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Microarray-based method for screening of immunogenic proteins from bacteria.

Hoppe S, Bier FF, von Nickisch-Rosenegk M - J Nanobiotechnology (2012)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fraunhofer Institute for Biomedical Engineering, Branch Potsdam, Am Mühlenberg 13, 14476 Potsdam, Germany. sebastian.hoppe@ibmt.fraunhofer.de

ABSTRACT

Background: Detection 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.

Results: Four 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.

Conclusions: The 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.

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Detection of immunogenic proteins on microarrays. The cell lysates are spotted onto HaloLink™ slides (shaded area) with immobilized ligand (curved lines). The HaloTag® (pink sector) is fused to the N-terminus of the protein of interest (purple sector) and attaches covalently to the ligand. Thus, the protein of interest is covalently attached to the surface prior to screening, whereas other proteins from the cell lysate are unbound and washed away after the coupling reaction. Subsequently, primary antibodies (blue) bind to the protein of interest. Afterwards, secondary antibodies (black) conjugated with a fluorophore (yellow) bind to the respective primary antibody. The fluorescent dye is used for detection of positive spots identifying sites of potential immunogenic proteins in the process.
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Figure 1: Detection of immunogenic proteins on microarrays. The cell lysates are spotted onto HaloLink™ slides (shaded area) with immobilized ligand (curved lines). The HaloTag® (pink sector) is fused to the N-terminus of the protein of interest (purple sector) and attaches covalently to the ligand. Thus, the protein of interest is covalently attached to the surface prior to screening, whereas other proteins from the cell lysate are unbound and washed away after the coupling reaction. Subsequently, primary antibodies (blue) bind to the protein of interest. Afterwards, secondary antibodies (black) conjugated with a fluorophore (yellow) bind to the respective primary antibody. The fluorescent dye is used for detection of positive spots identifying sites of potential immunogenic proteins in the process.

Mentions: In this paper, we describe a method to covalently attach different HaloTag® fusion proteins on HaloLink™ slides (see Figure 1) and consequently perform an immunoscreening using polyclonal antibodies in a microarray format, which is a suitable method for high-throughput applications such as screening of entire expression libraries (see Figure 2). This method reduces the pitfall of cross-reactive signals normally encountered with screenings and leads to a more rapid detection of immunogenic proteins compared to conventional methods.


Microarray-based method for screening of immunogenic proteins from bacteria.

Hoppe S, Bier FF, von Nickisch-Rosenegk M - J Nanobiotechnology (2012)

Detection of immunogenic proteins on microarrays. The cell lysates are spotted onto HaloLink™ slides (shaded area) with immobilized ligand (curved lines). The HaloTag® (pink sector) is fused to the N-terminus of the protein of interest (purple sector) and attaches covalently to the ligand. Thus, the protein of interest is covalently attached to the surface prior to screening, whereas other proteins from the cell lysate are unbound and washed away after the coupling reaction. Subsequently, primary antibodies (blue) bind to the protein of interest. Afterwards, secondary antibodies (black) conjugated with a fluorophore (yellow) bind to the respective primary antibody. The fluorescent dye is used for detection of positive spots identifying sites of potential immunogenic proteins in the process.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368735&req=5

Figure 1: Detection of immunogenic proteins on microarrays. The cell lysates are spotted onto HaloLink™ slides (shaded area) with immobilized ligand (curved lines). The HaloTag® (pink sector) is fused to the N-terminus of the protein of interest (purple sector) and attaches covalently to the ligand. Thus, the protein of interest is covalently attached to the surface prior to screening, whereas other proteins from the cell lysate are unbound and washed away after the coupling reaction. Subsequently, primary antibodies (blue) bind to the protein of interest. Afterwards, secondary antibodies (black) conjugated with a fluorophore (yellow) bind to the respective primary antibody. The fluorescent dye is used for detection of positive spots identifying sites of potential immunogenic proteins in the process.
Mentions: In this paper, we describe a method to covalently attach different HaloTag® fusion proteins on HaloLink™ slides (see Figure 1) and consequently perform an immunoscreening using polyclonal antibodies in a microarray format, which is a suitable method for high-throughput applications such as screening of entire expression libraries (see Figure 2). This method reduces the pitfall of cross-reactive signals normally encountered with screenings and leads to a more rapid detection of immunogenic proteins compared to conventional methods.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fraunhofer Institute for Biomedical Engineering, Branch Potsdam, Am Mühlenberg 13, 14476 Potsdam, Germany. sebastian.hoppe@ibmt.fraunhofer.de

ABSTRACT

Background: Detection 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.

Results: Four 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.

Conclusions: The 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.

Show MeSH
Related in: MedlinePlus