Development Of A Multi-colloidal Gold Immunochromatographic Strip For Rapid Detection Of Escherichia Coli O157:H7and Shigella Boydii

This article was prepared by immunization of BALB/c mice to produce the monoclonalantibody of Escherichia coli O157:H7, and then use it to produce immune colloidal gold teststrip. Shigella boydii colloidal gold test strip also produced by the Shigella boydii monoclonalantibodies which have purchased; successfully developed a joint detection of Escherichia coliO157:H7and Shigella boydii colloidal gold test strip, and the three kinds of strip wasmeasured the sensitivity, specificity and analog carrier to verify the performance of the teststrip. The results were as follows:(1)Fusing the Myeloma cells SP2/0and Escherichia coli O157:H7immune mice gettingspleen, by screened three secreting anti Escherichia coli O157:H7monoclonal antibodyhybridoma cell lines D3, E7, B9. D3and B9the subclass was IgG1. the subclass of E7wasIgG2a,and light chain subtype were κ. Measured by SDS-PAGE D3, E7and B9heavy chainsare45kd, light chains were26kd,30kd and25kd;(2)By Escherichia coli O157:H7test strip performance testing optimal combination ofantibody screening: The test strip detects antibodies with D3, capture antibody with E7, whichthe detect antibodies conjugated colloidal gold optimum pH is7.5-8.0and the optimumbinding capacity is24μg/mLby a single factor experiment;(3) Escherichia coli O157:H7test strip sensitivity measurement experiments havedetermined the sensitivity is106CFU/mL. And Escherichia coli O157:H7cross-reactivityexperiments have determined the specificity of the test strip is good and no cross-reactivityinterference influence the results of determination. Escherichia coli O157:H7contaminatedstrip simulation experiments show that for commercial bread, milk, jelly each25g (mL) wereadded about200CFU EHEC O157: H7, after8h,10h’s enrichment cultured,it can be detected;(4)By a single factor experiment, the detect antibodies of Shigella boydii conjugatedcolloidal gold optimum pH is7.5and the optimum binding capacity is21.6μg/mL;(5)It’s level of sensitivity is106CFU/mL through Colloidal gold test strip’s sensitivitymeasurement test of Shigella boydii; Through the cross-reactivity experiments it hasSpecificity with no cross-reactivity which can interfere with the test strip results. Thesimulation experiment carrier showed that Shigella boydii can be detected after8,10,10hours when200CFU Shigella boydii was added to commercial bread, milk and jelly of25g/mL respectively;(6)The sensitibity level of Escherichia coli O157:H7and Shigella boydii is106CFU/mLby the fast joint detection experiment. Through the cross-reactivity experiments it has Specificity with no cross-reactivity which can interfere with the test strip results. TheSimulation experiment carrier showed that Escherichia coli O157:H7and Shigella boydii canbe detected after8,10,10hours when200CFU Escherichia coli O157:H7and Shigellaboydii were added to commercial bread, milk and jelly of25g/mL respectively;The Studies show that performance of antibodies is good, we have succeeded todeveloped Escherichia coli O157:H7test strip, Shigella boydii strip and the two bacterias’combined detection technology. The specificity and sensitivity of single-check test strip andmulti-check test strip are comparative to the overseas products. they can be widely used ingovernmental foodborne pathogens regulatory authorities and food companies.