Study On The System Of Chemiluminescent Magnetic Enzyme-Linked Immunoassay

Chemiluminescent magnetic enzyme-Linked immunoassay is a new analytical technique, which integrates chemiluminescence with magnetic separation technique and immunoassay. Advantages of this assay include high sensitivity, fast detection speed, excellent specificity and easy automation.We have used this assay to detect Escherichia coli O157:H7 in food and freeβ-hCG in serum. The experimental parameters were optimized and the results were compared with other analytical assays. The experimental contents are as follows:一,Preparation of immunomagnetic microbeadsAmino-modified magnetic immunobeads were activiated by glutaraldehyde to form the carboxyl groups on the magnetic immunobeads'surfaces. These groups can conjugate with selected antibodies'amino groups to produce immunomagnetic microbeads which were endowed with the capacity to capture and detect target antigens.二,Detection of Escherichia coli O157:H7 in food samples based on chemiluminescent magnetic enzyme-linked immunoassayWe have used the established method to detect Escherichia coli O157:H7 in pork samples. Alkaline phosphatas(eALP) is utilized as a labeled reagent of Escherichia coli O157:H7 antibody, which can react with a chemiluminescent reagent, 3(2′spiroadamantane) 4 - methoxy - 4(3′′phosphoryloxy) phenyl - 1, 2 dioxetane(AMPPD) in buffer solution and emit photons. The limit of detection was 8.5×10~4 /mL. The linear detection range was 1.0×10~5～5.0×10~7 /mL. The intra- and extra- assay CVs were 14.8% and 20.0%, respectively and the correlation efficiency between this method and traditional numerical method was 0.981. This assay can be theoretically and practically applied to detect the concentration of Escherichia coli O157:H7 in variety of samples.三,Detection of freeβ-hCG in clinical serum based on chemilunescent magnetic enzyme-linked immunoassayWe have established a new chemilunescent magnetic enzyme-linked immunoassay to sensitively and specifically detect freeβ-hCG in clinical serum. The measurement was based on a sandwich enzymed-linked immuno-reaction separated by magnetic micro-sphere in the aid of a planar magnetic field. The enzyme, alkaline phosphatase(ALP)is utilized as a labeled reagent of the anti-β-hCG antibody, which react with an excellent chemiluminescent reagent, AMPPD in buffer solution. The emitted photons were detected and were directly proportional to the concentration of the freeβ-hCG in serum. The sensitivity of the assay was 10 times higher than that of the spectrophotometry. The linear detection range was 0.45~185.2 mIU/mL. The intra- and extra- assay CVs were both below 12%. The recovery percentage was in the range of 84%~120% when the method was applied to detect the concentration of freeβ-hCG in serum samples. The correlation efficiency between this method and the spectrophotometry immunoassay was 0.955. We conclude that chemilunescent magnetic enzyme-linked immunoassay is more accurate and precise than spectrophotometry immunoassay. Thus it can be practically applied to detect the concentration of freeβ-hCG in the clinical serum.