变抗体靶向表位的抗原性。为了在细胞与固相基质结合后更好地保存细胞，同时仍保留抗原性和形态，筛选了一系列固定剂和储存程序，以确定哪些最适合细胞ELISA。通过比较甲醇、洗涤缓冲液（WB）、汉克斯平衡盐溶液（HBSS）和HBSS中的0.5%福尔马林的相对细胞结合能力和随后的细胞形态来进行检测。考虑到所有变量，用0.5%福尔马林固定能最好地维持细胞抗原性、形态、结合能力，并且结果一致。比较固定后立即使用的细胞和在-80℃储存长达12个月后使用的固定细胞，以确定长期储存是否会影响抗原性。由于冷冻细胞和新鲜细胞在统计学上显示出相同的阳性与阴性比率以及抗体结合的一致性，因此确定福尔马林固定细胞的长期冷冻储存不会对抗体与细胞表面决定簇的结合能力产生不利影响。
