Wykorzystanie wybranych, rekombinowanych białek w serodiagnostyce zakażeń wywoływanych przez werotoksyczne pałeczki Escherichia coli (VTEC) u ludzi
[Use of selected recombinant proteins in serodiagnosis of infections caused by verotoxigenic Escherichia coli (VTEC) in humans]

Streszczenie

Przeprowadzone badania wykazały, że uzyskane we własnym zakresie rekombinowane białka intymina, werotoksyna 2b i Tir pałeczek E. coli można wykorzystać jako swoiste antygeny w rutynowej serodiagnostyce zakażeń VTEC, przebiegających zwłaszcza jako zespół hemolityczno-mocznicowy.

Najbardziej swoistym serologicznym markerem zakażenia pałeczkami VTEC jest obecność w surowicy osób chorych przeciwciał klasy IgG i/lub IgA dla rekombinowanego białka Tir.

Abstract

Introduction: Verocytotoxin-producing E. coli (VTEC) are a significant cause of haemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS) in humans. Because VTEC isolates are usually present in patients’ feces for only a limited period of time serodiagnosis based on the purified antigens have become the useful tool for laboratory diagnosis and monitoring of prevalence of VTEC infections. The aim of the present study was to evaluate the usefulness of in-house obtained recombinant proteins Tir, intymin and verocytotoxin 2b of E. coli as highly specific antigens in ELISA performed in the serodiagnosis of infections caused by VTEC in humans.

Materials and Methods: The study population, used for characterization of the humoral immune response to the recombinant proteins, consisted of 37 patients suspected for VTEC infection, mainly with clinical manifestation of HUS. Additionally serum samples from 78 clinically healthy persons and 96 patients with different bacterial infections (control group) were tested. Recombinant proteins 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).

Results: The antibodies against recombinant proteins were detected using the ELISA in  about half of the tested patients suspected in clinical investigation for VTEC infection. Most of the antibodies belong to the IgG and IgA class of immunoglobulins. Statistical analysis of the results showed that the frequency of detecting antibodies among patients with HUS was significantly higher in relation to the clinically healthy persons. However, the percentage of positive results in the control group were also much higher than in healthy persons what may indicate for presence of non-specific reactions. The least non-specific response was detected by ELISA with the protein Tir as antigen.

Conclusions: The study showed that recombinant proteins Tir, intimin and verocytotoxin 2b of E. coli may be used as antigens in routine diagnosis of VTEC infections. The most specific antigen is a recombinant protein Tir.

Key words: VTEC, recombinant proteins, intymin, verocytotoxin 2b, protein Tir, ELISA, HUS