Enzyme-linked immunoassay of rat tumor necrosis factor alpha (TNF-α)

Enzyme-linked immunoassay of rat tumor necrosis factor alpha (TNF-α)

Kit instruction manual

Read this manual carefully before use. This ELISA kit is based on the principle of double antibody sandwich technology to detect rat tumor necrosis factor alpha (TNF-α), which can only be used for research purposes, not for medical diagnosis.

Uses: For the determination of tumor necrosis factor alpha (TNF-α) in rat serum, plasma and related liquid samples.

working principle

This kit uses biotin double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the level of rat tumor necrosis factor alpha (TNF-α) in the sample. Add rat tumor necrosis factor alpha (TNF-α) to the enzyme-labeled wells pre-coated with rat tumor necrosis factor alpha (TNF-α) monoclonal antibody; incubate; after incubation, add biotin-labeled TNF-α antibody. It is combined with streptavidin-HRP to form an immune complex. After incubation and washing, the unbound enzyme is removed, and then substrates A and B are added to produce a blue color, which is converted into the final under the action of acid Yellow. The color depth is positively correlated with the concentration of rat tumor necrosis factor alpha (TNF-α) in the sample.

Kit composition

Kit composition

48 hole configuration

96-well configuration

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Instructions

1 serving

1 serving

Sealing film

2 pieces (48)

2 pieces (96)

sealed bag

1

1

Enzyme coated plate

1 × 48

1 × 96

Store at 2-8 ℃

Standard product: 480ng / L

0.5ml × 1 bottle

0.5ml × 1 bottle

Store at 2-8 ℃

Standard dilution

1.5ml × 1 bottle

1.5ml × 1 bottle

Store at 2-8 ℃

Streptavidin-HRP

3 ml × 1 bottle

6 ml × 1 bottle

Store at 2-8 ℃

Biotin-labeled anti-TNF-α antibody

0.5ml × 1 bottle

1 ml × 1 bottle

Store at 2-8 ℃

Developer A liquid

3 ml × 1 bottle

6 ml × 1 bottle

Store at 2-8 ℃

Developer B liquid

3 ml × 1 bottle

6 ml × 1 bottle

Store at 2-8 ℃

Stop solution

3ml × 1 bottle

6ml × 1 bottle

Store at 2-8 ℃

Concentrated washing liquid

(20ml × 20 times) × 1 bottle

(20ml × 30 times) × 1 bottle

Store at 2-8 ℃

Reagents and equipment needed but not provided

1. 37 ℃ thermostat.

2. Standard specification microplate reader.

3. Precision pipettes and disposable tips

4. Distilled water,

5. Disposable test tube

6. Absorbent paper

Precautions

1. The kit taken from 2-8 ° C should be equilibrated at room temperature for at least 30 minutes before opening the kit. If the enzyme-coated plates are not used up after opening, the slats should be stored in sealed bags.

2. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid experimental errors.

3. Strictly follow the instructions, and the test results must be determined by the microplate reader.

4. To avoid cross-contamination, avoid repeated use of the tip and sealing film in your hand.

5. Unused other reagents should be packed or covered. Do not mix reagents of different batches. Use before warranty.

6. Substrate B is sensitive to light and avoid prolonged exposure to light.

Washing method

Manual plate washing method: throw away the liquid in the enzyme label plate; place a few layers of absorbent paper on the experimental table, the enzyme label plate is forced to pat several times downward; inject at least 0.35ml of the diluted washing solution into the well and soak 2 minutes. Repeat this process several times as needed.

Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used proficiently

Specimen requirements

1. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.

2. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ° C, but repeated freezing and thawing should be avoided.

Operating procedures

1. Dilution of standard products: (This kit provides one original standard product, users can dilute in a small test tube according to the following chart.)

240 ng / L

Standard No. 5

Add 120μl of original standard to 120μl of standard dilution

120 ng / L

Standard 4

120μl Standard No. 5 is added to 120μl standard dilution

60 ng / L

Standard 3

120μl No. 4 standard is added to 120μl standard dilution

30 ng / L

Standard No. 2

120μl Standard No. 3 is added to 120μl standard dilution

15 ng / L

Standard 1

Add 120μl of Standard No. 2 to 120μl of Standard Diluent

2. Determine the number of slats required based on the number of substitute samples and the number of standard products. It is recommended to make multiple holes for each standard and blank hole. Each sample is determined according to its own quantity, and those that can use multiple holes can be used for multiple holes.

3. Sample addition: 1) Blank wells, blank control wells do not add samples, biotin-labeled anti-TNF-α antibody, streptavidin-HRP, only add developer A & B and stop solution. ; 2) Standard well: add 50ul of standard, Streptavidin-HRP50ul (biotin antibody has been integrated in the standard beforehand, so it is not added); 3) Sample test well: add 40ul of sample, then add 10ul of anti-TNF-α antibody and 50ul of streptavidin-HRP, covered with a sealing film, gently shake to mix, and incubate at 37 ° C for 60 minutes.

4. Mixing solution: Dilute 30 times concentrated washing liquid with distilled water 30 times and reserve.

5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds and then discard, repeat this 5 times, pat dry.

6. Color development: Add color developer A50ul to each well, and then add color developer B50μl, mix gently, and develop at 37 ° C in the dark for 10 minutes.

7. Stop: add 50μl of stop solution to each well to stop the reaction (at this time, the blue will turn to yellow).

8. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be performed within 10 minutes after adding the stop solution.

9. Calculate the linear regression equation of the standard curve according to the concentration of the standard product and the corresponding OD value, and then calculate the corresponding sample concentration on the regression equation according to the OD value of the sample. It can also be calculated using various application software.

Summary of operating procedures:

Detection range: 8ng / L → 240 ng / L.

Specification: 96T / box

Storage: 2-8 ° C.

Valid period: 6 months (2-8 ℃).

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