Optimization Of Photonic Crystal Hydrogen Microbeads And Applications In Multiplex Platelet Specific Antibody Detection

First part: Optimization working conditions of photonic crystal hydrogelBackground: Multiple analysis technology has been widely used in the field of biological analyst, especially in the gene sequence analysis and Protein function analysis. Multiple analysis offers a high throughput, favourable sensitivity and specificity multiplexed analysis technology for large-scale screening. The technology had been applied multiplex detection for cancer markers in State Key Laboratory of Bioelectronics Southeast University. On this basis, we investigated a new multiplex analyst based on photonic crystal technology.Method: Taking human Ig G as the target protein to be inspected,in agarose gel we firstly fixed Goat antihuman Ig G antibody,after washing and closed,sequentially adding standard antigen and fluorescence labeled antibody,then incubated and washed,and used inverted fluorescence microscope to obtain images and detect fluorescence intensity. The best modified antibody agarose gel concentration, the optimum time, concentration of sodium hydroxide, epichlorohydrin concentration as well as the most suitable antibody concentration is determined according to the fluorescence intensity.Result: The fluorescence intensity could reach the maximum under the optimum condition: 4% agarose hydrogel, 3 hours of reaction time, 1.0mol/L Na OH, 10% epichlorohydrin and 0.01mg/ml antibody. The density of coating antibody could reach the maximum.Conclusion: The study optimizating of antibody modification reaction conditions based on the photonic crystal agarose gel improved the detection sensitivity of suspension array.Second part: Simultaneous detection of platelet-specific antibodies onphotonic crystal-encoded suspension arrayBackground: Platelet specific antibody is produced from the dysfunction of immune system which can cause a variety of clinical illness such as immune thrombocytopenia(ITP), post-transfusion purpura(PTP), refractoriness to platelet transfusion(RPT) and so on. Besides, appearance of platelet antibodies is also related with allergenic transplant rejection and respiratory distress after transfusion. Platelet antibodies involve associated antibody adsorbed on the surface of platelet and specific antibody which specified on platelet membrane glycoprotein. The detection of platelet specific antibodies is critical for the diagnosis of ITP.Method: Based on the optical characteristics of photonic crystal hydrogel microspheres and the principle of combination between antigen and antibody, a new method of multiplex analyst for platelet specific antibody is put forward. Monoclonal antibodies and purified platelet membrane glycoproteins were applied in multiplex detection respectively. Parallel Mo Ab immobilization of platelet antigen(MAIPA) was used as a reference test. The multiplex assay having comparable or better sensitivity and specificity compared to the MAIPA and more rapid is established.Result: The microspheres array coated monoclonal antibodies showed greater sensitivity than MAIPA in detecting anti-GPIIb/IIIa antibodies(P<0.05) and similar sensitivity as MAIPA in detecting anti-GPIb/IX antibodies(P>0.05). The MAIPA and multiplex analyst had similar specificity. The microspheres array coated platelet glycoprotein showed parallel sensitivity and specificity with MAIPA.Conclusion: The multiplex analyst is a rapid, sensitive and efficient method for detecting platelet-specific antibody and may have potential application in clinical practice.