Immobilization of antigen

M.S. Sanford, P.T. Charles, S.M. Commisso, J.C. Roberts and D.W. Conrad, Photoactivatable cross-linked polyacrylamide for the site-selective immobilization of antigens and antibodies, Chem. Mater., 10 (1998) 1510—1520. 

In 2003, Dai et al. [80] reported a strategy for a reagentless immunosensor for the determination of carcinoma antigen-125 (CA-125) based on the immobilization of antigen and the direct electrochemistry of HRP bound to an antibody. The immunosensor was prepared by immobilizing CA-125 with titania sol-gel on a GCE by the vapour deposition method. The incubation of the immunosensor in phosphate buffer solution containing HRP-labelled CA 125 antibody leads to the formation of an HRP-modified surface. The immobilized HRP displays its direct electrochemistry with a rate constant of 3.04 1.21 s . With a competition mechanism, a differential pulse voltam-metric determination method for CA-125 was established by the peak current decrease of the immobilized HRP. The current decrease resulted from the... 

The nonspecific adsorptimi or nonspecific binding always occurs in the microchip due to the multiple biological components of the whole blood environment. So far, blocking buffers have been used to prevent the problem. However, it will also impact some specific bindings and decreases the detection limit. Another problem is unstable immobilization of antigen or antibody. Improved surface chemistry and new materials are needed to solve these problems [17]. 

Immobilization of antigen through a diazotization-coupling reaction Competitive assay... 

Immunoaffinity chromatography utilizes the high specificity of antigen—antibody interactions to achieve a separation. The procedure typically involves the binding, to a soHd phase, of a mouse monoclonal antibody which reacts either directly with the protein to be purified or with a closely associated protein which itself binds the product protein. The former approach has been appHed in the preparation of Factor VIII (43) and Factor IX (61) concentrates. The latter method has been used in the preparation of Factor VIII (42) by immobilization of a monoclonal antibody to von WiHebrand factor [109319-16-6] (62), a protein to which Factor VIII binds noncovalenfly. Further purification is necessary downstream of the immunoaffinity step to remove... 

Competitive immunoassays may also be used to determine small chemical substances [10, 11]. An electrochemical immunosensor based on a competitive immunoassay for the small molecule estradiol has recently been reported [11]. A schematic diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG was physisorbed onto the surface of an SPCE. This was used to bind monoclonal mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a standard solution of estradiol (E2), followed by a solution of AP-labeled estradiol (AP-E2). The E2 and AP-E2 competed for a limited number of antigen binding sites of the immobilized anti-estradiol antibody. Quantitative analysis was based on differential pulse voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the enzyme substrate 1-naphthyl phosphate by AP-E2. The analytical range of this sensor was between 25 and 500pg ml. 1 of E2. 

---