| The aim of this study was to establish a dot immunogold filtration assay detecting hepatitis A. Reaction principle is that NCM coated with anti-human IgM can capture IgM in human serum by filtration, then HAV antigen and colloidal gold labeled with anti-HAV was added. Positive results were developed with red dot. The whole process was completed within few minutes without extra appliance and device.Although there were so many preparation methods of colloidal gold, proper method could be chosen according to granule diameter. In this trial, citromalic acid trisodium recovery method was employed to prepare 15nm colloidal gold. Transmission electron microscope was used to determine the quality of colloidal gold. It was found that the diameter of granule was 15.5?O.Snm without anomalous shape. Scan outcome of colloidal gold between 400 and 700nm with ultraviolet spectrophotometer was used to appraise the quality of colloidal gold. It was found that absorption hump was 519nm,and the width of hump was narrow. Trials of optimal protein protection and optimal labeling pH value showed that optimal protein protection amount was 12ug/ml colloidal gold and optimal labeling pH value 8.2.Further study was carried out to improve test quality and decrease reagent cost. Improvement of technology and reaction device, optimization of different reagents applying amount and consummating measures of quality control were involved.Reaction device was improved by diminution of diameter and increase indepth. The reaction device improved was convenient and the liquid reagents filtered through well. The technology was improved by simplification of assembling process of reaction board. Previous assembling process was preparing membrane, coating, washing, marking, blocking, washing, and assembling. Current assembling process was that preparing membrane, washing, and assembling. Blocking was completed while detecting serum sample. The assembling cycle was shorted from 24h to 30min. The previous reagents cost was about 2.10 yuan(Y). The cost of anti-human IgM used in this assay was 1.10 yuan(Y) which accounted for 51 percent of total cost. That of antigen was about 0.7 yuan(Y) and accounted for 33 percent. After improvement in device and technology, the applying amount of anti-human IgM was 1/20 of previous study, reagents cost was 0.07 yuan(Y); that of HAV antigen was 1/2 of previous study, about 0.35 yuan(Y). Immunogold probe had something to do with detecting quality while amount was "ODl,2drops" with clear dot and lower background. After optimization the reagents cost was 0.79 yuan(Y), which accounted for 40 percent of previous study, 66 percent of ELISA. Test quality was improved significantly. Compared with domestic ELISA kits, sensitivity was increased from 74.07% to 93.5%; specificity from 61.90% to 98%. Statistic test showed significant difference. Quality controlling measures included reagents titer and quality controlling dot in kits. It was carried out by combination of ELISA and this assay. It was found that both the antigen and immunogold probe was effective. Quality controlling dot in reaction hole made reaction results easier to read.Most domestic reports of DIGFA were compared with ELISA. ABBOTT kit was adopted as standard to evaluate DIGFA and ELISA in this study. Thesensitivity, specificity, positive predictive value, negative predictive value, accuracy of DIGFA were 93.48%, 96%, 95.6%, 94.12%, 94.79%, respectively and ELISA were 95.65%, 94%, 93.62%, 95.92%, 94.79%, respectively. Compared with ELISA, the same index of DIGFA was 91.49%, 95.92%, 95.56%, 92.16%, 93.75%, respectively. Statistic test showed no significant difference. It proved that this assay could be served as diagnostic and screening reagents. Rheumatoid factor trial and blocking trial were negative. The same blood received both hemolysis and serum segregation was determined with DIGFA, the detection results of two treatments were similar, which indicated that this assay could determine blood. Repeated determination to simil... |
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