Primena 7C2C5 monoklonskog antitela u razvoju ELISA testova za otkrivanje infekcije sa Trichinella spp. i izolaciju komponenti parazita koje nose imunodominantni epitop

Abstract

Trihineloza je parazitska infekcija izazvana nematodoma iz roda Trichinella, pre svega Trichinella spiralis (T.spiralis). Svi sisari, neke ptice i reptili su prijemčivi na infekciju sa jednom ili vise vrsta iz roda Trichinella, ali samo ljudi razvijaju kliničku sliku bolesti. Infekcija ima kosmopolitsku distribuciju, prisutna je na svim kontinentima osim Antartika, gde do sada nije zabeležen nijedan slučaj. Do infekcije ljudi dolazi konzumiranjem nepregledanog, sirovog ili nedovoljno termički obradjenog mesa i mesnih prerađevina dobijenih od inficiranih domaćih i divljih životinja, a najčešće su u pitanju domaće svinje, konji i divlje svinje. Na polju imunoparazitologije, praćenje incidencije i prevalencije infekcije parazitom iz roda Trichinella kod ljudi i životinja bazira se, pored ostalog, i na primeni in vitro dijagnostičkih testova koji detektuju specifična antitela u serumu inficiranog domaćina. Ovakvo paraćenje je od izuzetnog značaja zbog činjenice da ova parazitoza predstavlja ozbiljan zdravstveni i ekonomski problem širom sveta, a naročito u zemljama centralne i istočne Evrope. Ključni problemi u imunodijagnostici infekcije izazvane sa Trichinella spp., kako kod ljudi tako i kod životinja, jesu visok nivo nespecifičnosti postojećih testova, zatim nepostojanje univerzalnih testova koji bi bili primenljivi u detekciji Trichinella-specifičnih antitela kod više vrsta domaćina, nepostojanje testova za detekciju infekcije mnogih vrsta potencijalnih domaćina, pre svega različitih vrsta divljači. Takođe, veliki nedostatak postojećih testova jeste i niska senzitivnost testova u početnim fazama infekcije, odnosno visok procenat lažno negativnih rezultata koji se u testu dobijaju u periodu 2-4 nedelje nakon infekcije. Korišćenjem monoklonskog antitela (mAt) 7C2C5, specifičnog prema epitopu jedinstvenom za mišićne larve roda Trichinella, razvijen je kompetitivni enzimski imunoesej (c-ELISA test) koji omogućava brzu detekciju Trichinella-specifičnih antitela u serumima poreklom od različitih vrsta domaćina (ljudi, svinja, konja). Test koristi jedno antitelo - mAt 7C2C5 (HRP-konjugovano), kao kompetitivni i detektujući reagens u isto vreme, bez korišćenja sekundarnih antitela specifičnih prema vrsti koju detektuju, čime je omogućena identifikacija Trichinella-specifičnih antitela bez obzira na njihov izotip ili vrstu domaćina koji produkuje antitela od interesa. Primenljivost testa dokazana je na serumima ljudi inficiranih sa T. spiralis i T. britovi, serumima prirodno i eksperimentalno inficiranih svinja sa T. spiralis i T. pseudospiralis i serumima eksperimentalno inficiranih konja (T. spiralis)...

Trichinellosis is parasitic infection caused by nematodes from the genus Trichinella, predominantly T. spiralis. A broad range of mammals, birds and reptiles are susceptible to infection with one or more species of the genus Trichinella, but only humans become clinically affected. The infection has a cosmopolitan distribution except Antarctica, where no case has been recorded so far. The main sources of human disease (trichinellosis) are pork, game and horse meat (or products) that contain infective muscle larvae. In the field of immunoparasithology, estimation of incidence and prevalence of Trichinella infection in humans and animals, is of extreme importance having in mind the fact that this infection presents serious problem worldwide, and especially in countries of Central and East Europe. The key problems in immunodiagnosis of Trichinella spp. infection in humans as well as in animals are low specificity of commercialy aveliable tests and leckness of universal tests that can be applied for detection of Trichinella-specific antibodies in multiple host species. Also, the important weakness of the existing tests is their low sensitivity in the initial stage of the infection, since the high percentage of false negative results is obtained during the period of 2-4 weeks post infection (p.i). Application of the monoclonal antibody (mAt) 7C2C5, specific for an epitope unique to the muscle larvae of the whole Trichinella genus (epitope is present on molecular triplet with 45, 49 and 53 kDa), we have developed a competitive (c)-ELISA that enables a rapid detection of Trichinella-specific antibodies in the sera originating from different host species (human, swine and horse). The test employs a single antibody, mAt 7C2C5 (HRP labeled), as both the competing and detecting reagent, which allows the detection of specific antibodies irrespective of their isotype or host origin and infected with either Trichinella spiralis or Trichinella britovi. This novel c-ELISA exhibited 100% specificity and sensitivity, as confirmed by the Western blot test. The assay is easy-to-use (one incubation step), and the 45-minute time required for the procedure is shorter than in any other ELISA format. The applicability of the test was demonstrated on sera from humans infected with T. spiralis and T. britovi, pigs infected with T. spiralis and T. pseudospiralis and sera of experimentally infected horses (T. spiralis). The applicability of the test was also demonstrated on the limited number of sera from experimental infected wild boars (T. spiralis infection). This test could be useful for both the detection and surveillance of Trichinella infections. An analysis of one of the potential markers for early diagnosis of human trichinosis such as Trichinella-specific IgE antibodies in human sera would be possible by the development of a reliable test for its detection...