Desenvolvimento e padronização do Dot-ELISA usando peptídeos recombinantes para o diagnóstico sorológico de Neospora caninum

Abstract

Pesq. Vet. Bras. 34(8):723-727, agosto 2014 723 RESUMO.-A neosporose é reconhecida como uma das maiores causas de aborto e perdas neonatais em bovinos de leite e corte em todo o mundo. Nos últimos anos esta doença tem atraído o interesse de pesquisadores com foco na epidemiologia e métodos eficazes de diagnóstico desta doença. No presente estudo objetivou-se desenvolver e pa- Neosporosis is recognized as a major cause of abortion and neonatal loss in cattle worldwide, both for dairy cattle and beef cattle. In recent years this disease has attracted the interest of researchers and studying the epidemiology and effective methods of diagnosis of this disease. The present study aimed to develop and standardize on a Dot-ELISA for the serological diagnosis of Neospora caninum by using recombinant peptide as antigen for the development of a diagnostic kit used on the field. The recombinant antigen (rNcGRA1) was designed based on the method of reverse genetics derived antigenic epitopes of dense granules protein of N. caninum and synthesized by GenScript (USA). It was produced by the fermentation in yeasts Pichiapastoris KM71. The serological technique was used for the Dot-ELISA detection of IgG specific for N. caninum in which 0.22μm nitrocellulose membranes were sensitized with 1μL of antigen and subsequently the plasmas were diluted in a washing solution and incubated for 1 hour. The results will revealed by the addition of Protein G labeled with peroxidasse for 30 minutes, followed by the developing solution based on 3,3'-Diaminobenzidine (DAB). Soon after standardization tested 44 bovine plasmas were diagnosed by indirect immunofluorescence assay (IFA), agreeing with the results on a 95.5% and a sensitivity and specificity of 100% and 92% respectively. In regard to the diagnostic kit for the Technology Platform Rapid Flow-Through Miriad ® , the peptide presented rNcGRA1 visible markings to react with positive plasma, and showed no markings using the negative plasma. This study is the first to use recombinant peptides and prove to be efficient for the serological diagnosis of cattle naturally infected.

Transplante de microbiota intestinal e o Clostridium difficile: indicações e diagnóstico laboratorial

“…Além do ELISA convencional existe o teste DOT-ELISA, que atualmente está em contrapartida a coprocultura da prática clínica, sendo uma técnica baseada na sensibilização de membranas de nitrocelulose com antígeno, e posterior adição de um anticorpo marcado com peroxidase, para ocorrência de reação com formação de cor. O princípio é semelhante ao ELISA; no entanto, não requer aparelhagem sofisticada (Blanco et al, 2014).…”