Development of Dromedary Antibody-based Enzyme Linked Immunosorbent Assay for Detecting Chikungunya virus Infections
Abstract
Background: Chikungunya virus (CHIKV) is an arthropod-borne Togavirus belonging to the genus Alphavirus that is responsible for sporadic worldwide outbreaks of Chikungunya fever, an acute febrile illness often associated with severe polyarthralgia. In Kenya, Chikungunya virus is of great epidemiological concern, with the last major outbreak occurring in 2016 in North Eastern Kenya. Reliable detection of CHIKV infections is key to controlling this re-emerging pathogen, for which no cure currently exists. Current diagnostic methods for CHIKV employ a combination of tests, particularly immunologic, serologic or virologic techniques. However, the independent scientific reviews on the validity and sensitivity of currently available commercial assays have been conflicting.
Objective: This study aimed to develop and validate a dromedary antibody-based enzyme linked immunosorbent assay for detecting Chikungunya virus infections.
Methods: To produce sufficient antigen for camel immunization, Chikungunya virus (strain Lamu 33) was propagated in confluent C6-36 E2 cells using Cytodex microcarrier system. Purified and inactivated CHIKV immunogen was used to inoculate two camels reared at the University of Nairobi farm in Kibwezi, Kenya. Camel serum samples collected over the entire immunization period were assayed for the presence of anti-Chikungunya IgG by indirect ELISA. Purification of camel Heavy Chain IgG antibodies was performed by lectin affinity chromatography on protein A and protein G-Sepharose columns; then conjugated with horse radish peroxidase (HRP). The HRP-conjugated camel Heavy Chain IgG2 and IgG3 were optimized for ELISA, with optical density measured using a microplate reader set at 492nm. A total of 188 human sera samples were assayed using the developed dromedary-based enzyme linked immunosorbent assay to determine Chikungunya virus infections.
Results: The sensitivity of the dromedary HCAb IgG2 assay was 91.3% (95% CI: 0.831 - 0.994); while that for HCAb IgG3 assay was 95.7% (95% CI: 0.898 - 1.01). The specificity of HCAb IgG2 assay was 92.3% (95% CI: 0.879 - 0.967); while the specificity of HCAb IgG3 method was 90% (95% CI: 0.851 - 0.949). For HCAb IgG2 and IgG3 based assays, the positive predictive values were 79.2% and 75.8 % respectively; while the negative predictive values were 97% and 98.4% for HCAb IgG2 and IgG3 based assays respectively.
Conclusion: The camel antibody based assay was found to be reliable assay with very good sensitivity and specificity, and can be deployed for detection of Chikungunya virus infections.
Key words: Chikungunya, ELISA, camel antibodies, diagnosis
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