8isoPGF2a ELISA 试剂盒

1. Goat Anti-Rabbit Antibody Coated Plate : One 96-well strip plate.
2. Anti-8-iso-PGF2α Antibody : One 20 μL tube of anti-8-iso-PGF2α rabbit IgG.
3. Sample Diluent : One 50 mL bottle.
4. Neutralization Solution : One 20 mL bottle.
5. 10X Wash Buffer : One 100 mL bottle.
6. Substrate Solution : One 12 mL amber bottle.
7. Stop Solution (Part. No. 310808): One 12 mL bottle.
8. 8-iso-PGF2α Standard : One 25 μL tube of 200 μg/mL 8-iso-PGF2α in DMSO.
9. 8-iso-PGF2α-HRP Conjugate : One 70 μL tube of 8-iso-PGF2α-HRP conjugate.

1. Protein samples such as purified protein, plasma, serum, cell lysate
2. Deionized water
3. 5 μL to 1000 μL adjustable single channel precision micropipettes with disposable tips
4. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
5. Bottles, flasks, and conical or microtubes necessary for reagent preparation
6. Reagents and materials necessary for sample extraction and purification
7. Multichannel micropipette reservoir
8. Plate orbital shaker or rotator 3
9. Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length)

• Quantify 8-isoprostane in about 3 hours
• Suitable for use with urine, plasma, serum, or cell lysates
• 8-iso-PGF2alpha included as positive control

• 1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate to 1X with deionized water. Stir to homogeneity.
• Anti-8-iso-PGF2α Antibody: Immediately before use, dilute the Anti-8-iso-PGF2α Antibody 1:1000 with Sample Diluent.
• 8-iso-PGF2α-HRP Conjugate: Immediately before use, dilute the conjugate 1:80 with Sample Diluent. Only prepare enough of the diluted conjugate for the number of wells immediately used.
• Substrate Solution: Prior to use, warm the Substrate Solution to room temperature. Note: Do not store diluted Anti-8-iso-PGF2α Antibody, 8-iso-PGF2α-HRP Conjugate, or 8-iso- PGF2α Standard solutions.

Hydrolysis of lipoprotein or phospholipid coupled 8-iso-Prostaglandin F2α (8-iso-PGF2α) is required to measure both free and esterified isoprostane. To hydrolyze this ester bond, the sample is usually treated with 2N NaOH at 45 °C for 2 hours. • Serum, plasma, tissue lysate samples: Use 1 part of 10N NaOH for every 4 parts of liquid sample. After incubation at 45 °C for 2 hours, add 100 μL of concentrated (10N) HCl p er 5 0 0μ L of hydrolyzed sample. The sample could turn milky after this addition. Centrifuge the samples for 5 minutes at 12,000 rpm in a microcentrifuge. The clear supernatant can be used in the assay or stored at -20 °C or below for future use. Before assaying, check to be sure each neutralized sample is in the pH range of 6-8. If it is not, adjust the pH to this range by adding 100 μL of the sample to 100 μL of the provided Neutralization Solution. • Urine samples: Acid hydrolysis of urine samples is necessary to break the bonds which hold lipid and non-lipid components together prior to ELISA. Urine sample is acidified to pH 3.0 by adding 1/10 volume of 1N HCl (Example: Add 100 μL of 1N HCl to 1 mL of urine sample). Acidified urine sample should be further diluted in PBS or Sample Diluent 1:4 to 1:8 before ELISA. 4 Preparation of 8-iso-PGF2α Standards

1. Prepare fresh standards by diluting the 8-iso-PGF2α Standard from 200 μg/mL to 0.2 μg/mL in Sample Diluent for a 1:1000 final dilution. (Example: Add 5 μL of 8-iso-PGF2α Standard stock tube to 4.995 mL of Sample Diluent)
2. Prepare a series of the remaining 8-iso-PGF2α standards according to Table
   1. Standard 8-iso-PGF2α Standard 8-iso-PGF2α Standard Tubes (μL) Sample Diluent (μL) (pg/mL) 1 5 μL of Standard Stock 4995 μL 200,000 2 250 μL of Tube #1 750 μL 50,000 3 250 μL of Tube #2 750 μL 12,500 4 250 μL of Tube #3 750 μL 3,125 5 250 μL of Tube #4 750 μL 781 6 250 μL of Tube #5 750 μL 195 7 250 μL of Tube #6 750 μL 49 8 0 μL 200 μL 0 Table
      1. Preparation of 8-iso-PGF2α Standard Curve. Note: Do not store diluted 8-iso-PGF2α Standard solutions.

Note: Each 8-iso-PGF2α Standard and unknown samples should be assayed in duplicate or triplicate. A freshly prepared standard curve should be used each time the assay is performed.

1. Add 100 μL of the diluted Anti-8-iso-PGF2α Antibody to the Goat Anti-Rabbit Antibody Coated Plate. Incubate 1 hour at 25 °C on an orbital shaker.
2. Remove the antibody solution from the wells. Wash wells 5 times with 300 μL 1X Wash Buffer per well. After the last wash, empty the wells and tap microwell plate on absorbent pad or paper towel to remove excess wash solution. Note: Thorough washing is necessary to remove all of the azide present in the antibody solution.
3. Combine 55 μL of the 8-iso-PGF2α standard or sample and 55 μL of 8-iso-PGF2α-HRP conjugate in a microtube and mix thoroughly. Transfer 100 μL of the combined solution per well. A well containing Sample Diluent can be used as a control. Incubate 1 hour at 25 °C on an orbital shaker.
4. Remove the combined solution from the wells. Wash 5 times with 300 μL of 1X Wash Buffer per well. After the last wash, empty wells and tap microwell plate on absorbent pad or paper towel to remove excess wash solution. 5
5. Add 100 μL of Substrate Solution to each well. Incubate at room temperature for 10-30 minutes on an orbital shaker.
6. Stop the enzyme reaction by adding 100 μL of Stop Solution to each well. Results should be read immediately (color will fade over time).
7. Read absorbance of each well on a microplate reader using 450 nm as the primary wave length.