Development and Evaluation of an Antigen Capture Enzyme-LinkedImmunosorbent Assay (AC-ELISA) for the Diagnosis of African Swine fever

Abstract

African swine fever (ASF) is a viral haemorrhagic fever of pigs with devastating impact on pig production and household income in Africa. Lack of a vaccine and treatment for ASF has increased the dependence on accurate diagnosis as basis for control and possible eradication of the disease. The aim of this study therefore, was to develop and evaluate an in-house sandwich enzyme linked immune sorbent assay (ELISA) using antibodies raised against African swine fever virus (ASFv) isolates from Uganda for diagnosis of ASF. The ASFv was grown in pig alveolar macrophages, the infected cells harvested, lysed and the virus precipitated using polyethylene glycol 6000. The virus was purified on Sepadex G-200 column equilibrated with 50mM Tris-HCl PH 7.2 containing 0.15M NaCl and viral proteins separated by SDS-PAGE. The target protein (vp73), was quantified and used to immunize rabbits to produce polyclonal antibodies against it. The purified immunoglobulin IgG (rabbit anti ASF-vp73) was used for antigen capture in sandwich ELISA. Eighty eight (88) known positive and 176 known negative pig serum samples were used to evaluate assay performance. The diagnostic sensitivity of the ELISA was 82.95% (95% CI, 78-100%), diagnostic specificity was 96.59% (95% CI, 90–100%). Positive and negative predictive values were 92.4% and 91%, respectively. The inter samples coefficient of variation of raw optical density values for known positive samples in different runs was <10% (range 1.1-7.8), while intra sample coefficient of variation ranged from 0.6-5.5% between runs. The developed antigen capture ELISA has a high diagnostic sensitivity and specificity and is therefore good for detection of active ASF infection. However, the developed assay should be further validated using larger sample size under different laboratory conditions and referenced serum samples from different ASF endemic countries.