A simple, rapid one-step ELISA using antibody-antibody complex

The enzyme-linked immunosorbent assay (ELISA) is one of the most frequently employed assays for clinical diagnostic testing and biological research. However, its time-consuming operation is a major drawback. The present work aims to establish a one-step ELISA method through the preparation of a primary antibody (Ab)-secondary Ab complex (Ab-Ab complex) in hopes of realizing more sensitive and faster detection of the trace amount of antigen (Ag). By controlling the mole ratio of the primary Ab to the secondary Ab, one-step ELISA can be successfully achieved. Compared with the traditional ELISA, the one-step ELISA could not only improve the detection sensitivity to 1 ng mL(-1) , but also reduce the operating time by 30%. Moreover, the signal intensity can be controlled by adjusting the ratio of the secondary Ab in the complex or by changing the color development time. This technique is further optimized to detect trace amounts of proteins adsorbed onto poly(N-vinylpyrrolidone) (PVP)-modified silicon surfaces (Si-PVP), and the results are close to the radiolabeling method. It is concluded that the simple one-step ELISA can be used for the rapid detection of trace amount of protein. The method holds promise for the clinical detection of trace Ag in solution and on low-adsorption material surfaces.