| ObjectiveThe E.coli O157:H7 polyclonal antibody Ig G was selected as the target strain for this experiment,and the most suitable concentration of the nanogold rod-labelled antibody was screened.This study will lay the experimental foundation for the sensitive and specific detection of E.coli O157:H7 and provide a scientific basis for further clinical detection of pathogenic microorganisms.Methods1.A whole bacteriological antigen of E.coli O157:H7 was prepared and immunised in rabbits to obtain antibodies against E.coli O157:H7.The antibody potency and specificity were measured by ELISA indirectly,the resulting antiserum was purified by affinity chromatography and the purification was identified by SDS-PAGE.2.The gold crystal seed growth method was used to polymerize gold nanorods and label different concentrations of mercapturized E.coli O157:H7 polyclonal antibody Ig G.The changes in the red peak shift of the longitudinal plasmon resonance absorption peak on the surface of the nanorods were observed by UV spectrophotometer scanning to screen the optimal concentration of the nanorods labeled antibody.3.Nanogold rod-labelled antibodies at different concentrations of E.coli O157:H7whole bacterium antigen,observation of the changes in the red peak shift of the longitudinal plasmon resonance absorption peak on the surface of the nanogold rods scanned by UV spectrophotometer,screening of the best antigen concentration for the detection of E.coli O157:H7 by nanogold rod-labelled method;nanogold rod-labelled antibodies at the best concentration for the detection of common pathogenic bacteria The specificity of the whole-bacteria antigen was verified by the concentration of the antigen.4.The results of the nano-gold stick labelling method for the detection of the whole bacterium antigen of E.coli O157:H7 were compared with the immuno-classical ELISA assay to explore the sensitivity of nano-gold stick labelling for the detection of E.coli O157:H7 polyclonal antibody Ig G at optimal concentrations.Results1.The whole bacteriological antigen of E.coli O157:H7 was successfully prepared and high potency,high purity E.coli O157:H7 polyclonal antibody Ig G was obtained.2.The red peak of the longitudinal plasma absorption peak on the surface of the nanogold rod labeled with E.coli O157:H7 polyclonal antibody Ig G antibody at a concentration of 50μg/ml was shifted to a maximum of 15.78 nm.The wavelength pattern and peak shape of the nanogold rod were normal at this time,and 50μg/ml was determined to be the optimum concentration for the labeled antibody.3.When the polyclonal antibody Ig G of Escherichia coli O157:H7 labeled with gold nanorods was used to detect the whole bacterial antigen of Escherichia coli O157:H7with a concentration of 10 cfu/ml,the red peak shift was 0.92 nm,and the detection result was negative;When detecting the whole bacterial antigen of Escherichia coli O157:H7with a concentration of 10~2～10~8 cfu/ml,there was an obvious red peak shift.The maximum red peak shift was 10.37 nm,and the minimum red peak shift was 2.54 nm.The detection result was positive,and the minimum detection limit was 10~2 cfu/ml.When the detection concentration is 10~3 cfu/ml,the red peak shift is maximum.When detecting antigen at this concentration,the antigen antibody binding is optimal,and a whole cell antigen of 10~3cfu/ml is determined as the optimal detection antigen concentration.When the whole bacterial antigen of Escherichia coli,Staphylococcus aureus,Listeria monocytogenes,Shigella baumannii and Salmonella enteritidis with 10~3 cfu/ml concentration detected by the polyclonal antibody Ig G of Escherichia coli O157:H7labeled with gold nanorods,the red peak shifts were 0.84 nm,-2.98 nm,0.91 nm,1.46nm and 1.59 nm,respectively.All detected red peak shifts were≤2 nm,and the detection result was negative.4.The minimum concentration of the whole antigen of E.coli O157:H7 by ELISA is 10~4 cfu/ml;the minimum concentration of colloidal gold for the detection of E.coli O157:H7 is 1.14x10~3 cfu/ml,which is significantly more sensitive than the nano-gold rod labeling method for the detection of E.coli O157:H7.ELISA and colloidal gold assayConclusionsSuccessfully prepared nano gold rods with stable aspect ratio.A highly sensitive functionalized nanogold rod with an optimal concentration of 50μg/ml was selected for labeling polyclonal antibody Ig G with nanogold rods.To construct a gold nanorod labeled polyclonal antibody Ig G against Escherichia coli O157:H7 to detect the whole bacterial antigen of Escherichia coli O157:H7.Compared with colloidal gold detection method,ELISA method has higher sensitivity,simple operation,short detection time and low cost,providing new technical support for the sensitivity detection of E.coli O157:H7 infection. |
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