Development of an antibody capture assay for the detection and quantification of IgG antibody to HSV 1 and HSV 2

Abstract

Background and Aims: HSE is one of the most serious infections of the CNS with high death rates and intense sequelae in those who survive the disease. Death rates can fall only if the antiviral treatment is started right after the initial symptoms are experienced. Although PCR is the most common method in the diagnosis of HSE, it has some limitations. The aim of the project was to develop a capture enzyme-linked immunosorbent assay (ELISA) to detect and measure the HSV intrathecal antibody synthesis. Methods: The concentration of previously cultured virus antigen was measured by the BCA protein method. Then, the viral antigen was coupled with biotin-ester. The optimum concentration of the biotinylated virus antigen and rabbit anti-human IgG was determined. Next, the ELISA test was established to detect and measure the presence of HSV antibody by using 3 positive anti-HSV IgG serums and 1 negative anti-HSV IgG serum which served as control blank. During the development of the IgG antibody capture ELISA, non-specific binding was observed. Two further tests were done to determine whether the non-specific binding occurred due to the binding of the biotin labelled virus to: (1) the plastic well or (2) the rabbit anti-human lgG. Results: The viral protein concentration measured by the BCA protein assay was about 1606.9 μg/ml = 1.6 mg/ml. A biotinylated virus concentration of 1/500 μg/ml was chosen for use because it produced the highest measurement (OD 2.1) when incubated with the streptavidin-peroxidase enzyme. The optimal rabbit anti-human IgG concentration chosen was around 1/1000 μg/ml because it produced the highest measurement when the reaction was detected by the biotin-streptavidin complex in agreement with previous studies. The non-specific reaction was due to the binding of the biotinylated virus to the plastic well and to the rabbit anti-human IgG.