| Objective:To define a diagnostic approach for the identification with the dot immunoenzyme filtration assay for the antigens of N. meningitides Group-A, HSV-1, CMV and C. neoformans, which could be given experimental evidences to detect acute encephalitis/meningitis pathogens with the protein chip arrays.Methods:1 To determine the known N. meningitides Group-A with the dot immunoenzyme filtration assay through the following processes: Preparation of the antigen,and the antigen, antibody and universal polymerized HRP-Anti Ms/Rb IgG were diluted by half sequential dilution. Following that, the reaction concentration of corresponding antigen and antibody at each test points analyzed with special statistic software. determined with the matrix titration method. Then the optical density (OD) of each positive reaction was measured and analyzed with special statistic software. The optimum reaction combination of the titers and the response equation could be given by the statistical analysis.2 To determine the known HSV-1antigen, with the dot immunoenzyme filtration assay through the following processes: the antigen, antibody and universal polymerized HRP-Anti Ms/Rb IgG were diluted by half sequential dilution. Following that,the reaction concentration of corresponding antigen and antibody at each test points was determined with the matrix titration method. Then the optical density (OD) of each positive reaction is measured and analyzed with special statistic software. The optimum reaction combination of the titers and the response equation could be given by the statistical analysis.3 To determine the known CMV antigen, with the dot immunoenzyme filtration assay through the following processes: the antigen, antibody and universal polymerized HRP-Anti Ms/Rb IgG were diluted by half sequential dilution. Following that, the reaction concentration of corresponding antigen and antibody at the individual test points was determined with the matrix titration method. Then the optical density (OD) of each positive reaction was measured and analyzed with special statistic software. The optimum reaction combination of the titers and the response equation can be given by the statistical analysis.4 To determine the known C. neoformans antigen by the dot immunoenzyme filtration assay through the following processes: Preparation of the antigen. the antigen, antibody and universal polymerized HRP-Anti Ms/Rb IgG were diluted by half sequential dilution. Following that, the reaction concentration of corresponding antigen and antibody at each test points was determined with the matrix titration method. Then the optical density (OD) of each positive reaction was measured and analyzed with special statistic software. The optimum reaction combination of the titers and the response equation could be given by the statistical analysis.Result:1 The selection of best reaction conditions in testing N. meningitides Group-A antigen by dot immunoenzyme filtration assay was achieved by square titration. The best combined titers of antigen and anti-N. meningitides Group-A PcAb were as follows: anti-N. meningitides Group-A PcAb 0.1250μg/ ml (1:800) and universal polymerized HRP-Anti Ms/Rb IgG was concentration 1:10, which was the concentration of this experiment. N. meningitides Group A antigen was diluted further, and established the regression equation that Y=0.4564X+0.0728, The correlation coefficient (r) was 0.9212 (P< 0.05). The lowest antigen concentration was 33.7 ng/ml, and the contrast was negative, without showing any nonspecific reaction.The best combined titers of antigen and anti-N.menin- gitides Group-A McAb were as follows: anti-N meningitides Group-A McAb 0.3125μg/ml (1:320) and universal polymerized HRP-Anti Ms/Rb IgG are concentration 1:10, which was the concentration of this experiment. The antigen was diluted further, and established the regression equation that Y=0.4828X+0.0689. The correlation coefficient (r) was 0.9070(P<0.05). The lowest antigen concentration was 16.8ng/ml, and the contrast was negative, without showing any nonspecific reaction.2 The selection of best reaction conditions in testing HSV-1 antigen by dot immunoenzyme filtration assay was achieved by square titration. The best combined titers of antigen and anti-HSV-1 PcAb were as follows: anti-HSV-1 PcAb 8.4μg/ ml (1:100) and universal polymerized HRP-Anti Ms/Rb IgG were concentration 1:10, which was the concentration of this experiment. HSV-1 antigen was diluted further, and established the regression equation: Y=0.0984X+0.1310. The correlation coefficient (r) was 0.8884 (P<0.05). The lowest antigen concentration was 66.8 ng/ml, and the contrast was negative, without showing any nonspecific reaction.3 The selection of best reaction conditions in testing CMV antigen by dot immunoenzyme filtration assay was achieved by square titration. The best combined titers of antigen and anti-CMV PcAb were as follows: anti-CMV PcAb 12.5μg/m (1:160) and universal polymerized HRP-Anti Ms/Rb IgG were concentration 1:10, which is the concentration of this experiment. The antigen was diluted further, and established the regression equation that Y=0.0440X+0.0576. The correlation coefficient (r) was 0.9297 (P<0.05). The lowest antigen concentration was 55.7 ng/ml, and the contrast was negative, without showing any nonspecific reaction.4 The selection of best reaction conditions in testing C. neoformans antigen by dot immunoenzyme filtration assay was achieved by square titration. The best combined titers of antigen and anti-C. neoformans McAb were as follows: anti-C. neoformans McAb 2.5μg/ml (1:80) and universal polymerized HRP-Anti Ms/Rb IgG are concentration 1:10, which was the concentration of this experiment. The antigen was diluted further, and established the regression equation that Y=0.3528X+0.0693, The correlation coefficient (r) was 0.8889. (P<0.05). The lowest antigen concentration was 8.7 ng/ml, and the contrast was negative, without showing any nonspecific reaction.Conclusions:A method for the identification of the antigens of N. meningitides Group-A, HSV-1, CMV and C. neoformans had been preliminarily established. By using this method, the minimum detectable quantities of N. meningitides Group A antigen were 33.7 ng/ml and 16.8 ng/ml, detected by mouse anti-N. meningitides PcAb and McAb respectively. The minimum detectable quantities of the antigens of HSV-1, CMV, and C. neoformans were 66.8 ng/ml, 55.7 ng/ml, 8.7 ng/ml respectively. The method was high in sensitivity and specificity, and easy to use. Furthermore, the results obtained could be stored in the room temperature. It was proves to be a method that was suitable for extensive clinic applications and adapted to the national condition.
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