Production of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water

Revista da Sociedade Brasileira de Medicina Tropical

Silvia Cristina Osaki²  Adriana Oliveira Costa¹  Ludmilla Della Coletta Troiano²  Ernesto Renato Kruger²  Juliana Tracz Pereira¹  Nelson Luis Mello Fernandes²  Márcia Benedita De Oliveira Silva¹  Vanete Thomaz Soccol² 

[1] ,Universidade Federal do Paraná Programa de Pós-Graduação em Processos Biotecnológicos e Biotecnologia, Divisão de Biotecnologia Curitiba PR

关键词: Polyclonal antibodies;    Direct immunofluorescence;    Oocysts;    Water;    Anticorpos policlonais;    Imunofluorescência direta;    Oocistos;    Água;   

DOI  :  10.1590/S0037-86822011000500011

来源: SciELO

【 摘 要 】

INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20% of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.

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