Production, Characterization and Assay Application of a Purified, Baculovirus-Expressed, Serogroup Specific Bluetongue Virus Antigen
| Autor: | M. I. Sabara, L. Luo |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Serotype
medicine.drug_class Enzyme-Linked Immunosorbent Assay Antibodies Viral Monoclonal antibody Binding Competitive Bluetongue Sensitivity and Specificity Virus law.invention Diagnosis Differential Western blot Antigen Antibody Specificity law medicine Animals Antigens Viral General Veterinary General Immunology and Microbiology biology medicine.diagnostic_test Viral Core Proteins Antibodies Monoclonal General Medicine Virology Molecular Weight Polyclonal antibodies biology.protein Recombinant DNA Antibody Baculoviridae Bluetongue virus |
| Zdroj: | Transboundary and Emerging Diseases. 55:175-182 |
| ISSN: | 1865-1682 1865-1674 |
| DOI: | 10.1111/j.1865-1682.2008.01022.x |
| Popis: | The predominant serodiagnostic assay used in many countries to detect bluetongue virus (BTV) infections is a competitive enzyme-linked immunosorbent assay (c-ELISA) which employs two critical reagents: a cell culture-derived BTV antigen and group-specific monoclonal antibody (Mab). Ongoing difficulties have been reported by laboratories in the production and quality control of the native antigen reagent which relies on the presence of adequate molar quantities and appropriate presentation of the major BTV core protein VP7. To address this important issue, a recombinant baculovirus was constructed containing a cDNA copy of genome segment 7 of BTV serotype 11 and used to infect insect cells which, in turn, expressed high levels of theVP7 protein with an estimated molecular mass of 39 kDa. In its purified form, this recombinant protein could be detected by group-specific Mabs designated 3.17.A3 and 8A3B.6 produced against BTV serotypes 1 and 17, respectively, as well as by polyclonal bovine antibodies raised against North American and South African BTV serotypes. No reactivity was observed by Western blot analysis with these two Mabs suggesting that the common antigenic determinants, on the BTV VP7 protein, were mainly conformational. It was interesting to note that the purified recombinant VP7 protein demonstrated a greater degree of reactivity with Mab 8A3B.6 compared to that exhibited with Mab 3.17.A3 when evaluated in an ELISA. Due to its antigenic similarity to the native antigen, the recombinant protein was found to be a suitable replacement for use in a c-ELISA to detect BTV-specific antibodies with the added advantage that it could be consistently produced and was, therefore, amenable to quality control testing for purity, stability and other standards. |
| Databáze: | OpenAIRE |
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