CelluSpots™ peptide arrays for immunodiagnosis and kinase profiling



CelluSpots™ peptide arrays for immunodiagnosis and kinase profiling

O. Brandt1, E. Rossmann2,3 and R. Wallich3

1.INTAVIS Bioanalytical Instruments AG, Nattermannallee 1, 50829 Köln, Germany

2.Genzyme Virotech GmbH, Löwenplatz 5, 65428 Rüsselsheim, Germany

3.University of Heidelberg, Institute for Immunology, Im Neuenheimer Feld 305, 69120 Heidelberg, Germany




   INTRODUCTION    

Peptide arrays are useful tools to characterize antibodies, to determine sequence specificities of enzymes (e.g. kinases) or to find interaction partners to given peptide sequences. One popular format for such arrays is a cellulose sheet with hundreds of synthetic peptides bound to it. These SPOTarrays have been used successfully in a broad range of applications since their invention 15 years ago [1]. A drawback is the use of large reagent volumes and the limited throughput with only one copy of the library. The

method presented here for the production of identical arrays on multiple microscope slides overcomes these limitations [2,3]. The peptides are synthesized on a modified cellulose support which is dissolved in a cleavagemixture after the synthesis. The resulting solutions of peptide-celluloseconjugates are then spotted onto slides by conventional spotting techniques.

This allows the production of identical arrays that can be incubated with sample volumes of 150 μl or less. As application examples we show epitope mapping of several Borrelia antigens with human sera and determination of kinase specificity using a substrate library. It is possible to use detection methods like autoradiography, chemiluminescence or enzymatic color development which can be performed without expensive instrumentation.






   SERUM SCREENING    

Complete antigen spanning overlapping peptides are spotted as peptidecellulose-conjugates onto coated microscope slides. A three-dimensional layer is formed that exceeds a typical monolayer by a factor of 100. After incubation of the peptide arrays with sera, different detection methods like enzymatic color development or chemiluminescence can be used without expensive instrumentation.





Screening of serum samples with identical CelluSpots™ peptide arrays. After incubation and detection the color developed arrays are scanned followed by computer analysis with different cluster algorithms.







   RESULTS    

Several Borrelia antigens represented by overlapping 20mer peptides where spotted onto microscope slides covered with an inert white foil. Two identical blocks of 384 peptides are surrounded by red orientation marks. After an initial incubation with diluted human serum bound antibodies were detected by anti-IgG-peroxidase conjugates and DAB solution as substrate.




Results of 211 serum incubations on CelluSpots™ peptide arrays.Hierarchical cluster analysis for the serum samples are shown for all peptides.




Kinase substrates and consensus sequences of known kinase substrates from literature and databases were synthesized and spotted onto microscope slides. The resulting arrays were incubated with kinases and radioactively labeled ATP (ATP-gamma 32P). After several washing steps the dry slides were exposed to a phosphorimager screen and X-ray film.






   CONCLUSION    

CelluSpots™ peptide arrays are versatile tools for immunological screening. Hundreds of sera, kinases or other samples can be tested in parallel against defined sets of peptides in a convenient array format. Identical arrays of freely defined peptides can be made economically with the new method and it is easily applied to the development of diagnostic tests, vaccines or to search for suitable enzyme inhibitors. Some advantages of CelluSpots™ arrays are: low sample volume, high peptide loading, parallel testing of many different samples and versatile detection methods.






   REFERENCES    

[1] Frank, R. (1992) Tetrahedron, 48, 9217-9232.

[2] Beutling, U. et al. (2005) Kenes International, Geneva, pp.152-153.

[3] Dikmans, A. et al. (2006) QSAR Comb. Sci. 25(11), 1069-1080.



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