Simultaneous detection and differentiation of anti-Helicobacter pylori antibodies by flow microparticle immunofluorescence assay

Abstract

Helicobacter pylori is the key pathogen for gastroduodenal diseases. The clinical outcome of H. pylori infection is influenced by the presence of strain-specific virulence factors that are usually detected by the presence of specific anti-H. pylori antibodies in serum. Apart from the detection of these antibodies by enzyme-linked immunosorbent assay (ELISA), it is desirable to obtain additional information concerning the presence of certain virulence factors of H. pylori that are currently detected by immunoblot analysis. At present, the immunodiagnosis of an H. pylori infection includes two separate methods: ELISA and immunoblot analysis. Here, we report the development and evaluation of a new rapid flow microparticle immunofluorescence assay (FMIA) for detection of anti-H. pylori antibodies in human serum. The assay allows rapid qualitative and quantitative detection of anti-H. pylori antibodies by using crude antigen preparations as well as single recombinant antigens (urease A, urease B, CagA, and alkylhydroxy peroxide reductase) in the same sample with one measurement, and thus it combines the advantages of enzyme immunoassay and Western blot analysis. Seventy-five patient samples were analyzed by FMIA, ELISA, and Western blotting with respect to their immunoreactivity against crude H. pylori extracts and individual H. pylori antigens. Statistical analyses revealed an overall similarity of more than 90% among the results for FMIA, ELISA, and Western blot. Therefore, we conclude that FMIA is a powerful and time- and cost-saving assay system for the detection of antimicrobial antibodies, with higher sensitivity and a larger measurement range than ELISA.

FIG. 1.

Detection of antibodies against defined H. pylori antigens using Western blot analyses. (A) Commercially available Western blots were incubated with rabbit H. pylori antiserum. (B) Recombinant antigens prepared as described in Materials and Methods and separated by SDS-PAGE, blotted on nitrocellulose, and stained with rabbit H. pylori antiserum.

FIG. 2.

Detection of anti-H. pylori antibodies using FMIA. Antigen-coated microparticles were prepared as described in Materials and Methods. (A) Defined microparticle populations were separated by size (forward scatter) and FITC fluorescence (around 520 nm). (B) Specific anti-H. pylori immunoreactivity was analyzed by using phycoerythrin-labeled anti-human IgG antibodies. The fluorescence intensity around 560 nm correlated with the concentration of anti-H. pylori antibodies in the serum samples. For each microparticle population, a sample that was incubated with patient serum (shaded curve) and a sample that was incubated with buffer alone (negative control, open curve) are shown.

FIG. 3.

Determination of measurement range by the ELISA (A) and FMIA (B) systems. Three H. pylori-positive serum samples (solid symbols) and one negative sample (open squares) were diluted between 12- and 100,000-fold. The immunoreactivity was analyzed with the ELISA and FMIA.

FIG. 4.

Correlation between FMIA, ELISA, and histology. (A) Immunoreactivity against crude H. pylori extracts was measured with the ELISA and FMIA systems. The corresponding results were blotted (n = 75). Solid lines indicate the threshold values for FMIA and ELISA. Pearson correlation analyses of H. pylori-positive samples (solid circles) showed a significant correlation between ELISA and FMIA (dashed line, r = 0.8, P < 0.01). Samples with low immunoreactivity in the ELISA (<13 EU) were negative in the FMIA (open circles), whereas samples with intermediate reactivity in the ELISA (14 to 19 EU [open squares]) were in part above the FMIA threshold (solid vertical line). The results obtained by FMIA (B) and ELISA (C) measurements were compared to the corresponding histological findings. In all sera from patients with positive histology, we found anti-H. pylori antibodies, whereas some sera from patients with negative histology were positive in the FMIA and/or ELISA.

FIG. 5.

Comparative analysis of antibodies against single H. pylori antigens in Blot 1 (Viramed) and FMIA. ELISA-positive patient samples (n = 60) were analyzed in FMIA and Blot 1. The corresponding results were depicted in dot blots. The calculated FMIA threshold is indicated by a horizontal solid line. Only those samples that could be clearly evaluated in a Western blot were used for comparative analyses (urease A, 50 of 60 samples; urease B, 55 of 60 samples; CagA, 60 of 60 samples; APR, 43 of 60 samples).

FIG. 6.

Comparative analysis of antibodies against single H. pylori antigens in Blot 1 (Viramed) and Blot 2 (Biosens). Patient samples (n = 29) were each analyzed in Blot 1 and Blot 2. The corresponding results were depicted in dot blots. Only those samples that could be clearly evaluated in both Western blots were used for comparative analyses (urease A, 23 of 29 samples; urease B, 24 of 29 samples; CagA, 26 of 29 samples; APR, 23 of 29 samples).