O’Nyong-nyong fever (ONNF) is an acute, mosquito-borne, non-fatal illness characterized primarily by debilitating polyarthralgia and/or polyarthritis. It is caused by O’Nyong-nyong virus (ONNV), first isolated from a febrile patient in 1959 in Northern Uganda. O’Nyong-nyong virus has been associated with rapidly spreading epidemics in Africa recording more than 2 million cases to date. A key element for control of arbovirus transmission is early diagnosis of infections. The main challenges in ONNV diagnosis, include (i) lack of commercial kits and specific assays to distinguish the closely related group A alphaviruses, in which ONNV belongs, and (ii) lack of effective treatments and vaccines. This study aimed to (i) determine the best cell line for ONNV isolation and large-scale production of ONNV purified proteins, (ii) generate ONNV monoclonal and polyclonal antibodies and (ii) assembly of an ELISA-based assay for sensitive detection of ONNV. O’Nyong-nyong virus strain SG650 was isolated in Vero cells, purified by sucrose gradient centrifugation, and used as an immunogen in BALB/c mice and New Zealand White rabbit immunization. Mice that developed sufficient antibody titers against ONNV were sacrificed and their spleen cells fused with parental myeloma cells using hybridoma technology for generation of anti-ONN monoclonal antibodies (mAbs). Similarly, rabbits that developed sufficient antibody titer were sacrificed and their serum purified and used to generate polyclonal antibodies (pAbs). The five MAbs generated were characterized and their potential for diagnostics determined and confirmed using an indirect IgG ELISA and Focus Neutralization Assay (FRNT50). The polyclonal antibodies were either conjugated with horseradish peroxidase for antigen detection ELISA development, used as capture antibodies for antigen detection ELISA or used unconjugated as primary antibodies for FRNT assays. To the best of our knowledge, this is the first documented effort to generate mAbs and pAbs against ONNV strain SG650. Generated antibodies can be explored and utilized for development and evaluation of serological assays to facilitate fast and timely diagnosis of ONNV infections in the laboratory, conduct surveillance and enhance differential diagnostic capability of arboviral and undefined febrile illnesses.

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