Surgimiento del Apéndice D Dentro del Reglamento del ACI 318S-

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Figure 3.2. Lipid Peroxidation (LPO) Standard Curve

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3.10. Determination of 8–hydroxy–2'–deoxyguanosine (Wu et al. 2004;

Patel et al. 2007; Shen et al. 2007)

8–OHdG was determined quantitatively using a commercial ELISA kit obtained from Cell Biolabs, Inc. San Diego, CA.

3.10.1. Introduction

Free radicals and other reactive species are constantly generated in vivo and cause oxidative damage to bio–molecules, a process held in check only by the existence of multiple antioxidant and repair systems as well as the replacement of damaged nucleic acids, proteins and lipids. DNA is probably the most biologically significant target of oxidative attack, and it is widely thought that continuous oxidative damage to DNA is a significant contributor to the age–related development of the major cancers, such as those of the colon, breast, rectum and prostate.

Among numerous types of oxidative DNA damage, the formation of 8–hydroxy–2′–

deoxyguanosine (8–OHdG) is a ubiquitous marker of oxidative stress. 8–OHdG, one of the oxidative DNA damage by–products, is physiologically formed and enhanced by chemical carcinogens. During the repair of damaged DNA in vivo by exonucleases, the resulting 8–OHdG is excreted without further metabolism into urine.

3.10.2. Assay principle

The oxidative DNA damage ELISA kit is a competitive ELISA for the quantitative measurement of 8–OHdG. The unknown 8–OHdG samples or 8–OHdG standards were first added to an 8–OHdG/bovine serum albumin (BSA) conjugate pre–absorbed Elisa immunoassay (EIA plate). After a brief incubation, an anti–8–OHdG monoclonal antibody was added, followed by an HRP conjugated secondary antibody. The 8–

OHdG contents in unknown samples were determined by comparison with pre–

determined 8–OHdG standard curve.

3.10.3. Kit components

1. Ninety-six well protein binding plate (Part No. 231001): One strip well of 96–

well plate.

2. 8–OHdG conjugate (Part No. 232001): One 120 µl vial of 8–OHdG–BSA conjugate at 1.0 mg/ml in phosphate buffered saline (PBS).

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3. Anti–8–OHdG antibody (Part No. 232002): One 15 µl vial of anti–8–OHdG.

4. Secondary antibody, HRP Conjugate (Part No. 10902): One 50 µl vial.

5. Assay diluents (Part No. 310804): One 50 ml bottle.

6. 10X wash buffer (Part No. 310806): One 100 ml bottle.

7. Substrate solution (Part No. 310807): One 12 ml amber bottle.

8. Stop solution (Part No. 310808): One 12 ml bottle.

9. 8–OHdG standard (Part No. 232003): One 100 µl vial of 2 µg/ml 8–OHdG in 1X PBS, 0.1% BSA.

3.10.4. Storage and stability of reagents

Both the 8–OHdG Standard and the 8–OHdG Conjugate were stored at –80 °C and all other components at 4 °C. They were stable at these temperatures until their expiration dates.

3.10.5. Preparation of reagents

1. 8–OHdG coated plate: 8–OHdG conjugate (1 mg/ml) was diluted to 10 µg/ml in 1X PBS. 100 µl of the 10 µg/ml 8–OHdG Conjugate was added to each well and incubated overnight at 4 °C. The 8–OHdG coating solution was removed and the wells were washed once with dH₂O. The plate was blotted on paper towels to remove excess fluid and 200 µl of assay diluents were added to each well and blocked for 1 hr at room temperature. The plate was transferred to 4

°C and the assay diluents were removed immediately before use.

2. 1X wash buffer: The 10X Wash Buffer Concentrate was diluted to 1X with deionised water, stirred to homogenise.

3. Anti–8–OHdG antibody: The anti–8–OHdG antibody was diluted in 1:500 with assay diluents immediately before use.

4. Secondary antibody: Secondary antibody was diluted in 1:1000 with assay diluents immediately before use.

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3.10.6. Preparation of standard curve

A dilution series of 8–OHdG standards in the concentration range of 0–20 ng/ml was prepared by diluting the 8–OHdG standard in assay diluents as shown below:

STD Tubes 8–OHdG STD Assay Diluents 8–OHdG

(µl) (µl) (ng/ml)

1 10 990 20

2 500 of Tube #1 500 10

3 500 of Tube #2 500 5

4 500 of Tube #3 500 2.5

5 500 of Tube #4 500 1.25

6 500 of Tube #5 500 0.625

7 500 of Tube #6 500 0.313

8 500 of Tube #7 500 0.156

9 500 of Tube #8 500 0.078

10 0 500 0

3.10.7. Preparation of samples

Plasma samples were diluted at least ten fold in assay diluents and used directly in the assay. Samples containing precipitates were centrifuged at 3000 g for ten minutes prior to use in the assay.

3.10.8. Assay protocol

1. All reagents were prepared and mixed thoroughly before use. Each 8–OHdG sample including unknown and standard were assayed in duplicate.

2. Fifty microlitres of unknown samples or 8–OHdG standard were added to the wells of 8–OHdG conjugate coated plate and incubated at room temperature for 10 minutes on an orbital shaker.

3. Fifty microlitres of the diluted anti–8–OHdG antibody were added to each well and incubated at room temperature for 1 hr on an orbital shaker.

4. Microwell strips were washed 3 times with 250 µl of 1X wash buffer per well with thorough aspiration between each wash. After the last wash, wells were emptied and microwell strips were tapped on paper towel to remove excess 1X wash buffer.

5. One hundred microlitres of the diluted secondary antibody–enzyme conjugates were added to all wells and incubated at room temperature for 1 hr on an orbital shaker.

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6. Microwell strips were washed 3 times according to step 4 above.

7. Substrate solution was warmed to room temperature.

8. One hundred microlitres of substrate solutions were added to each well including the blank well and incubated at room temperature for 15 minutes on an orbital shaker.

9. The enzyme reactions were stopped by addition of 100 µl of stop solution into each well including the blank well. Results were read immediately (Colour fades over time).

10. Absorbance of each microwell was read on a microplate reader at wavelength of 450 nm and optical reference wavelength of 620 nm.

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Figure 3.3. 8–OHdG ELISA Standard Curve 0.000

0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600 1.800 2.000

-0.500 0.000 0.500 1.000 1.500 2.000 2.500

Absorbance at 450 nm

Log 10 [8OHdG]