Designation of an in-house ELISA for detection of VZV IgG and determination of antibody avidity by use of diethylamine

• Published in: Pakistan journal of biological sciences 2007-10, Vol.10 (19), p.3306-3313
• Main Authors: Fard, A H Mohagheghi, Pam, J Vallely
• Format: Article
• Language: English
• Subjects: Antibody Affinity; Child; Enzyme-Linked Immunosorbent Assay - methods; Herpesvirus 3, Human - immunology; Humans; Immunoglobulin G - analysis; Immunoglobulin G - immunology; Leukemia - blood; Varicella-zoster virus
• ISSN: 1028-8880
• DOI: 10.3923/pjbs.2007.3306.3313

Herpesviruses are ubiquitous in nature and both humans and animals harbour different species of this diverse family. However, only 8 species have been so far recovered from human beings. Different techniques (molecular and non-molecular) have been employed for rapid and efficient diagnosis of herpesviruses especially in case ofimmunocompromised hosts who are considered as high risk individuals. An indirect in-house ELISA was standardized to measure IgG antibody against Varicella-Zoster Virus (VZV) in sera from different groups of individuals. In the process of optimization of in-house ELISA, optimal dilutions of antigen, serum, conjugate and monoclonal IgG antibody along with selection of efficient blocking buffer and washing steps of microtitre plates were studied by the help of checkerboarding. The results were calculated according to Specific Binding Ratio (SBR) and cut-off procedures. The efficiency of newly developed indirect in-house ELISA was attempted and results were compared with data previously obtained by the agency of commercial kits or other serological techniques. The latter helped to investigate different technical aspects of in-house ELISA. When accuracy of ELISA was confirmed the protocol was applied in screening of the sera from immunocompetent and immunocompromised hosts that facilitated examination of the clinical aspects of the hosts. Diethylamine (DEA) in 20, 35 and 70 mM concentrations were used in order to assay IgG antibody avidity. The IgG avidity index was calculated by dividing OD of each sera obtained with denaturant to OD of the same specimen without application of denaturants. Avidity indices proved to be an important tool to differentiate between primary and recurrent infections and between seropositivity and seronegativity.