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ELISA kit for antigenic diagnosis of Cysticerosis


BIO K 364/2

Código: BIO K 364/2. Categorías: , , .
   
Descripción

I – INTRODUCTION

Tænia saginata is the beef tapeworm. Cattle are its intermediate host. They harbour the tapeworm’s larval form, the cysticercus (Cysticercus bovis), in their muscles. Humans – its final hosts – are infected by ingesting infested undercooked beef. The zoonosis is currently more frequent in industrialised countries than Taenia solium infestations, for T. saginata is a cosmopolitan parasite. It is spread by desiccation of the worm’s gravid proglottids. The infectious eggs or proglottids that are shed by humans contaminate the soil and pasture on which cattle – the intermediate hosts – graze. Upon reaching the animal’s intestine, the hexacanth embryos that are released by digestion of the embryophore cross the gut wall and travel through the bloodstream until they reach muscle tissue, where they become encysted, giving rise to the cysticerci. Tænia solium is the pig tapeworm. Swine are its intermediate hosts and humans its final hosts. This tapeworm seems to have practically disappeared in industrialised countries because of the ease with which veterinary inspectors detect it. Taenia solium can cause a zoonosis. In some cases human beings can be the intermediate hosts of T. solium; they then harbour the cysticercoid larvae. This occurs most often in developing countries that are poor or lack effective veterinary inspections and where human beings live in contact with pigs and/or where sanitation is poor. When pigs are infested experimentally the circulating antigens are detectible as of 2-6 weeks after infestation and remain present for an observation period of six months. The minimum number of living cysts that can be detected with the kit is one cysticercus. Bio-X Diagnostics’s Cysticercosis Antigen ELISA Kit is an immunoenzyme test for detecting the viable cysticerci of various Taenia species (T. solium, T. saginata, T. hydatigena, and T. asiatica).

 

II – PRINCIPLE OF THE TEST

The entire 96-well microtitration plate has been sensitised with a monoclonal antibody specific for Taenia saginata (its secretory-excretory antigens). This antibody captures the cysticercal antigens obtained from serum samples. The test is indeed a sandwich antigen ELISA test, even though blood serum is used. These blood serum samples are treated with trichloroacetic acid and then neutralised. Next, the samples incubated on the microplate for one hour under shaking. After this incubation the plate is rinsed and the conjugate (a second monoclonal antibody specific for an antigenic determinant of Taenia saginata coupled to peroxidase) is added; then the plate is incubated for another hour at 21 +/- 3 °C. After a second rinse, the detection solution (single-component TMB solution) is added. If antigens from the cysticerci of Taenia spp. are present, the conjugate remains bound to the corresponding well and the enzyme catalyses the transformation of the colourless chromogen into a blue compound. The intensity of the colour is proportionate to the antigen titre in the serum samples. The reaction is then stopped by adding an acid solution and each sample’s optical density is read at 450 nm. A control antigen is provided with the kit to confirm the validity of the test results.

 

III – COMPOSITION OF THE KIT

– Microplates: Two 96-well microtitration plate. All the wells on each microplate have been sensitised with the antibody specific for Taenia spp. – Washing solution: One 100-ml bottle of 20-fold concentrated washing solution. The solution crystallises spontaneous when cold. If only part of the solution is to be used, bring the bottle to 21 +/- 3 °C until disappearance of all crystals. Mix the solution well and remove the necessary volume. Dilute the buffer twentyfold with distilled or demineralised water. – Trichloroacetic acid solution: One 30-ml bottle. This reagent must be stored between +2 and +8 °C. – Neutralising solution: One 30-ml bottle. This reagent must be stored between +2 and +8 °C. – Negative control: One vial containing serum negative for the presence of Taenia spp. cysticerci. – Positive control: One vial containing serum positive for the presence of Taenia spp. cysticerci. – Conjugate: One 25-ml vial of peroxidase-linked anti-Taenia spp conjugate. This reagent is ready to use. – Single-component TMB: One 25-ml vial of the chromogen tetramethylbenzidine (TMB). This reagent must be stored between +2 and +8 °C protected from light. It is ready to use. – Stopping solution: One 15-ml vial of the stop solution containing 1 M phosphoric acid

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