Microarray-based method for screening of immunogenic proteins from bacteria

Abstract

BackgroundDetection of immunogenic proteins remains an important task for life sciences as it nourishes the understanding of pathogenicity, illuminates new potential vaccine candidates and broadens the spectrum of biomarkers applicable in diagnostic tools. Traditionally, immunoscreenings of expression libraries via polyclonal sera on nitrocellulose membranes or screenings of whole proteome lysates in 2-D gel electrophoresis are performed. However, these methods feature some rather inconvenient disadvantages. Screening of expression libraries to expose novel antigens from bacteria often lead to an abundance of false positive signals owing to the high cross reactivity of polyclonal antibodies towards the proteins of the expression host. A method is presented that overcomes many disadvantages of the old procedures.ResultsFour proteins that have previously been described as immunogenic have successfully been assessed immunogenic abilities with our method. One protein with no known immunogenic behaviour before suggested potential immunogenicity.We incorporated a fusion tag prior to our genes of interest and attached the expressed fusion proteins covalently on microarrays. This enhances the specific binding of the proteins compared to nitrocellulose. Thus, it helps to reduce the number of false positives significantly. It enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability. We validated our method by employing several known genes from Campylobacter jejuni NCTC 11168.ConclusionsThe method presented offers a new approach for screening of bacterial expression libraries to illuminate novel proteins with immunogenic features. It could provide a powerful and attractive alternative to existing methods and help to detect and identify vaccine candidates, biomarkers and potential virulence-associated factors with immunogenic behaviour furthering the knowledge of virulence and pathogenicity of studied bacteria.

Chicken Anti-Campylobacter Vaccine – Comparison of Various Carriers and Routes of Immunization

“…Two highly immunogenic, extracytoplasmic and conserved C. jejuni proteins — CjaA (Cj0982c in the genome of C. jejuni NCTC11168) and CjaD (Cj0113 in the genome of C. jejuni NCTC11168), which are often tested as candidates for chicken anti- Campylobacter vaccination – were chosen for this study. The immunogenicity of CjaA and CjaD has been documented by our research group, as well as by others ( Wyszyńska et al, 2004 ; Buckley et al, 2010 ; Layton et al, 2011 ; Clark et al, 2012 ; Hoppe et al, 2012 ). Recently we have shown that GEM particles (Gram-positive Enhancer Matrix), from TCA-pretreated L. salivarius , can act as a surface display platform for C. jejuni antigens ( Bosma et al, 2006 ; Kobierecka et al, 2015 ).…”

Rapid Identification of Novel Immunodominant Proteins and Characterization of a Specific Linear Epitope of Campylobacter jejuni

“…As we have shown elsewhere, an approach using HaloTagH and specifically coated HaloLink TM slides is better suited to detect immunodominant proteins while reducing cross-reactivity to a minimum [13]. The HaloTagH provides several advantages to other commonly used tags as it enhances the amount of soluble proteins expressed [14] reducing the formation of inclusion bodies.…”

Leptospiral Outer Membrane Protein Microarray, a Novel Approach to Identification of Host Ligand-Binding Proteins

“…The utilization of protein microarrays to identify ligand-binding proteins is an innovative approach (51,60,73,106) that could serve as a useful tool for elucidating host-pathogen interactions. In the microbiology field, proteome microarrays have mostly been used for serological studies to identify targets of the human or animal immune response during course of infection with the goal of discovering diagnostic antigens (6,9,15,22,25,26,42,44,54,59,68,91,94,99,101,107). To date, a few reports have described protein microarrays as a tool to screen for proteins with host ligand-binding capacities, both studies focusing on Streptococcus (32,61).…”