GENERATION AND FUNCTIONAL CHARACTERIZATION OF CHICKEN-BASED IgY POLYCLONAL ANTIBODIES AGAINST BLACK MAMBA (Dendroaspis polylepis) SNAKE VENOM

Abstract

The life-threatening medical issues associated with snakebites have been a public health concern for decades at the global level. The Black mamba, D. polylepis, is one of the many venomous snakes found in Kenya. According to the Kenyan Ministry of Health data, 15,000 snakebites occur annually. For snakebites, antivenom immunotherapy is still the preferred course of action and different antivenoms are needed to treat the venom of different snake species. Traditionally, antivenoms for treatment are produced from horse or sheep but have complicated and expensive production issues. Alternative production approaches, such as using immunoglobulin Y (IgY) antibodies derived from chicken egg yolks, may overcome disadvantages with traditional antivenom manufacturing techniques. In many tropical and subtropical nations such as Kenya, diagnosis of snakebite in the health facilities is imperative. For the purpose of determining which species caused the bite, it is critical to detect and measure snake venom in the blood of envenomed patients. The study presents the generation of IgY antibodies, and development of an enzyme-linked immunosorbent assay (ELISA) against D. polylepis. In this current study, D. polylepis specific IgY polyclonal antibodies were purified from the egg yolks of chickens immunized with D. polylepis venom. These antibodies were subsequently assessed for their in-vivo neutralizing capacity vis-à-vis commercial antivenoms, PANAF Premium and VINS. Additionally, the study sought to develop a sensitive ELISA prototype for detection of D. polylepis venom in Kenya using generated chicken-based IgY polyclonal antibodies. The IgY antibodies were purified by ammonium sulfate precipitation and affinity-chromatography, with quality and specificity determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA. Furthermore, serum samples containing specific chicken-based IgY antibodies previously raised against D. polylepis venom toxins were used in the assay development. ELISA parameters were optimized, and developed assay assessed for applicability. The LD50 of D. polylepis was 0.54 mg/kg in chicks (via intramuscular route), and 0.34 mg/kg in mice (via intraperitoneal route), respectively. Pool of extracted IgY yielded 2.8 mg/mL concentration. Purified IgY under non-reducing and reducing conditions on SDS-PAGE xv exhibited a single-protein band of about 183 kDa and two bands (67 kDa and 25 kDa), respectively. The minimum-edematogenic dose was 0.05 µg. Anti-D. polylepis IgY antibodies and two antivenoms demonstrated the capacity to neutralize the toxic activities of D. polylepis venom. The median effective dose (ED50) for lethality neutralization were 41.36, 35.41 and 46.60 µL/3LD50, and for edema were 80±11.55, 60±18.26, 90±8.16 µL/6MED, respectively for IgY antibodies, VINS and PANAF antivenoms. There was no statistical differences among their neutralizing efficacies (P value = 0.320-0.859) The limit of detection (LoD) of the ELISA for neurotoxic venoms was 0.01 µg/mL. The developed assay identified venoms in blood of BALB/c mice and, distinguished D. polylepis venom from that of other snake species, as well as neurotoxic and cytotoxic venoms. This developed assay would be useful in helping physicians to diagnose and manage snakebite cases. The development of an effective IgY antibodies with higher titer represents a significant step towards antivenom production against D. polylepis. Diversity of the IgY antibodies development to capture other medically important snake venoms, molecularly characterize IgY, and development of venom detection kits with quick turnaround times based on liquid or lateral flow tests approach are recommend