Highly sensitive and specific solid-phase competitive assay utilizing a fusion protein

An in vitro solid-phase, competitive assay for detecting the presence of a peptide analyte in a biological sample, comprising

• contacting a fusion protein comprising a first peptide and a second peptide with a first antibody or fragment thereof which specifically binds to a site of the first peptide which is absent from the second peptide while the second peptide remains free, and allowing the formation of complexes of the first peptide and the first antibody or fragment thereof, the first antibody or fragment thereof being covalently bound to a poly(amino acid)-coated solid support at a site other than the first peptide binding site;
• adding thereto the biological sample suspected of comprising a peptide analyte, which binds specifically to a second antibody;
• adding thereto, in the presence of the biological sample, a second antibody or fragment thereof specifically binding to the analyte peptide and to a site of the second peptide that is absent from the first peptide, and allowing the formation of free complexes of the analyte and the second antibody or fragment thereof and solid supported complexes of the fusion protein and the second antibody or fragment thereof;
• and
• determining the amount of solid supported second antibody or fragment thereof present and comparing it to the amount of solid supported antibody or fragment thereof present in a control assay conducted by substituting an equivalent volume of buffer for the biological sample, whereby when the amount of peptide analyte in the sample increases the amount of solid supported second antibody decreases.