Analyzing antibody specificity with whole proteome microarrays

Abstract

Although approximately 10,000 antibodies are available from commercial sources, antibody reagents are still unavailable for most proteins. Furthermore, new applications such as antibody arrays and monoclonal antibody therapeutics 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 approximately 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.

Label‐Free Impedance Biosensors: Opportunities and Challenges

“…Because electrical signals are readily steered, it is possible to detect various analytes using a single readout circuit. Regardless of readout mechanism, multiplexed protein detection is complicated by cross-reactivities -a probe binds to multiple targets or vice versa -which severely limits the possible degree of multiplexing and is especially troublesome in real-world situations [86][87][88]. However, a panel of several biomarker measurements has far more diagnostic power than a single biomarker can provide [89].…”

Evaluation of Lionex TB kits and mycobacterial antigens for IgG and IgA detection in cerebrospinal fluid from tuberculosis meningitis patients

“…There are several reports of cross-reactive antibodies that may recognize very similar epitopes from unrelated targets and the antigen-specific antibodies detected in some CSF-OIM may be related to cross reactivity with other infectious microorganisms rather than previous mycobacterial latent infection (Michaud et al 2003, Predki et al 2005). In the CSF-OIM of the present series, which were infected with S. pneumoniae and Cryptococcus, a mycobacterial antigen-specific reaction was observed mainly for IgA-16 kDa and the Lionex kit.…”

A pooling-deconvolution strategy for biological network elucidation

“…We tested PI-Deconvolution using yeast proteome microarrays 11,12 , which contain 4,088 purified Saccharomyces cerevisiae proteins (as glutathione S-transferase fusions) immobilized on nitrocellulose-coated glass slides. For this purpose, 15 (~16=2 4 ) V5 epitope-tagged bait proteins were employed (Fig.…”