Characteristic band pattern in western blots for specific detection of anti- antibodies in different host species

Abstract

Western blot (Wb) is considered to be the gold standard test for Trichinella infection serology, since this method allows specifi c Trichinella antigens to be distinguished from cross-reactive antigens. This is not the case with widely used antibody assay techniques - indirect immunofl uorescence and ELISA - which are sensitive, but subject to crossreactions that make the interpretation of weakly positive results diffi cult. Application of Trichinella spiralis muscle larvae excretory-secretory (ES) antigens for the specifi c antibody detection in ELISA resulted in improved specifi city compared to that of crude worm extract that was previously in use, but since production of ES has not yet been standardized, differences among laboratories occur. For this reason, the Wb profi le of serum samples from different T. spiralis infected host species: human, horse, swine and dog, was investigated in the Serbian National Reference Laboratory for Trichinellosis (NRLT). The common feature of the obtained Wb profi les was the appearance of a triad of bands with molecular masses (Mw) of 45, 49, and 53 kDa. The very same triad was recognized by a monoclonal antibody (mAb) 7C2C5 specifi c for an immunodominant epitope unique to the muscle larvae stage of all species in the genus Trichinella. Inhibition studies confi rmed that mAb and anti-Trichinella antibodies from sera competed for the same parasite epitope. Based on the obtained results, the NRLT introduced the recognition of the above mention triad as the basis for specifi c anti-Trichinella antibodies detection in the sera of infected hosts.

Western blot (Wb) metoda se smatra zlatnim standardom u serološkoj dijagnostici trihineloze obzirom da ova metoda omogućava razlikovanje specifičnih Trichinella antigena od onih koji daju ukrštenu reakciju. Ovo nije slučaj sa drugim, u praksi češće korišćenim tehnikama baziranim na detekciji antitela – indirektna imunofl uorescencija i ELISA, koje su senzitivne ali podložne unakrsnoj reaktivnosti što otežava interpretaciju slabo pozitivnih rezultata. Primena ekskretorno-sekretornog (ES) antigena larvi u mišićima, Trichinella spiralis u detekciji specifičnih antitela u ELISA testu, rezultirala je povećanom specifičnošću testa u odnosu na prethodni koji je bio baziran na primeni totalnog solubilnog ekstrakta parazita. Ipak, obzirom da proizvodnja ES antigena još uvek nije standardizovana, postoje izvesne razlike između rezultata dobijenih u različitim laboratorijama. Iz ovog razloga, u Nacionalnoj Referentnoj Laboratoriji Srbije za Trihinelozu (NRLT) ispitivali smo Wb profi l uzoraka seruma različitih vrsta domaćina infi ciranih sa T. spiralis, i to ljudi, konja, svinja i pasa. Zajedničko svojstvo dobijenih Wb profi la bila je pojava tripleta traka koje odgovaraju proteinima sa molekulskim masama (Mw) od 45, 49, 53 kDa. Isti triplet prepoznaju i monoklonska antitela (mAt) 7C2C5 specifična za imunodominantni epitop karakterističan za stadijum mišićnih larvi svih vrsta parazita iz genusa Trichinella. Eksperimentalne studije inhibicije su pokazale da postoji kompeticija između mAt i anti-Trichinella antitela iz seruma za isti epitop parazita. Na osnovu dobijenih rezultata, NRLT uvodi prepoznavanje pomenutog tripleta traka kao osnovu za detekciju specifičnih anti-Trichinella antitela u serumima infi ciranih domaćina.