An immunoassay method for quantitative detection of proteins using single antibodies

• Published in: Analytical biochemistry Vol. 400; no. 2; pp. 213 - 218
• Main Authors: Zhou, Shengliang, Lu, Xiaojuan, Chen, Caifa, Sun, Dongxu
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-05-2010
• Subjects: Antibodies - immunology; Antibodies - metabolism; Antigen labeling; Antigens - chemistry; Enzyme-Linked Immunosorbent Assay - methods; Interleukin-6 - analysis; Interleukin-6 - genetics; Protein Array Analysis; Protein immunoassay; Protein microarray; Recombinant Proteins - analysis; Recombinant Proteins - genetics; SALRA; Protein microarray; SALRA; Protein immunoassay; Antigen labeling
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2010.01.038

A new immunoassay method called specific analyte labeling and recapture assay (SALRA) to quantitatively measure protein abundance was developed, and the assay conditions were optimized. The key features of this method include labeling the antigen bound to the capture antibody, eluting the labeled antigen, and recapturing it by the same capture antibody on the detection plate. The reporter molecules on the labeled antigen provide a convenient and reliable means for signal detection. We demonstrated that the dose–response curve of SALRA was comparable to that of sandwich enzyme-linked immunosorbent assay (ELISA) and better than that of the antigen direct labeling method. In addition, multiple proteins can be measured simultaneously by SALRA. Using the SALRA method, the detection limit for most of the cytokines tested was approximately 0.01
ng/ml. Further SALRA tests on interleukin 6 (IL-6) showed the linear dose–response was 3.3 to 0.01
ng/ml, the accuracy of the test was 71 to 91%, the intraassay variation was 3.6 to 7.4%, and the interassay variation was 3.8 to 10.0%. The applications of SALRA include quantitatively measuring proteins for which there are no ELISA tools available and providing a new platform for protein microarrays.