Skip to main content
SpringerLink
Log in

Analyzing antibody specificity with whole proteome microarrays

  • Letter
  • Published:

From Nature Biotechnology

View current issue Submit your manuscript

Abstract

Although approximately 10,000 antibodies are available from commercial sources, antibody reagents are still unavailable for most proteins1. Furthermore, new applications such as antibody arrays2,3,4,5 and monoclonal antibody therapeutics6,7 have increased the demand for more specific antibodies to reduce cross-reactivity and side effects. An array containing every protein for the relevant organism represents the ideal format for an assay to test antibody specificity, because it allows the simultaneous screening of thousands of proteins for possible cross-reactivity. As an initial test of this approach, we screened 11 polyclonal and monoclonal antibodies to ∼5,000 different yeast proteins deposited on a glass slide and found that, in addition to recognizing their cognate proteins, the antibodies cross-reacted with other yeast proteins to varying degrees. Some of the interactions of the antibodies with noncognate proteins could be deduced by alignment of the primary amino acid sequences of the antigens and cross-reactive proteins; however, these interactions could not be predicted a priori. Our findings show that proteome array technology has potential to improve antibody design and selection for applications in both medicine and research.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1: Fluorescent images of antibody probes of the yeast proteome microarray.
Figure 2: Analysis of anti-Hda1 binding to the yeast proteome microarray.
Figure 3: Analysis of anti-Pep12 binding to the yeast proteome microarray.

Similar content being viewed by others

References

  1. Kusnezow, W. & Hoheisel, J.D. Antibody microarrays: promises and problems. Biotechniques (Suppl.), 14–23 (2002).

  2. Schweitzer, B. et al. Multiplexed protein profiling on microarrays by rolling-circle amplification. Nat. Biotechnol. 20, 359–365 (2002).

    Article  CAS  Google Scholar 

  3. Haab, B.B., Dunham, M.J. & Brown, P.O. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biol. 2, research0004.1–0004.13 (2001).

    Article  Google Scholar 

  4. Knezevic, V. et al. Proteomic profiling of the cancer microenvironment by antibody arrays. Proteomics 1, 1271–1278 (2001).

    Article  CAS  Google Scholar 

  5. Moody, M.D., Van Arsdell, S.W., Murphy, K.P., Orencole, S.F. & Burns, C. Array-based ELISAs for high-throughput analysis of human cytokines. Biotechniques 31, 186–190, 192–194 (2001).

    Article  CAS  Google Scholar 

  6. Huston, J.S. & George, A.J. Engineered antibodies take center stage. Hum. Antibodies 10, 127–142 (2001).

    Article  CAS  Google Scholar 

  7. Pastan, I. & Kreitman, R.J. Immunotoxins in cancer therapy. Curr. Opin. Investig. Drugs 3, 1089–1091 (2002).

    CAS  PubMed  Google Scholar 

  8. Yu, C.J., Lin, Y.F., Chiang, B.L. & Chow, L.P. Proteomics and immunological analysis of a novel shrimp allergen, Pen m 2. J. Immunol. 170, 445–453 (2003).

    Article  CAS  Google Scholar 

  9. Zhu, H. et al. Global analysis of protein activities using proteome chips. Science 293, 2101–2105 (2001).

    Article  CAS  Google Scholar 

  10. Zhu, H. & Snyder, M. Protein arrays and microarrays. Curr. Opin. Chem. Biol. 5, 40–45 (2001).

    Article  CAS  Google Scholar 

  11. Robertson, L.S. & Fink, G.R. The three yeast A kinases have specific signaling functions in pseudohyphal growth. Proc. Natl. Acad. Sci. USA 95, 13783–13787 (1998).

    Article  CAS  Google Scholar 

  12. Tokiwa, G., Tyers, M., Volpe, T. & Futcher, B. Inhibition of G1 cyclin activity by the Ras/cAMP pathway in yeast. Nature 371, 342–345 (1994).

    Article  CAS  Google Scholar 

  13. Ho, Y. et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183 (2002).

    Article  CAS  Google Scholar 

  14. von Mollard, G.F., Nothwehr, S.F. & Stevens, T.H. The yeast v-SNARE Vti1p mediates two vesicle transport pathways through interactions with the t-SNAREs Sed5p and Pep12p. J. Cell Biol. 137, 1511–1524 (1997).

    Article  CAS  Google Scholar 

  15. Ito, T. et al. A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc. Natl. Acad. Sci. USA 98, 4569–4574 (2001).

    Article  CAS  Google Scholar 

  16. Holthuis, J.C., Nichols, B.J., Dhruvakumar, S. & Pelham, H.R. Two syntaxin homologues in the TGN/endosomal system of yeast. EMBO J. 17, 113–126 (1998).

    Article  CAS  Google Scholar 

  17. Miceli, R.M., DeGraaf, M.E. & Fischer, H.D. Two-stage selection of sequences from a random phage display library delineates both core residues and permitted structural range within an epitope. J. Immunol. Methods 167, 279–287 (1994).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Protometrix, 688 East Main Street, Branford, 06405, Connecticut, USA

    Gregory A Michaud, Michael Salcius, Fang Zhou, Rhonda Bangham, Jaclyn Bonin, Hong Guo, Paul F Predki & Barry I Schweitzer

  2. Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, 06520-8103, Connecticut, USA

    Michael Snyder

Authors
  1. Gregory A Michaud
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael Salcius
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fang Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rhonda Bangham
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jaclyn Bonin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hong Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Michael Snyder
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Paul F Predki
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Barry I Schweitzer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Barry I Schweitzer.

Ethics declarations

Competing interests

The authors are employees of Protometrix.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Michaud, G., Salcius, M., Zhou, F. et al. Analyzing antibody specificity with whole proteome microarrays. Nat Biotechnol 21, 1509–1512 (2003). https://doi.org/10.1038/nbt910

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt910

  • Springer Nature America, Inc.

This article is cited by

Search

Navigation