ABSTRACT
Development of a rapid, on-site detection tool for snakebite is highly sought after, owing to its clinically and forensically relevant medicolegal significance. Polyvalent antivenom therapy in the management of such envenomation cases is finite due to its poor venom neutralization capabilities as well as diagnostic ramifications manifested as untoward immunological reactions. For precise molecular diagnosis of elapid venoms of the big four snakes, we have developed a lateral flow kit using a monoclonal antibody (AB1; IgG1 - κ chain; Kd: 31 nM) generated against recombinant cytotoxin-7 (rCTX-7; 7.7 kDa) protein of the elapid venom. The monoclonal antibody specifically detected the venoms of Naja naja (p < 0.0001) and Bungarus caeruleus (p<0.0001), without showing any immunoreactivity against the viperidae snakes in big four venomous snakes. The kit developed attained the limit of quantitation of 170 pg/µL and 2.1 ng/µL in spiked buffer samples and 28.7 ng/µL and 110 ng/µL in spiked serum samples for detection of N. naja and B. caeruleus venoms, respectively. This kit holds enormous potential in identification of elapid venom of the big four snakes for effective prognosis of an envenomation; as per the existing medical guidelines.
Subject(s)
Colorimetry/methods , Cytotoxins/analysis , Elapidae/immunology , Immunoassay/methods , Immunotoxins/analysis , Snake Venoms/analysis , Animals , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/immunology , Bungarus/genetics , Bungarus/physiology , Cytotoxins/genetics , Cytotoxins/immunology , Elapid Venoms/analysis , Elapid Venoms/genetics , Elapid Venoms/immunology , Elapidae/physiology , Immunotoxins/genetics , Immunotoxins/immunology , Naja naja/immunology , Naja naja/physiology , Snake Venoms/immunology , Viperidae/immunology , Viperidae/physiologyABSTRACT
Oxytetracycline (OTC), one of the largely used antibiotic in veterinary practice has been banned due to its potential side effects. Development of a field applicable and affordable kit to detect OTC will help to eliminate such milk from human consumption. An aptamer has been designed (27 nt; Kd = 29.2 ± 19.4 nM) through rational truncation. OTC interacts with this aptamer in G rich regions as confirmed by molecular modelling and circular dichroism spectroscopy. To develop a lateral flow based aptasensor, OTC was conjugated with a 7 kDa carrier protein to immobilize onto the nitrocellulose membrane. Using 0.125 µM aptamer-gold conjugate, assay could visually detects upto 5 ng/mL of OTC in spiked milk within 10 mins [Limit of quantitation (LOQ)-0.254 ± 1.62 ng/mL; permissible limit 100 ng/mL]. It showed no cross reactivity with components of milk and data correlated with analysis done through HPLC.
