OCENA PRZYDATNOŚCI IMMUNOENZYMATYCZNEGO ODCZYNU ELISA DO WYKRYWANIA PRZECIWCIAŁ DLA LIPOPOLISACHARYDU PAŁECZEK KLEBSIELLA RHINOSCLEROMATIS U CHORYCH NA TWARDZIEL
[APPLICATION OF ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) FOR DIAGNOSIS OF ANTIBODIES TO LIPOPOLYSACCHARIDE OF KLEBSIELLA RHINOSCLEROMATIS IN PATIENTS WITH RHINOSCLEROMA]

Streszczenie

Odczynem ELISA poszukiwano swoistych przeciwciał dla antygenu 2a pałeczek K. rhinoscleromatis w 65 próbkach surowicy uzyskanych od osób z klinicznym podejrzeniem twardzieli. Próbki te przysłano w latach 1970-2009 do rutynowych badań odczynem wiązania dopełniacza. Obecność przeciwciał klasy IgA i IgG na poziomie diagnostycznie znamiennym wykryto w 33 (50,8%) a przeciwciał klasy IgM w 28 (43,1%) próbkach surowicy. Przeprowadzone badania wykazały wysoką czułość i swoistość odczynu ELISA z użytym antygenem w poszukiwaniu przeciwciał dla pałeczek twardzieli.

Abstract

The ELISA were performed on polystyrene microtiter plates (Nunc, MaxiSorp) coated with LPS (2a antigen) at the final concentration of 10 μg/ml. The antigen was extracted from Klebsiella rhinoscleromatis Rh32 by the trichloroacetic acid and separated by ethanol (Boivin method). The antibodies against the LPS were detected by ELISA in serum samples collected from 65 patients suspected in clinical investigation for rhinoscleroma in Poland from 1970 to 2009. Additionally, the specificity of
the antigen was tested using serum sample of immunized rabbit and 30 sera of patients from control group, with high level of antibodies to different bacterial pathogens. All serum samples were diluted 1:100. The concentrations of IgA, IgG and IgM antibodies were expressed as optical density (OD) measured at the wavelength of 450 nm. The cut-off limit of serum antibodies was set at mean antibody OD determined in the sera of 30 blood donors exceeded by three standard deviations. The presence
of IgA and IgG antibodies were detected by ELISA in 33 (50,8%) and IgM in 28 (43,1%) of patients. Most of the serum samples (75%) with high level of specific antibodies were obtained from patients before 1980. On the other hand antibodies to K. rhinoscleromatis were detected only in 2 (6,7%) patients from the control group and none of blood donors. In conclusion, our home–made ELISA,
based on purified LPS of K. rhinoscleromatis showed high specificity and sensitivity in the diagnosis of antibodies to K. rhinoscleromatis in comparison to the complement fixation test. The presence of high level of specific IgA, IgG and IgM antibodies in the sera obtained in different stages of disease may showed that during the rhinoscleroma is permanent stimulation of antibody production.