Detection of H. pylori antibody profile in serum by protein array

Abstract

Aim: To detect multiple H. pylori antibodies in serum samples of individuals who carry H. pylori by protein array.

Methods: Recombinant H. pylori antigens, urease B subunit (UreB), vacuolating toxin A (VacA) and cytotoxin associated gene A protein (CagA), were prepared and immobilized in matrixes on nitrocellulose membrane by robotics to bind the specific immunoglobulin G (IgG) antibodies in serum. Staphylococcus protein A (SPA) labeled by colloid gold was used to integrate the immuno-complex and gave red color signal. The scanner based on charge-coupled device (CCD) could collect the image signal and convert it into digital signal.

Results: When human IgG was printed on the membrane in increasing concentrations and incubated with immunogold, a linear dose response curve was obtained and the detection limit for IgG was about 0.025 ng. The cutoff values, which were defined as the mean grey level plus 3 times of standard deviation, were 27.183, 28.546 and 27.402, for anti-UreB IgG, anti-CagA IgG and anti-VacA IgG, respectively, as 400 human serum samples with negative H. pylori antibodies were detected by the protein array. When 180 serum samples from patients in hospital were employed for detection of IgG against UreB, CagA and VacA, the sensitivity of the protein array was 93.4%, 95.4%, 96.0%, and the specificity was 94.8%, 94.4% and 97.5%, respectively, as compared with the results obtained by ELISA. The assay also showed high reproducibility, uniformity and stability, and the results were available within 30 min.

Conclusion: The protein array is a very practical method for rapid detection of multiple antibodies in serum samples. It is especially useful for large scale epidemiological investigation of the infection of H. pylori.

Figure 1

A: Schematic map of protein distribution on the protein array. Four squares were involved and different proteins were printed in different areas: UreB in upper left area; VacA in upper right area; CagA in left bottom area; R.M (10 ng) in the upper row of right bottom area for negative control (Ctrl), H. IgG in the middle row for positive control and at the bottom row for strong positive control; B: positive results of anti-UreB IgG, anti-CagA IgG and anti-VacA IgG detected by the protein array.

Figure 2

Longitudinal section of the protein array apparatus. The shell of the apparatus was made of plastic materials. A window was opened on the top of the outer shell and the margin of the nitrocellulose membrane printed with antigens was sealed to the inner surface of the window. Under the nitrocellulose membrane was the macromolecular bibulous material.

Figure 3

The mean grey level of the spot against various amount of H.IgG immobilized on the protein array. The concentrations of human IgG spotted from lane 1 to lane 6 are as follows: 1.25, 2.5, 5, 10, 20, 40 μg/mL. The optimized amount of SPA binding to colloid gold was 12 μg/mL. The data in the plot were obtained from the image of CCD by averaging the mean grey levels of the 6 replicate spots.