Regan S, Cama V, Sterling C R
Department of Veterinary Science, University of Arizona, Tucson 85721.
J Protozool. 1991 Nov-Dec;38(6):202S-204S.
Simple modifications to a recently published merozoite purification procedure (Bjorneby et al., J. Immunol. 145:298, 1990) increased yields 3- to 5-fold. Calves were infected with 2.5 x 10(8) Cryptosporidium parvum oocysts and sacrificed 65 h post-infection. The ilium and caecum were removed. The tissue was sieved through a large strainer (2 mm2) to produce a homogeneous suspension. Red blood cells were removed by differential centrifugation (600 g); merozoites remained in the supernatant. The merozoites were pelleted (2,100 g) and washed in modified Hank's balanced salt solution deficient in Mg+2 and Ca+2. Percoll purification (density 1.070 g/ml and centrifugation speed of 22,000 g for 30 min) yielded 8 x 10(8) merozoites. Nineteen monoclonal antibodies (MAb) detected by either an enzyme-linked immunosorbent assay or an immunofluorescence assay, have been generated against the merozoite stage. Gels of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver-stained showed that sporozoites and merozoites have many common lower molecular weight proteins. Western blots of sporozoite and merozoite antigens reacted with anti-sporozoite MAb showed several cross-reacting antigens shared by these life-cycle stages.
对最近发表的裂殖子纯化程序（Bjorneby等人，《免疫学杂志》145:298，1990年）进行简单修改后，产量提高了3至5倍。给小牛接种2.5×10⁸个微小隐孢子虫卵囊，感染后65小时宰杀。取出髂骨和盲肠。将组织通过大滤网（2平方毫米）过筛以产生均匀的悬浮液。通过差速离心（600克）去除红细胞；裂殖子留在上清液中。将裂殖子沉淀（2100克），并在缺Mg²⁺和Ca²⁺的改良汉克平衡盐溶液中洗涤。经Percoll纯化（密度1.070克/毫升，离心速度22000克，30分钟）得到8×10⁸个裂殖子。已经通过酶联免疫吸附测定或免疫荧光测定检测到19种针对裂殖子阶段产生的单克隆抗体（MAb）。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离并用银染色的蛋白质凝胶显示，子孢子和裂殖子有许多共同的低分子量蛋白质。与抗子孢子单克隆抗体反应的子孢子和裂殖子抗原的蛋白质免疫印迹显示，这些生命周期阶段共有几种交叉反应抗原。
