Description

8-OHdG (8-Hydroxydeoxyguanosine) ELISA Kit

This ELISA kit uses Competitive-ELISA as the method. The microtiter plate provided in this kit has been pre-coated with 8-OHdG. During the reaction, 8-OHdG in the sample or standard competes with a fixed amount of 8-OHdG on the solid phase supporter for sites on the Biotinylated Detection Ab specific to 8-OHdG. Excess conjugate and unbound sample or standard are washed from the plate, and Avidin conjugated to Horseradish Peroxidase (HRP) are added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of 8-OHdG in the samples is then determined by comparing the OD of the samples to the standard curve.

Assay type	Competitive-ELISA
Format	96T
Assay time	2.5h
Reactivity	Universal
Detection Method	Colormetric
Detection Range	1.56-100 ng/mL
Sensitivity	0.94 ng/mL
Sample Volume	50uL
Sample Type	Serum, plasma and other biological fluids

Specificity

This kit recognizes 8-OHdG in samples. No significant cross-reactivity or interference between 8-OHdG and analogues was observed.

Typical Data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration(ng/mL)	O.D	Average
100	0.398 0.454	0.426
50	0.51 0.562	0.536
25	0.734 0.724	0.729
12.5	1.014 1.034	1.024
6.25	1.387 1.385	1.386
3.13	1.746 1.718	1.732
1.56	1.993 1.993	1.993
0	2.325 2.337	2.331

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level 8-OHdG were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level 8-OHdG were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/mL)	4.77	8.88	45.84	4.81	9.56	43.89
Standard deviation	0.24	0.49	2.27	0.34	0.49	1.94
C V (%)	5.03	5.52	4.95	7.07	5.13	4.43

Recovery

The recovery of 8-OHdG spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	90-105	97
EDTA plasma (n=5)	92-105	98
Cell culture media (n=5)	84-95	90

Linearity

Samples were spiked with high concentrations of 8-OHdG and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	97-111	96-109	96-111
1:2	Average (%)	103	101	103
1:4	Range (%)	94-105	89-105	101-113
1:4	Average (%)	99	96	106
1:8	Range (%)	93-108	97-114	98-110
1:8	Average (%)	99	104	104
1:16	Range (%)	96-111	93-108	93-107
1:16	Average (%)	102	99	99

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro ELISA Plate(Dismountable)	8 wells X12 strips	-20'C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100X)	1 vial, 120 uL
Concentrated HRP Conjugate (100X)	1 vial, 120 uL	-20'C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4'C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25X)	1 vial, 30 mL
Substrate Reagent	1 vial, 10 mL	4'C(shading light)
Stop Solution	1 vial, 10 mL	4'C
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

8-OHdG (8-Hydroxydeoxyguanosine) ELISA Kit (UNES00018) Assay procedure

1. Set standard, test sample and control (zero) wells on the pre-coated plate and record their positions. It is recommended to measure each standard and sample in duplicate. Note: add all solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensure solutions do not foam when adding to the wells.

2. Add 50µL of Standard, Blank or Sample to their respective wells. The blank well is added with Sample / Standard dilution buffer.

3. Immediately add 50 µL of Biotin-detection antibody working solution to each well.

4. Cover with a plate seal and gently tap the plate to ensure thorough mixing. Incubate for 45minutes at 37°C.

5. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.

6. Add 100µL of HRP Conjugate working solution to each well and over with a plate seal.Incubate for 30 minutes at 37°C.

7. Repeat the aspiration/wash process 5 times according to step 5.

8. Add 90µL of the Substrate reagent to each well and cover with a new plate seal. Incubatefor approximately 15 minutes at 37°C and protect from light. The reaction time can beshortened or extended according to the colour change, but not by more than 30 minutes. Whenapparent gradient appears in standard wells, terminate the reaction.

9. Stop: Add 50µL of Stop Solution to each well (wells will develop a yellow color immediately).Note: Adding the stop solution should be done in the same order as the substrate solution.

10. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm. In advance, preheat the instrument and set the testing parameters.

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