Competitive assay formats for high-throughput affinity arrays.
The authors describe a novel method for the quantitation of differential levels of biomolecules using unlabeled samples and protein-binding arrays for assessing differential expression. Traditional affinity arrays, whether in microplates or protein microarrays, suffer from a few common problems-a shortage of characterized antibodies and highly variable affinities for those available. Also, the assayed proteins could be present in a wide range of concentrations and physicochemical properties, so that it becomes an onerous task to optimize assay conditions for each antibody-antigen pair. Currently, this restricts parallel affinity assays to a low number of carefully selected antibodies and restricts the development of highly multiplexed parallel affinity assays. A displacement strategy allows the use of a much wider range of antibodies, reducing the requirement for matched affinities. The competitive assays described here also show a much higher tolerance for nonspecific background noise. The range of assayed protein concentrations is only limited by the sensitivity of the detection system used.
Barry
Richard
R
Oxford GlycoSciences (UK) Ltd., Abingdon, United Kingdom.
Diggle
Tricia
T
Terrett
Jonathan
J
Soloviev
Mikhail
M
D000915:Antibody Affinity; D000918:Antibody Specificity; D001667:Binding, Competitive; D005260:Female; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D007118:Immunoassay / Q000379:methods*; D007668:Kidney / Q000378:metabolism; D008403:Mass Screening; D020411:Oligonucleotide Array Sequence Analysis; D010053:Ovary / Q000378:metabolism; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D020136:Hydrogel, Polyethylene Glycol Dimethacrylate