A Burkholderia pseudomallei protein microarray reveals serodiagnostic and cross-reactive antigens

Abstract

Understanding the way in which the immune system responds to infection is central to the development of vaccines and many diagnostics. To provide insight into this area, we fabricated a protein microarray containing 1,205 Burkholderia pseudomallei proteins, probed it with 88 melioidosis patient sera, and identified 170 reactive antigens. This subset of antigens was printed on a smaller array and probed with a collection of 747 individual sera derived from 10 patient groups including melioidosis patients from Northeast Thailand and Singapore, patients with different infections, healthy individuals from the USA, and from endemic and nonendemic regions of Thailand. We identified 49 antigens that are significantly more reactive in melioidosis patients than healthy people and patients with other types of bacterial infections. We also identified 59 cross-reactive antigens that are equally reactive among all groups, including healthy controls from the USA. Using these results we were able to devise a test that can classify melioidosis positive and negative individuals with sensitivity and specificity of 95% and 83%, respectively, a significant improvement over currently available diagnostic assays. Half of the reactive antigens contained a predicted signal peptide sequence and were classified as outer membrane, surface structures or secreted molecules, and an additional 20% were associated with pathogenicity, adaptation or chaperones. These results show that microarrays allow a more comprehensive analysis of the immune response on an antigen-specific, patient-specific, and population-specific basis, can identify serodiagnostic antigens, and contribute to a more detailed understanding of immunogenicity to this pathogen.antigen discovery ͉ melioidosis ͉ diagnostic ͉ antigen prediction

Identification of Potential Serodiagnostic and Subunit Vaccine Antigens by Antibody Profiling of Toxoplasmosis Cases in Turkey

“…1B). We aimed to down-select the number of exons to ϳ2000 using a bioinformatic filtering process based on antigenic features seen in other proteome-wide serological screens in bacteria (63,64). First, genes lacking a mass spectroscopy profile were excluded to enrich for functional genes.…”

“…Meanwhile, the array employed here represents only a fraction of the nearly 8000 genes encoded in the T. gondii genome. Although the protein microarray strategy has been used extensively in bacteria (45,47,53,63,64,67,68), its application to antigen discovery in eukaryotic genomes is relatively novel, having been applied previously only to Plasmodium falciparum (46,69). Of the 15 known serodiagnostic antigens listed above, the smallest exons of four were represented on the current chip, namely GRA1 (070250_1 and _2), GRA2 (027620_2), GRA5 (086450_1), and MIC5 (077080_1).…”

Plasmodium vivax Pre-Erythrocytic–Stage Antigen Discovery: Exploiting Naturally Acquired Humoral Responses

“…Our protein microarray method has been used successfully to identify novel antigens for several different organisms, including Francisella tularensis, 36 Burkholderia pseudomallei, 49 and P. falciparum. 32 In this study, we did not detect robust responses to PvCSP, the immunodominant sporozoite antigen; only 8 of 60 exposed donors displayed detectable levels of antibodies against PvCSP.…”

A multiplexed immunoassay system based upon reciprocating centrifugal microfluidics

“…The pressure differential, P c , induced at a high angular speed of 7000 rpm 21 drives the liquid downstream, over the antigen array, and into the pressure chamber resulting in compression of the air trapped in the pressure chamber. 27 The high rotational acceleration rate (10 000 rpm/s) prevents the liquid from priming the siphon valve at this point. Next, the angular speed is gradually reduced to decrease the centrifugal force and release the stored pneumatic energy.…”

“…The system is based on a novel technique introduced recently by us for reciprocating fluid samples on a CD to enhance mixing in a miniaturized bioassay chamber. 27 It works by using the centrifugal acceleration field acting upon a liquid within a rotating disk to generate and store pneumatic energy for later use in reversing the direction of flow of the liquid as centrifugal acceleration is reduced (by reducing angular frequency). Through a sequence of steps increasing and decreasing centrifugal acceleration (Fig.…”