Study On The Combined Method For The Pathogenic Antigen Assay In Acute Infectious Central Nervous System-related Diseases

Purpose:To define a triple diagnostic approach for the identificationwith dot immunobinding assay method (dot-Iba) of the antigensof Mycobacterium tuberculosis, Neisseria meningitidis A-groupand Streptococcus pneumoniae in acute bacterial meningitis.Method:1.To determine the known Mycobacterium tuberculosisantigen (BCG) with dot immunobinding assay method throughthe following processes: Collection of the top-layer liquid of theexperimental BCG vaccine after ultrasonic splitting andcentrifugation (20,000 rpm) under low temperature to allow it tohave a quantitative protein concentration of 1.34mg/ml. Thesolution is then diluted by using the doubling sequential dilutionmethod to varying concentrations ranging from 1:800-1:204800;rabbit anti-Mycobacterium tuberculosis polyclonal antibody(4mg/ml) is diluted to a concentration of 1:200-1:6400 while theuniversal polymerized HRP-Anti Ms/Rb IgG is diluted to aconcentration of 1:10-1:40. Following that, the reactionconcentration of corresponding antigen and antibody at theindividual test points is determined with matrix titration method.The same determination work is made at each test pointrepeatedly for 6 times. Through gross inspection, all those informs of brown spots or in circles can be identified as positive,and those that cannot be identified are regarded as negative. Andeach test is made with negative contrast. The optical density(OD) of each positive reaction point is measured with the MoticMed 6.0 digital medical image analyzing system (A) fordetermining the mean OD at each point. Through data analysis,selection is made of the optimum reaction combination of thetiters of the rabbit anti-Mycobacterium tuberculosis PPDpolyclonal antibody and the universal polymerized HRP-AntiMs/Rb IgG. After the determination of the titers of the two kindsof antibodies, the antigens at different solution concentrationsare measured. Based on the data obtained and the analysis of thevariation of OD in relation to the content of antigen, a standardcurve can be plotted for deriving the regression equation andcorrelation coefficient.2.To determine the known Neisseria meningitidis A-groupantigen with dot immunobinding assay method through thefollowing processes: Collection of the top-layer liquid of themeningococcus (A group) after ultrasonic splitting andcentrifugation (20,000 rpm) under low temperature to allow it tohave a quantitative protein concentration of 0.23mg/ml. Thesolution is then diluted by using the doubling sequential dilutionmethod to varying concentrations ranging from 1:25-1:6400;mouse anti-Neisseria meningitidis A-group monoclonalantibody (0.1mg/ml) is diluted to a concentration of 1:20-1:640while the universal polymerized HRP-Anti Ms/Rb IgG is dilutedto a concentration of 1:10-1:40. Following that, the reactionconcentration of corresponding antigen and antibody at theindividual test points is determined with matrix titration method.The same determination work is made at each test pointrepeatedly for 6 times. Through gross inspection, all those informs of brown spots or in circles can be identified as positive,and those that cannot be identified are regarded as negative. Andeach test is made with negative contrast. As for the reading ofdata, determination of the titer of antibody reactions and theplotting of the standard curve, refer to the previous section.3.To determine the known Streptococcus pneumoniaeantigen with dot immunobinding assay method through thefollowing processes: Collection of the top-layer liquid of thepneumococcus after ultrasonic splitting and centrifugation(20,000 rpm) under low temperature to allow it to have aquantitative protein concentration of 1.37mg/ml. The solution isthen diluted by using the doubling sequential dilution method tovarying concentrations ranging from 1:400-1:102400; the mouseanti-Streptococcus pneumoniae monoclonal antibody (0.5mg/ml)is diluted to a concentration of 1:100-1:3200 while the universalpolymerized HRP-Anti Ms/Rb IgG is diluted to a concentrationof 1:10-1:40. Following this, the same processes as those asdescribed in the previous sections are taken.4.The antigens of Mycobacterium tuberculosis (BCG),Neisseria meningitidis A-group and Streptococcus pneumoniaeare respectively spotted on the different regions of anitro-fibrous film together with negative contrast. Theabovementioned three kinds of antigens are then determinedunder different concentrations by using the triplex identificationmethod.Results:1.For the determination of the Mycobacterium tuberculosis(BCG) antigen, the optimal reaction combinations of theconcentrations of rabbit anti-Mycobacterium tuberculosis PPDpolyclonal antibody and the universal polymerized HRP-AntiMs/Rb IgG are 1:200 (20μg/ml) and 1:10 respectively. Throughfurther dilution of the antigens, a regression equation and thecorrelation coefficient can be derived﹙Y=0.2663X-0.0252,r=0.9823, P<0.05﹚. The contrast serums are all found to benegative, without showing any nonspecific reaction, and theminimum quantity of antigen detected is 3.65ng/ml.2.For the determination of the Neisseria meningitidisA-group antigen, the optimal reaction combinations of theconcentration of the mouse anti-Neisseria meningitidis A-groupmonoclonal antibody and the universal polymerized HRP-AntiMs/Rb IgG are 1:160 (0.625μg/ml) and 1:10 respectively.Through further dilution of the antigens, a regression equationand the correlation coefficient can be derived (Y=0.0532X-0.0106, r=0.9349, P<0.05). The contrast serums are all found tobe negative, without showing any nonspecific reaction, and theminimum quantity of antigen detected is 36ng/ml.3.For the determination of the streptococcus pneumoniaeantigen, the optimal reaction combinations of the concentrationof the mouse anti-Streptococcus pneumoniae monoclonalantibody and the universal polymerized HRP-Anti Ms/Rb IgGare 1:800 (0.625μg/ml) and 1:10 respectively. Through furtherdilution of the antigens, a regression equation and thecorrelation coefficient can be derived (Y=0.1207X-0.0232,r=0.9692, P<0.05). The contrast serums are all found to benegative, without showing any nonspecific reaction, and theminimum quantity of antigen detected is 13.4ng/ml.4.With the use of the combined measuring method, theMycobacterium tuberculosis (BCG) antigen (1:400), Neisseriameningitidis A-group antigen (1:25) and Streptococcuspneumoniae antigen (1:800) are spotted on the different regionsof a nitro-fibrous film and then an analysis is made with rabbitanti-Mycobacterium tuberculosis PPD polyclonal antibody(1:200) and universal polymerized HRP-Anti Ms/Rb IgG (1:10).The test result shows no cross reaction, and the respectivenegative contrast serum shows no sign of nonspecific reaction.The same cases are true when the test is made with mouseanti-Neisseria meningitidis A-group monoclonal antibody(1:200) and universal polymerized HRP-Anti Ms/Rb IgG (1:10)as well as the mouse anti-Streptococcus pneumoniaemonoclonal antibody (1:800) and universal polymerized...