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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 M├╝hlenberg 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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Related in: MedlinePlus

Contrast values from all replicates. The contrast values (n = 5) are presented as a box-whisker-plot where the square represents the arithmetic mean and the horizontal line the median. The whiskers enclose 90% of all values (95% - 5%) and 98% of the values fall between the crosses. CjaA, hisJ, flaC and peb1a show positive signals above the cutoff throughout the slides with only one exception for flaC due to an outlier in the cutoff. On the other hand, gapA, argC and pyrC show negative results, i.e. contrast values significantly below the respective cutoff values for all the slides. The remaining proteins pal and pseB are in the range of the cutoff values.
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Figure 7: Contrast values from all replicates. The contrast values (n = 5) are presented as a box-whisker-plot where the square represents the arithmetic mean and the horizontal line the median. The whiskers enclose 90% of all values (95% - 5%) and 98% of the values fall between the crosses. CjaA, hisJ, flaC and peb1a show positive signals above the cutoff throughout the slides with only one exception for flaC due to an outlier in the cutoff. On the other hand, gapA, argC and pyrC show negative results, i.e. contrast values significantly below the respective cutoff values for all the slides. The remaining proteins pal and pseB are in the range of the cutoff values.

Mentions: To validate the results from one slide technical replicates (n = 5) were analyzed. Summing up the results of all slides, a box-whisker-plot was composed, see Figure 7. The replicates emphasized the results from the one slide above. CjaA, hisJ, flaC and peb1a were clearly above the respective cutoffs and led to positive signals in all the slides. On the contrary, gapA, argC and pyrC were significantly below the cutoff. The tendency of pal and pseB to fall within close proximity of the cutoff is observed throughout the replicates.


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

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

Contrast values from all replicates. The contrast values (n = 5) are presented as a box-whisker-plot where the square represents the arithmetic mean and the horizontal line the median. The whiskers enclose 90% of all values (95% - 5%) and 98% of the values fall between the crosses. CjaA, hisJ, flaC and peb1a show positive signals above the cutoff throughout the slides with only one exception for flaC due to an outlier in the cutoff. On the other hand, gapA, argC and pyrC show negative results, i.e. contrast values significantly below the respective cutoff values for all the slides. The remaining proteins pal and pseB are in the range of the cutoff values.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Contrast values from all replicates. The contrast values (n = 5) are presented as a box-whisker-plot where the square represents the arithmetic mean and the horizontal line the median. The whiskers enclose 90% of all values (95% - 5%) and 98% of the values fall between the crosses. CjaA, hisJ, flaC and peb1a show positive signals above the cutoff throughout the slides with only one exception for flaC due to an outlier in the cutoff. On the other hand, gapA, argC and pyrC show negative results, i.e. contrast values significantly below the respective cutoff values for all the slides. The remaining proteins pal and pseB are in the range of the cutoff values.
Mentions: To validate the results from one slide technical replicates (n = 5) were analyzed. Summing up the results of all slides, a box-whisker-plot was composed, see Figure 7. The replicates emphasized the results from the one slide above. CjaA, hisJ, flaC and peb1a were clearly above the respective cutoffs and led to positive signals in all the slides. On the contrary, gapA, argC and pyrC were significantly below the cutoff. The tendency of pal and pseB to fall within close proximity of the cutoff is observed throughout the replicates.

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 M├╝hlenberg 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