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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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Western Blot of purified protein. CjaA and hisJ show strong bands of the corresponding sizes (31 kDa and 28 kDa) after removal of the HaloTag®. The remaining proteins cannot be detected in western blot by visual means.
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Figure 8: Western Blot of purified protein. CjaA and hisJ show strong bands of the corresponding sizes (31 kDa and 28 kDa) after removal of the HaloTag®. The remaining proteins cannot be detected in western blot by visual means.

Mentions: Further verification of the results was performed by using a standard western blot experiment to test for immunogenicity. Figure 8 shows the results of the investigated proteins after purification with HaloLink™ magnetic beads was performed prior to SDS-PAGE and blotting. Only two of the investigated immunogenic proteins (cjaA, and hisJ) show strong visible bands in western blot matching the expected sizes. The three remaining immunogenic proteins, peb1a, flaC and pal, cannot be distinguished in western blot analysis as well as all the other proteins. Additionally, a Dotblot was performed (Figure 9) with the purified proteins, which showed clear positive signals for cjaA and hisJ, a rather weak signal for peb1a and extremely low signals for flaC and pal.


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

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

Western Blot of purified protein. CjaA and hisJ show strong bands of the corresponding sizes (31 kDa and 28 kDa) after removal of the HaloTag®. The remaining proteins cannot be detected in western blot by visual means.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Western Blot of purified protein. CjaA and hisJ show strong bands of the corresponding sizes (31 kDa and 28 kDa) after removal of the HaloTag®. The remaining proteins cannot be detected in western blot by visual means.
Mentions: Further verification of the results was performed by using a standard western blot experiment to test for immunogenicity. Figure 8 shows the results of the investigated proteins after purification with HaloLink™ magnetic beads was performed prior to SDS-PAGE and blotting. Only two of the investigated immunogenic proteins (cjaA, and hisJ) show strong visible bands in western blot matching the expected sizes. The three remaining immunogenic proteins, peb1a, flaC and pal, cannot be distinguished in western blot analysis as well as all the other proteins. Additionally, a Dotblot was performed (Figure 9) with the purified proteins, which showed clear positive signals for cjaA and hisJ, a rather weak signal for peb1a and extremely low signals for flaC and pal.

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