The Development Of Immune Protein Chip Based On The Soybean Peroxidase

Protein chips are constructed by immobilizing different kinds of protein molecules on a solid surface, since each protein molecule are ordered array, each molecule is located at different positions. According to the location of different molecules we can detecte a large number of target protein. The present multianalyte immunoassays displayed some advantages, such as shorten analysis time, smaller sample consumption, more accurate diagnosis, lower cost per test and higher sensitivity. All these advantages provided the availability of this system to carry out large-scale screening of multiple serum markers in clinical diagnosis. The details are summarized as follows:(1) A novel chemiluminescent (CL) imaging microtiter plates for detection of four biomarkers related to Down’s syndrome screening was developed and evaluated. To enhance the sensitivity of CL immunosensing, soybean peroxidase (SBP) was used to instead of horseradish peroxide (HRP) as label enzyme. The microtiter plates were fabricated by simultaneously immobilizing of four capture monoclonal antibodies:anti-inhibin-A, anti-unconjugated oestriol (anti-uE3), anti-alpha-fetoprotein (anti-AFP) and beta anti-HCG (anti-β-HCG), on nitrocellulose membrane (NC) to form immunosensing microtiter wells, adding the target antigens, corresponding biotin-antibody and SBP-SA bioconjugates triggered the formation of sandwich immunocomplex, the CL reaction were triggered after adding chemiluminescent substrate solution, presenting an array-based chemiluminescence imaging assay method for high throughput detection of four kinds of target antigens.Under a sandwiched immunoassay, the CL signals on each sensing sites of the microtiter plates were collected by a charge-coupled device (CCD), presenting an array-based chemiluminescence imaging method for detection of four target antigens in a well at the same time.(2) On the basis of microtiter plates, enzyme-linked immunosorbent assay kits were fabricated by simultaneously immobilizing of free-PSA (f-PSA) and total-PSA (t-PSA) capture antibody on nitrocellulose (NC) membrane. And the two kinds of antigens (f-PSA and t-PSA) could be tested in per detection unit for 3 times at the same time.