Peroxidase conjugate preparation for the diagnosis of enzootic bovine leucosis by Elisa kit
Journal Title: Бюлетень "Ветеринарна біотехнологія" - Year 2018, Vol 32, Issue
Abstract
Introduction. Enzootic bovine leucosis (EBL) is one of the most important viral diseases of the cattle in Ukraine. The control and eradication measures of EBL are based on carrying out of regular serological monitoring. Enzyme-linked immunosorbent assay (ELISA) is a highly sensitive and specific method widely need for EBL serological diagnostics. The goal of the work. To prepare a protein G peroxidase conjugate for the ELISA Kit for the diagnosis of EBL. Materials and methods. Preparation of the ELISA conjugate based on recombinant protein G from Streptococcus spp. and horseradish peroxidase were performed using the periodate method. Purification of the obtained resulting conjugate was carried out by chromatography using Sephadex G-25, the selected fractions belonged to the first peak. The specificity of the protein G-peroxidase conjugate was verified using an indirect solid phase ELISA. For this purpose, recombinant antigens of Bovine leukemia virus were absorbed on polystyrene plates. Test samples of blood serum and a sample dilution solution was added to each well. Horseradish peroxidase conjugate with recombinant G protein of Streptococcus spp. was used as a detection agent. The reaction was performed using TMB chromogen solution «NeA-Blue» (CLINICAL Science Products Inc., USA). The measurements were carried out with an automatic spectrophotometer «Labsistems» in the two-wave mode: the first filter – 450 nm, the second – 620 nm. Results of research and discussion. The prepared peroxidase conjugate was titrated to obtain the optimal working dilution by multiple successive dilutions. The optimal dilution of the ELISA conjugate, which was 1:32000 – 1:64000, was determined by multiple sequential dilutions during IFF exposure with pre-identified positive and negative serum bovine leukosis. With this dilution, the high ratio of the optical density values of the positive and negative sera is 28.1 and 32.6, respectively. Conclusions and prospects for further research. Analyzing the results of the conjugate titration, it was concluded that the optimal dilution of the immune enzyme conjugate for the developed ELISA Kit for the diagnosis of bovine leukemia of this series was 1:64000. In further experiments with working dilution of the conjugate, it is necessary to assess the specificity and sensitivity of the test kit. For this purpose, intraindustral panel of samples contained positive and negative reference sera and blood sera from non-infected animals will be used.
Authors and Affiliations
Yaroslav Khomenko, Iryna Mushtuk, Olena Ayshpur, Yevgen Mintsyuk
Keywords
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- EP ID EP407693
- DOI 10.31073/vet_biotech32(2)-72
- Views 111
- Downloads 0
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