3.4.9.1. Principle
Principle guiding indirect ELISA was employed. A specific double stranded DNA (dsDNA) sequence containing the NF-kB response element immobilized onto the bottom of wells of a 96 well plate. NF-kB contained in a nuclear extract specifically binds to the NF-kB response element. NF-kB (p65) is then detected by addition of a specific primary antibody (detecting antibody) directed against NF-kB (p65). A secondary antibody conjugated to emzyme (Horse Radishperoxidase (HRP) to bind the detecting antibody. On addition of the chromogenic substrate (peroxide) the enzyme converts the substrate to a detectable form inducing a colour change. The intensity of the colour is directly propotional to the quantity of NF-kB present in the nuclear extract.
3.4.9.2. Test Procedure
Indirect ELISA method was employed. The procedure is as described by the manufacturer (Rockland Immunochemicals (Gilbertsville, Pennsylvania, USA). The plates and buffers were equilibrated to room temperature prior to opening. One hundred microliter (100µL) complete Transcription Factor Binding Assay Buffer (CTFB) was added to the Blank wells (Blk); 100 µL of CTFB was added to Non-specific Binding wells (NSB). Eighty microliter (80µL) of CTFB was added to Competitor wells (CI) prior to adding 10µL of competitor double stranded DNA (dsDNA) to the wells. Ninety microliter (90µL) of CTFB was added to sample wells (U1U44) -prior to adding 10µL of nuclear extract to the wells. Ninety microliter (90µL) of CTFB was added to positive Control wells prior to adding 10µL of positive control to appropriate wells.
The plate was covered with a seal and incubated for 1 hour at room temperature without agitation. The plates were emptied and washed 5 times with 200µL of 1X wash buffer. After each wash, the plate was emptied in the sink. After the final wash (i.e. 5th wash), the plate was tapped on a paper towel to remove any residual wash buffer.
Addition of Anti-NF-kB (p65) Primary Antibody: Anti-NF-kB (p65) antibody was diluted 1:100 in 1X antibody binding buffer (ABB). Total volume of 100µL of diluted Anti-NF-kB (p65) antibody was added to each well except the Blank (Blk) wells. The adhesive cover was used to seal the plate. The plate was incubated for 1 h at room temperature without agitation. The plate was emptied and the wells washed 5 times with 200µL of 1X wash buffer. After each wash, the
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contents of the plate were emptied into the sink. After the final wash (i.e. 5th wash), the plate was tapped 5 times on a paper towel to remove any residual wash buffer.
Addition of the HRP Goat anti-Rabbit conjugated Secondary Antibody: The HRP-conjugated secondary antibody was diluted 1:100 in 1X Antibody binding buffer (ABB) and 100 µL antibody was added to each well except the Blank (Blk) wells. The adhesive cover was used to seal the plate, incubated for 1 hour at room temperature without agitation. The plate was emptied and washed 5 times with 200µL of 1X wash buffer. After each wash, the contents of the plate were empty into the sink. After the final wash, the plate was tapped 5 times on a paper towel to remove any residual wash buffer. The plate was developed by adding to each well 100µL of developing solution which has been equilibrated to room temperature. The plate was incubated for 45 minutes at room temperature with gentle agitation protected from light. The plate was allowed to turn medium to dark blue prior to adding stop solution. Absorbance was read at 450nm within 5 minutes of adding the stop solution. The plate reader was blanked according to the manufacturer‘s requirements using the blank wells.
3.4.10. 8-hydroxy-2-deoxy Guanosine (8-OH-dG) Assay (StressMarq Biosciences Inc. USA) 3.4.10.1 Principle
This assay is based on the competition between hydroxy-2-deoxy guanosine (OH-dG) and OH-dG-acetylcholinesterase (AChE) conjugate (OH-dG Tracer) for a limited amount of 8-OH-dG Monoclonal Antibody. Because the concentration of the 8-8-OH-dG Tracer is held constant while the concentration of 8-OH-dG varies, the amount of 8-OH-dG Tracer that is able to bind to the 8-OH-dG Monoclonal Antibody will be inversely proportional to the concentration of 8-OH-dG in the well. This antibody-8-OH-dG complex binds to goat polyclonal anti-mouse IgG that has been previously attached to the well. Ellman‘s Reagent (which contains the substrate to 8-OH-dG-acetylcholinesterase AChE) is added to the well. The product of this enzymatic reaction has a distinct yellow color and absorbs strongly at 412 nm. The intensity of this color, determined spectrophotometrically, is proportional to the amount of 8-OH-dG Tracer bound to the well, which is inversely proportional to the amount of free 8-OH-dG present in the well during the incubation.
129 3.4.10.2. Preparation of the standard for use in EIA
One hundred microliter (100μl) of the 8-OH-dG standard (Catalog# SKC-120C) was transferred into a clean test tube, and diluted with 900μl distilled water. The concentration of this solution (the bulk standard) will be 30ng/ml. This was stored at 4°C. Clean test tubes were numbered 1 - 8. Then 900μl EIA Buffer was aliquoted to tube 1 and 500μl EIA Buffer to tubes 2-8. 100μl of the bulk standard (30ng/ml) was transferred to tube 1 and mixed thoroughly. The standard was serially diluted by removing 400μl from tube 1 and placing in tube 2; this was mixed thoroughly, then 400μl was removed from tube 2 and place it into tube 3 and mixed thoroughly. This process was repeated till tube 8.
3.4.10.3. Test Procedure
The procedure is as described by the manufacturer StressMarq Biosciences Inc. USA. One hundred microliter (100μl) Enzyme Immunoassay (EIA) Buffer was added to Non-Specific Binding (NSB) wells, while 50μl EIA Buffer was added to Maximum Binding wells. Upto 50μl of 8-hydroxy-2-deoxy Guanosine Standard from tube 8 was added to both of the lowest standard wells (S8), while 50μl from tube 7 was added to each of the next two standard wells (S7). This was continued until all the standards were aliquoted. Then 50μl of sample was added per well.
Each dilution was assayed in duplicate. Fifty microliter (50μl) of 8-hydroxy-2-deoxy Guanosine AChE Tracer was added to each well except the Total Activity (TA) and the Blank (Blk) wells, then 50μl 8-hydroxy-2-deoxy Guanosine Monoclonal Antibody was added to each well as well, except in the Total Activity (TA), the Non-Specific Binding (NSB), and the Blank (Blk) wells.
The plate was covered with plastic film and incubated for 18 hours at 4°C. Ellman‘s Reagent was reconstituted immediately before use (20 ml of reagent is sufficient to develop 100 wells) distilled water. The well was emptied and rinsed five times with Wash Buffer. Two hundred microliter (200μl) of Ellman‘s Reagent was added to each well and 5μl of tracer to the Total Activity wells. The plate was covered with plastic film. The plate was developed with a flat cover in the dark. The bottom of the plate was wiped with a clean tissue to remove fingerprints and dirt. The plate cover was removed being careful to keep Ellman‘s Reagent from splashing on the cover. Any loss of Ellman‘s Reagent will affect the absorbance readings. The plate was read at 420nm wavelength and the concentration of each sample was determined using the standard curve plot.
130 3.4.11. TNF-alpha Assay (ABCAM USA)
3.4.11.1 Principle
The principle of sandwich ELISA was applied. Plate coated with a capture antibody can capture native antigen (TNF-α) in the test sample if any in other to immobilize the antigen on the walls of the plate. Subsequently, detecting antibody directed against (TNF-α) can specifically bind to the immobilized antigen. The detecting antibody is probed with enzyme-linked secondary antibody (horseradish peroxidase conjugated antibody). On addition of the chromogenic substrate (3,3‘,5,5‘-tetramethylbenzidine (TMB) the enzyme converts to the substrate to a detectable form inducing a colour change. The intensity of the colour is directly propotional to the quantity of (TNF-α) present in the serum sample.
3.4.11.2. Preparation of TNF-alpha Standard
Tumour Necrosis Factor-alpha TNF-alpha standard sample was reconstitute by adding 100μL of standared diluents buffer to stock vial to get 800pg/ml stock Standard 1 using pipette. This was mix thoroughly and gently and left at room temperature for 10 minutes. Other tubes were labeled from 2 to 8. 100μL of diluents buffer was added into each tube. Standard 2 was prepared by adding 100μL of Standard 1 to tube 2 and mix thoroughly. Standard 3 was prepared by adding 100μL of Standard 2 to tube 3 and mix thoroughly. Standard 4 was prepared by adding 100μL of Standard 3 to tube 4 and mix thoroughly. Standard 5 was prepared by adding 100μL of Standard 4 to tube 5 and mix thoroughly. Standard 6 was prepared by adding 100μL of Standard 5 to tube 6 and mix thoroughly to get concentrations of 800pg/ml, 400pg/ml, 200pg/ml, 100pg/ml, 50pg/ml, 25pg/ml, 12.5pg/ml and 6.25pg/ml.
3.4.11.3 Assay Procedure
The procedure is as directed by ABCAM USA. All reagents were brought to room temperature (18-25°C) prior to use. One hundred micro-litre (100μL) of each standard and sample was added into appropriate wells. The wells were covered and incubated for 2 hours 30 minutes at room temperature, enabling antigen (TNF-alpha)-capture antibody complex. The solutions were discarded and washed 4 times, by filling each well with 1X Wash Solution (300μL) using a multi-channel Pipette. After the last wash, any remaining wash buffer was removed by decanting and the plate blotted against clean paper towels. One hundred microliterr (100μL) of 1X Biotinylated TNF alpha Detection Antibody was added to each well and incubated for 1hour at room temperature with gentle shaking. The solution was decanted and the wash repeated. One hundred microliter (100μL) of 1X HRP-Streptavidin solution was added to each well. The plate
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was incubated for 45 Minutes at room temperature with gentle shaking. The solution was decanted and the wells washed. Then 100μL of TMB One-Step Substrate Reagent was added to each well and incubated for 30 Minutes at room temperature in the dark with gentle shaking, and 50μL of Stop Solution added to each well. The wells were read at 450Nm immediately. Standard curve was used to determine the amount of TNF-a in the samples.