Sadler A M, Krausa P, Marsh S G, Heyes J M, Bodmer J G
Tissue Antigen Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London, UK.
J Immunol Methods. 1992 Apr 27;149(1):11-9. doi: 10.1016/s0022-1759(12)80043-3.
A microenzyme-linked immunosorbent assay (microELISA) method has been developed using an Astroscan 2100 system automated plate reader which was initially designed for tissue typing by a two colour fluorescent microcytotoxicity assay. A 96-well plate ELISA used for screening mouse monoclonal antibodies raised against surface HLA antigens has been modified for use with the Astroscan plate reader and 72-well typing trays. The existing substrate 4-methylumbelliferyl-beta-D-galactoside (4MUG) has been replaced with fluorescein-di-beta-D-galactopyranoside (FDG), to provide a wavelength (530 nm) detectable by the Astroscan or other automated plate readers designed for reading microcytotoxicity assay plates. The assay volumes have also been reduced tenfold for use with Terasaki microtest plates. The assay now has the major advantage of requiring only 5 microliters of test supernatant allowing hybridomas to be screened earlier during a fusion and on a wider cell panel. The use of the large panel which includes B lymphoblastoid cell lines (B-LCL) and mouse L cell transfectants expressing HLA genes, reduces the length of time the hybridomas need to be kept in tissue culture before selection. Other advantages include the reduction in the number of target cells required, smaller volumes of reagents throughout the assay and the ability to screen cytotoxic as well as non-cytotoxic monoclonal antibodies. The sensitivity of this microELISA proved to be comparable with the original assay and so provides an efficient screening method for monoclonal antibodies.
一种微酶联免疫吸附测定（microELISA）方法已经利用Astroscan 2100系统自动酶标仪开发出来，该酶标仪最初设计用于通过双色荧光微细胞毒性测定进行组织分型。一种用于筛选针对表面HLA抗原产生的小鼠单克隆抗体的96孔板ELISA已被修改，以便与Astroscan酶标仪和72孔分型板一起使用。现有的底物4-甲基伞形酮基-β-D-半乳糖苷（4MUG）已被荧光素-二-β-D-吡喃半乳糖苷（FDG）取代，以提供一个可被Astroscan或其他设计用于读取微细胞毒性测定板的自动酶标仪检测到的波长（530nm）。测定体积也减少了十倍，以用于Terasaki微量测试板。该测定现在的主要优点是只需要5微升测试上清液，从而能够在融合过程中更早地对杂交瘤进行筛选，并且可以在更广泛的细胞板上进行筛选。使用包括B淋巴母细胞系（B-LCL）和表达HLA基因的小鼠L细胞转染体的大细胞板，减少了杂交瘤在选择前需要在组织培养中保存的时间。其他优点包括所需靶细胞数量减少、整个测定过程中试剂体积更小以及能够筛选细胞毒性和非细胞毒性单克隆抗体。这种microELISA的灵敏度被证明与原始测定相当，因此为单克隆抗体提供了一种有效的筛选方法。
