Pamela M. Khasandi

Caprylic acid (CA) fractionation of serum is a simple and cost-effective method of producing high-quality immunoglobulins. While standardised procedures exist for CA purification of IgG for various animals, no published protocol exists for baboon IgG. This study aimed to optimise an efficient protocol for purifying IgG from baboon serum using CA through a stepwise one-factor-at-a-time (OFAT) approach. The effects of serum pH, CA concentration, stirring time and intensity, dialysis buffer, and lyophilisation were evaluated based on the protein content, with SDS-PAGE profiles and albumin–globulin ratios distinguishing IgG from residual albumin. Serum at pH 5.0 with 7% CA (v/v) produced the highest yield, minimising albumin while maximising IgG content. Lower pH (4.0–4.5) and CA (5–6%) reduced protein content, while a higher pH (5.5–6.0) and CA (8–15%) increased protein, but with elevated albumin and contaminants. Stirring serum vigorously at 1200 rpm for 60 min provided effective precipitation of non-IgG proteins. Lower intensities and shorter times resulted in higher albumin and residual proteins, while excessive stirring caused protein denaturation. Dialysis buffer composition had little impact, while lyophilisation significantly enhanced IgG concentration. The optimal protocol involved serum at pH 5.0, 7% CA (v/v), vigorous stirring (1200 rpm) for 60 min, and dialysis against sodium phosphate buffer (pH 7.4) followed by lyophilisation. The resulting IgG enrichment and purity were comparable to commercial-grade products. This study thus established optimal conditions for the purification of baboon IgG with CA, which could be used to support research in this animal model of immunology.

Antivenom administration is currently the only therapy for snakebite envenoming. However, in sub-Saharan Africa, inadequate quality control systems have led to deficits in the availability, accessibility, efficacy and safety of regionally available antivenoms, which, in turn, hinder snakebite treatment and management in the region. To address this impediment to snakebite treatment in Kenya, this study aimed to assess the preclinical neutralising potencies of four different antivenoms previously or currently available in Kenya (SAIMR polyvalent, AFRIVEN, PANAF-Premium^TM and Inoserp^TM) against key snakes of medical importance in the region, towards establishing a national antivenom quality control laboratory. Venoms were extracted from the Kenyan “big five” medically important snake species: Naja ashei, Naja pallida, Naja nigricollis, Dendroaspis polylepis and Bitis arietans, and their lethal potencies were determined using a murine median lethal dose (LD₅₀) assay. In vitro immunological assays (ELISAs and immunoblotting) and an established preclinical murine in vivo neutralisation assay (median effective dose [ED₅₀]) were used to assess the immunoglobulin-binding and venom-neutralising efficacies of the test antivenoms. In vitro assays revealed high venom-binding titres of SAIMR polyvalent, AFRIVEN and PANAF-Premium^TM, and reactivity to a wide range of venom proteins across the different snake venoms. Contrastingly, Inoserp^TM antivenom had low binding titres and poor reactivity to the snake venom proteins. These findings were aligned with the in vivo results, where SAIMR polyvalent, AFRIVEN and PANAF-Premium^TM showed potent venom-neutralising efficacies against all the tested snake venoms, while Inoserp^TM had low potency and failed to neutralise the lethal effects of N. ashei, N. pallida and D. polylepis venoms at the manufacture-claimed doses. Based on these robust preclinical results, we conclude that SAIMR polyvalent, AFRIVEN and PANAF-Premium^TM antivenoms offer considerable potential for the treatment of envenoming by diverse medically important snakes in Kenya. The observed deficiencies with the Inoserp^TM product highlight the importance of (i) robust, independent preclinical antivenom efficacy testing and (ii) the value of establishing a quality control laboratory to inform local regulatory and procurement decision making.