3.2.7.2.4.3 Serum superoxide dismutase (SOD)

The ELx800 absorbance micro plate reader from BioTek (USA) instruments was used to measure the absorbance of SOD in serum. The solar bio human superoxide dismutase assay kit, 96-Well plate, Cat. No. (EHu 00036), was used to measure SOD level in human serum.

Principle of the assay

Superoxide anions (O2) are generated from the conversion of xanthine to uric acid and hydrogen peroxide by xanthine oxidase (XOD), converts NBT to NBT-diformazan. NBT-diformazan (Chromagen Solution) absorbs light at 550nm.SODs reduce superoxide ion concentrations and thereby lower the rate of NBT-diformazan formation. The extent of reduction in the appearance of NBT diforma-zan is a measure of SOD activity present in your experimental sample (Robak and Gryglewski, 1988).

Procedures of assay

Procedures of SOD assay included the following steps:

1. 30 μl of sample dilution was added to the testing sample well, and then 20 μl testing sample was added.

2. After plate had been closed with plate closure membrane, it was incubate of 30 min at 37℃.

3. Wells were washed three times with washing solution using micro plate strip washer, and finally were dried by pat.

4. 50 μl streptavidin (horseradish peroxidase) HRP-Conjugate reagent were added per each well, except blank well.

5. The plate was then incubated for 30 min at 37℃, while shaking.

6. Wells were then washed with washing buffer using micro plate strip washer, and finally were dried by pat.

7. 50µl of Chromogen solution A and 50µl of Chromogen solution B were added to each well.

8. The plate was incubated for 15min at 37C°.

9. Then 50µl stop solution (Sulphuric acid) was added to each well, which stopped the reaction (the blue color change to yellow color). The absorbance was read at 450 nm after stop solution added within 15min, using micro plate reader.

3.2.7.2.4.4 Serum catalase

The NWLSS™ Catalase activity assay can be measured by monitoring the consumption of H2O2 substrate at 240 nm.

The procedure of CAT assay (briefly)

Catalase activity assay kit (Northwest Life Science Specialties, LLC-CAT01), was used for determining CAT activity colorimetrically according to the method of (Aebi, 1984) as the following:

1. 10 µL of sample or standard was added to the bottom of a micro-centrifuge tube or deep well microplate.

2. 190 µL of Working Assay Cocktail was pipetted into each tube or Microplate well. By capping/inverting or by repeated pipetting was quickly mixed.

3. The solution was incubated for exactly 2 minutes.

4. 200 µL of Stop Solution was added in the same manner and order used in Step 2. Microplate Reader is used to measure the absorbance at 650 nm for CAT in liver and brain homogenate.