Ocena przydatności uzyskanych we własnym zakresie rekombinowanych białek Yop pałeczek Yersinia enterocolitica jako wysoce swoistych antygenów w odczynach ELISA i recom-dot wykonywanych w kierunku serodiagnostyki jersiniozy
[Usefulness of in-house obtained recombinant proteins Yop of Yersinia enterocolitica as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis]

STRESZCZENIE

Uzyskane we własnym zakresie rekombinowane białka wydzielnicze YopD, YopB, YopE i VAg pałeczek Y. enterocolitica wykorzystano jako wysoce swoiste antygeny w odczynie ELISA i recom-dot. Najwyższą przydatność w serodiagnostyce jersiniozy u ludzi wykazało rekombinowane białko YopD,

które może z powodzeniem zastąpić w odczynie ELISA preparat białek wydzielniczych Yop uzyskanych w warunkach natywnych. Opracowany zestaw recom-dot może być przydatny jako referencyjny test potwierdzający wynikiuzyskane odczynem ELISA z natywnymi antygenami pałeczek Y. enterocolitica.

ABSTRACT

Introduction: Proper analysis of the human immune response is crucial in the laboratory diagnosis of many bacterial infections. The current serological diagnosis of yersiniosis often is carried out using ELISA with native antigens. However, recombinant proteins increase the specificity of the serological assays, particularly in patients with chronic, nonspecific infections. The aim of the present study was to evaluate the usefulness of in-house obtained recombinant proteins Yop of Yersinia enterocolitica as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis. Materials and Methods: Recombinant YopD, YopB, YopE and V-Ag proteins of Y.enterocolitica were expressing in E. coli BL21 (DE3) using the pET-30 Ek/LIC expression vector (Novagen). Purification was accomplished by immobilized metal (Ni2+) affinity column chromatography (His-trap). The proteins were used as antigens in standard ELISA and recom-dot assay, which was performed on nitrocellulose strips. The study population, used for characterization of the humoral immune response to the recombinant proteins, consisted of 74 patients suspected for Y. enterocolitica infection and 41 clinically healthy blood donors. Some of the results obtained by ELISA and recom-dot were compared with results obtained by commercial western-blot Yersinia (Virotech). Results: In the group of patients suspected for yersiniosis in clinical investigation the most positive results were obtained in ELISA with the recombinant protein YopD (IgA respectively 25 (42.4%), IgG 41 (69.5%), IgM 24 (40.7%). The percentage of positive results in the group of blood donors did not exceed 10.0% in IgG and 5.0% in IgA/IgM classes of immunoglobulin. The results obtained in the recom-dot assay showed that among 74 tested serum samples obtained from individuals suspected of yersiniosis the most common IgA,

IgG and IgM antibodies were found for recombinant protein YopD (respectively IgG in 60.8%, IgA in 37.8% and IgM in 33.8% of serum samples). IgG antibodies to the protein V-Ag were observed in 32.4%, protein YopB in 27.0% and for the protein YopE in 18.9% serum samples. Immunoglobulin A, and M for the recombinant proteins were found much less frequently than IgG antibodies (respectively 12.2% and 10.8% for V protein-Ag in 10.8% and 14.9% protein and YopB 2,7% and 10.8% for the protein YopE). Further studies showed that results obtained in recom-dot assay with recombinant protein YopD were comparable with the results of commercial western-blot Yersinia. Conclusions: The study showed that in-house obtained recombinant proteins can be used as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis. The most useful was the protein YopD.