Lie O, Vartdal F, Funderud S, Gaudernack G, Olsaker I, Frøysadal E, Ugelstad J, Thorsby E
National Veterinary Institute, Oslo, Norway.
Anim Genet. 1988;19(2):75-86. doi: 10.1111/j.1365-2052.1988.tb00794.x.
This paper describes a totally new immunomagnetic (IM) technique adapted to serological BoLA typing. The basic technique has recently been developed by Vartdal et al. (1986) for serological HLA typing. The main advantage is that bovine mononuclear cells (e.g. T-cells and possibly their subsets, B-cells and monocytes) can be quickly and specifically isolated with high yield and viability from whole blood in a one-step procedure. This is achieved by magnetic separation of rosettes formed between the cells and superparamagnetic monosized polystyrene microspheres (Dynabeads TM) coated with cross-species reactive monoclonal antibodies (MAbs) specific for various human T-cell antigens or for HLA class II monomorphic epitopes. The cells are isolated within 5 min after a 5-min incubation at 4 degrees C. Magnetic separation of rosettes with a strong cobalt-samarium magnet eliminates all the laborious centrifugation steps necessary with conventional procedures. The isolated cells, still attached to the particles, are available for microcytotoxic assay. This is carried out within 55 min, including a two-step application of alloantiserum and complement and addition of acridine orange/ethidium bromide for the staining of viable (green) and dead (red) cells. The high viability of isolated cells gives a very low background kill compared with the conventional cytotoxic assay. The IM typing technique is also superior in sensitivity to the conventional technique as standardized for the international BoLA comparison test. The IM technique is likely to have its greatest impact on class II typing; class II positive cells being separated very efficiently. Polymorphic HLA class II MAbs detected likely polymorphic BoLA class II epitopes.
本文描述了一种全新的适用于血清学牛白细胞抗原（BoLA）分型的免疫磁（IM）技术。该基本技术最近由瓦尔达尔等人（1986年）开发用于血清学人类白细胞抗原（HLA）分型。其主要优点是，牛单核细胞（如T细胞及其可能的亚群、B细胞和单核细胞）可以通过一步法从全血中快速、特异性地分离出来，且产量高、活力强。这是通过磁分离细胞与超顺磁性单尺寸聚苯乙烯微球（Dynabeads TM）形成的花环来实现的，这些微球包被有针对各种人类T细胞抗原或HLA II类单态表位的跨物种反应性单克隆抗体（MAbs）。在4℃孵育5分钟后，细胞在5分钟内被分离出来。用强钴钐磁体对花环进行磁分离消除了传统方法所需的所有繁琐的离心步骤。分离出的细胞仍附着在颗粒上，可用于微量细胞毒性测定。这在55分钟内完成，包括两步应用同种异体抗血清和补体，并添加吖啶橙/溴化乙锭用于活细胞（绿色）和死细胞（红色）的染色。与传统细胞毒性测定相比，分离细胞的高活力导致背景杀伤率非常低。在国际BoLA比较试验标准化的情况下，IM分型技术在灵敏度方面也优于传统技术。IM技术可能对II类分型影响最大；II类阳性细胞能被非常有效地分离。检测到的多态性HLA II类MAbs可能识别多态性BoLA II类表位。
