ABSTRACT
Bites by spiders from Loxosceles genus often lead to a wide variance in envenomation profile of patients and diagnosis is difficult due to the number of diseases that mimic loxoscelism. In such a context, it is of interest to consider the design of standardized recombinant colorimetric antibodies for diagnosis and specific detection of individual circulating toxins in biological fluids of envenomed patients. We have previously prepared a monoclonal murine IgG (LiMab7) that reacts with Loxosceles intermedia venom components of 32-35kDa and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a colorimetric bifunctional protein consisting in the corresponding single-chain antibody fragment (scFv) fused to alkaline phosphatase (AP) of Escherichia coli. The immune tracer was tested in two different types of immunoassays and it proved to be efficient in both. Thus, this recombinant fusion protein (scFv-LiMab7/AP) can be used for rapid and specific immunotitration of L. intermedia venom with a linear range of 39-20000ng/mL and a detection limit of 39ng/mL without any cross-reaction.
Subject(s)
Brown Recluse Spider/physiology , Immunoassay/methods , Neurotoxins/analysis , Phosphoric Diester Hydrolases/analysis , Skin/metabolism , Spider Bites/diagnosis , Spider Venoms/analysis , Alkaline Phosphatase/genetics , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Neutralizing/metabolism , Diagnosis, Differential , Humans , Mice , Neurotoxins/immunology , Phosphoric Diester Hydrolases/immunology , Protein Engineering , Recombinant Fusion Proteins/metabolism , Reference Standards , Sensitivity and Specificity , Single-Chain Antibodies/genetics , Single-Chain Antibodies/metabolism , Skin/pathology , Spider Bites/immunology , Spider Venoms/immunologyABSTRACT
The brown recluse spider, Loxosceles reclusa Gertsch & Mulaik, is a well-known venomous spider common in the south-central United States where infestations can reach thousands of individuals in a single structure. Bites from this spider pose a risk of dermonecrotic lesions (loxoscelism) or, rarely, more serious systemic effects. The heat tolerance of this spider is understudied but may offer an alternative pest control solution to pesticides or fumigation, both of which have their disadvantages. We subjected brown recluse spiders to increasing temperatures to establish the upper lethal temperature (LT). Using probit analysis to generate probability of mortality at increasing temperatures, we then exposed adult spiders to the observed LT50 to simulate whole-room heat treatment. Laboratory results predict exposure to 48°C for 130 min will achieve 100% mortality of adult spiders. Field tests need to be conducted to determine the efficacy of heat treatment in a variety of real-world situations.
