The test specimens (blood type O healthy human serum and antibodies to blood types A and B) were then mixed with the oxidant dilutions and saline (control) at a 1?:?1 ratio, and the mixtures were incubated at 37C for 30?min. serum incubated at 37C for 30?min [9, 10]. Three immunological detection methods, including precipitation reactions, agglutination reactions, and enzyme immunoassays, were DIAPH2 employed to determine antigen-antibody-binding activity and to assess the effects of oxidative stress on total serum antioxidant capacity in the immune system. 2. Materials and Methods 2.1. Precipitation Reaction (Double-Diffusion Test) 2.1.1. Treatment of Test Specimens Oxidants (20?mM potassium permanganate solution, 20?mM iodine solution, and 50?mM hydrogen peroxide solution) were diluted at a 1?:?1 ratio using distilled water to obtain five concentrations. The test specimen (a rabbit polyclonal anti-human whole-serum antibody) was diluted with saline at a 1?:?1 ration, followed by mixing with the oxidant dilutions at 1?:?1 ratios. The mixed samples and saline (control) were incubated at 37C for 30?min. The pH of the test specimen was determined, and if the pH was outside of 7.2 to 7.5, new reagents were prepared. 2.1.2. Double-Diffusion Test Seven holes were inserted into the prepared agar plate and 20?= ?0.696+ 2.47, where represents the reaction intensity and represents the concentration. When = 1.15 and = 1.9, ID50 concentration of potassium permanganate is 1.9?mM. 2.2. Agglutination Reaction 2.2.1. Treatment of Test Specimens Here, 20?mM potassium permanganate solution and 20?mM iodine solution were diluted at a 1?:?3 ratio with distilled water in order A-366 to prepare five dilutions. Then, 50?mM hydrogen peroxide solution was similarly diluted at a 1?:?1 ratio to obtain five dilutions. The test specimens (blood type O healthy human serum and antibodies to blood types A and B) were then mixed with the oxidant dilutions and saline (control) at a 1?:?1 ratio, and the mixtures were incubated at 37C for 30?min. The pH of the test specimen was determined, and if the pH was outside of 7.2 to 7.5, new reagents were prepared. 2.2.2. Agglutination Reaction In 10 small test tubes, the treated samples were diluted 10-fold at 1?:?1 ratios, followed by addition of 0.1?mL 2% red blood cells (type A) to each tube. The tubes were shaken to make sure that the mixtures were homogeneous, and all the tubes were centrifuged at 2000?rpm for 2?min. 2.2.3. Interpretation of Results Agglutination (100%) was scored as 4+, 75% agglutination as 3+, 50% agglutination as 2+, and 25% agglutination as 1+. In the case of minimal agglutination, the background turbidity was scored as W+. If no agglutination and no hemolysis were observed, all cells were defined as free and scored as 0. To simplify calculations, serum-dilution factors (1?:?2, 1?:?4, 1?:?8, etc.) were transformed into a logarithmic scale (lg?2, 2?lg?2, 3?lg?2, etc.). The square of the oxidant concentration and the corresponding aggregation-response intensity was determined as a measure of the effects of oxidants on antibody activity in the agglutination reaction (Figure 1): represents the aggregation strength in a dilution. Changes in were arranged from the largest to the smallest; therefore, all of the values should be considered together. The area under the curve was a better indicator for A-366 evaluating the effects of oxidant concentration on antibody activity as compared to value. Open in a separate window Figure 1 Schematic diagram of the degree of aggregation calculated as the total area under the plotted line. For example, the area, = and are constants, represents the temperature in kelvin, and = 0.05 was used as the inspection level. 3. Results 3.1. Precipitation Reaction Fifteen parallel tests were performed, with the results indicating that potassium permanganate, iodine, and hydrogen peroxide all exhibited inhibitory effects on A-366 antibody activity in the precipitation.