Teixeira Weslen Fabricio Pires, Coelho Willian Marinho Dourado, Nunes Cáris Maroni, Meireles Marcelo Vasconcelos
Faculdade de Medicina Veterinária, Universidade Estadual Paulista, Araçatuba, SP, Brazil.
Rev Bras Parasitol Vet. 2011 Oct-Dec;20(4):269-73. doi: 10.1590/s1984-29612011000400003.
The aim of this study was to produce a conjugate containing anti-Cryptosporidium parvum polyclonal antibodies and standardize a Direct Immunofluorescence Assay (DIF) for detecting C. parvum oocysts in fecal samples from calves. In order to obtain anti-C. parvum polyclonal antibodies, two New Zealand rabbits were immunized with a purified solution of C. parvum oocysts and Freund's adjuvant. Purification of the immunoglobulin G (IgG) fraction was performed by means of precipitation in ammonium sulfate and chromatography using a DEAE-cellulose column. The anti-C. parvum polyclonal antibody titer was determined by means of the enzyme-linked immunosorbent assay (ELISA). The rabbit anti-C. parvum IgG fraction was conjugated with fluorescein isothiocyanate and standardization of the DIF was performed using various dilutions of conjugate on slides positive for C. parvum oocysts. The cross-reactivity of the anti-C. parvum conjugate was tested using oocysts of Cryptosporidium serpentis, Cryptosporidium andersoni, Escherichia coli, Eimeria sp., and Candida sp. An anti-C. parvum conjugate was successfully produced, thus allowing standardization of DIF for detection of Cryptosporidium oocysts in fecal samples. Cross-reactivity of anti-C. parvum polyclonal antibodies with C. andersoni and C. serpentis was also observed.
本研究的目的是制备一种含有抗微小隐孢子虫多克隆抗体的偶联物，并标准化一种直接免疫荧光测定法（DIF），用于检测犊牛粪便样本中的微小隐孢子虫卵囊。为了获得抗微小隐孢子虫多克隆抗体，用纯化的微小隐孢子虫卵囊溶液和弗氏佐剂对两只新西兰兔进行免疫。通过硫酸铵沉淀和使用DEAE-纤维素柱的色谱法对免疫球蛋白G（IgG）组分进行纯化。通过酶联免疫吸附测定法（ELISA）测定抗微小隐孢子虫多克隆抗体效价。将兔抗微小隐孢子虫IgG组分与异硫氰酸荧光素偶联，并在微小隐孢子虫卵囊阳性载玻片上使用不同稀释度的偶联物对DIF进行标准化。使用蛇形隐孢子虫、安氏隐孢子虫、大肠杆菌、艾美耳球虫和念珠菌的卵囊测试抗微小隐孢子虫偶联物的交叉反应性。成功制备了抗微小隐孢子虫偶联物，从而实现了用于检测粪便样本中隐孢子虫卵囊的DIF标准化。还观察到抗微小隐孢子虫多克隆抗体与安氏隐孢子虫和蛇形隐孢子虫的交叉反应性。
