Enhancing sensitivity in colorimetric immunoassay by using secondary antibody-gold nanoparticle conjugate

A lateral flow colorimetric immunoassay (LFA) based on gold nanoparticles (AuNP) for detecting the antibody-antigen interaction and gold enhancement for amplifying the specific binding signal has been developed. Influenza A was used as the model study to determine the performance of the system. Nucleoprotein (NP) of the influaza A virus was selected as the targeting molecule. Two different sizes of AuNP conjugated to antibody against NP were prepared and applied to the assay. The 1st conjugate was AuNP immobilized with an anti-NP antibody and secondary-immobilized with biotinylated bovine serum albumin (B-BSA). The 2nd conjugate was AuNP immobilized with an anti-biotin antibody. The detection sensitivity of the enhanced sensitivity LFA increased about 3-folds and 8-folds compared with conventional LFA and commercial influenza A LFA, respectively. The signal intensity of the test spot/line at the capture test zone was increased dramatically. With this method, nucleoprotein of influenza A virus can be detected within 15 minutes and was highly reproducible. The results indicated that the technique can facilitate the more sensitive LFA test for diagnosis of many diseases.