Rat Insulin-like Growth Factor-1 (IGF-1) ELISA Kit Instructions

This kit is for research use only Detection range: 78 pg / ml -5,000 pg / ml
Specificity: This kit can detect IGF-1 in rats and does not cross-react with other related proteins.
Validity: 6 months Expected application: ELISA method for quantitative determination of IGF-1 content in rat serum, cell culture supernatant or other related liquids.

Explanation
1. Kit storage: -20 ℃ (when not in use for a long time); 2-8 ℃ (when used frequently).
2. The concentrated washing liquid will be salted out at low temperature, and it can be heated and dissolved in the water bath when diluted.
3. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.
4. The well of the ELISA plate just opened may contain a little water-like substance. This is normal and will not have any impact on the experimental results.
Overview Insulin like growth factor 1 (IGF-1), also known as growth hormone stimulating hormone c, is a relative molecular mass of 7600

Of single-chain non-sugar polypeptides. It was first discovered that its function is to regulate the growth of somatic cells, and then it was found to be an important cell growth and differentiation process

Regulatory protein. In mammals, the mature IGF-1 structure is quite conservative, and its protein structure is in rats, pigs, cattle, and canines

100% the same. IGF-1 is mainly secreted by the liver and exists in the blood and other body fluids in the form of a conjugate with IGFBP.
Experimental principle The kit uses double antibody sandwich enzyme-labeled immunoassay to determine the level of IGF-1 in the specimen. Coat the microplate with purified antibody to make a solid phase antibody

, Add IGF-1 antigen, biotinylated anti-rat IGF-1 antibody, and HRP-labeled avidin to the microwells coated with mAb in sequence, wash thoroughly

After cleaning, the substrate TMB is used for color development. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. colour

The depth is positively correlated with IGF-1 in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated.
Kit composition and reagent preparation
1. Enzyme-linked plate: one piece (96 wells)
2. Standard product (lyophilized product): 2 bottles.
3. Sample diluent: 1 × 20ml / bottle.
4. Test dilution A: 1 × 10ml / bottle.
5. Test diluent B: 1 × 10ml / bottle.
6. Detection solution A: 1 × 120ul / bottle (1: 100)
7. Detection solution B: 1 × 120ul / bottle (1: 100)
8. Substrate solution: 1 × 10ml / bottle.
9. Concentrated washing solution: 1 × 30ml / bottle, each bottle is diluted 25 times with distilled water.
10. Stop solution: 1 × 10ml / bottle (2N H2SO4).

Reagents and equipment needed but not provided
1. Standard specification microplate reader.
2. High-speed centrifuge.
3. Electric constant temperature incubator
4. Clean test tubes and Eppendof tubes
5. Series adjustable pipettes and tips. When testing more samples at one time, it is best to use a multi-channel pipette.
6. Distilled water, volumetric flask, etc.
Collection and preservation of specimens
1. Supernatant of cell culture: collect the supernatant after centrifugation, and store the specimen at -20 ℃, and avoid repeated freezing and thawing.
2. Serum: Please leave the specimen at room temperature for 2 hours or overnight at 4 ° C, centrifuge at 1000 xg for 20 minutes, take the supernatant for testing, or place the specimen at-

Store at 20 ℃, but avoid repeated freezing and thawing.
3. Plasma: EDTA or heparin can be used as an anticoagulant, centrifuge at 1000 xg for 20 minutes within 30 minutes after blood collection, and the supernatant can be taken for detection, or the

Store the specimen at -20 ℃, but avoid repeated freezing and thawing.
Note: Hemolysis of specimens will affect the final test results, so hemolysis specimens should not be tested.
Dilution principle of specimens:
First of all, we should know the approximate content of the sample to be tested through literature search, and determine the appropriate dilution factor. Only diluted to within the range of the standard curve

, The test result is accurate. Detailed records should be made during the dilution process. When the concentration is finally calculated, the concentration of the specimen is diluted by "N" times

The degree should be multiplied by "N".
Standard dilution principle: Each bottle of standard is diluted with sample diluent to 1ml before use. After it is capped, it is allowed to stand for more than 10 minutes, and then repeatedly inverted /

Rubbing to help dissolve, its concentration is 10,000 pg / mL, after serial dilution, diluted to 5,000 pg / mL, 2,500 pg / mL,

1,250 pg / mL, 625 pg / mL, 312 pg / mL, 156 pg / mL, 78 pg / mL, the sample dilution is directly used as the standard concentration 0

pg / mL, prepared within 15 minutes before use.
Dilution principle of test solution A:
Before use, dilute with Test Solution A Diluent. Before dilution, prepare according to the pre-calculated total amount required for each experiment (100ul per well)

When preparing, 0.1-0.2ml should be prepared. If 10ul detection solution A plus 990ul detection dilution A is prepared, mix gently.

Prepared within time.
Dilution principle of test solution B:
Before use, dilute with test solution B diluent. Before dilution, prepare according to the pre-calculated total amount required for each experiment (100ul per well)

When preparing, 0.1-0.2ml should be prepared. If 10ul detection solution A plus 990ul detection dilution A is prepared, mix gently.

Prepared within time.
Before starting the experiment, please configure all reagents in advance. When diluting the reagents or samples, they should be mixed evenly. Try to avoid foaming when mixing. Every test

A standard curve should be made. If the sample concentration is too high, dilute with sample diluent to make the sample meet the detection range of the kit.
1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. Add 100ul of sample diluent to blank wells, and add standard or

The sample to be tested is 100ul. Be careful not to have bubbles. Add the sample and add the sample to the bottom of the well of the microplate. Try not to touch the wall of the well.

The plate was covered or covered, and reacted at 37 ° C for 120 minutes.
To ensure the validity of the experimental results, please use a new standard solution for each experiment.
2. Discard the liquid and spin dry without washing. Add 100ul of detection solution A working solution to each well (take 1ul detection solution A and add 99ul detection dilution

Prepare the ratio of solution A, mix gently, and prepare within one hour before use), 37 ℃, 60 minutes.
3. After incubating for 60 minutes, discard the liquid in the well, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, 350ul / per well, spin dry.
4. Add 100ul of detection solution B working solution (same as the detection A working solution) to each well at 37 ℃ for 60 minutes.
5. After incubating for 60 minutes, discard the liquid in the wells, spin dry, wash the plate 5 times, soak for 1-2 minutes each time, 350ul / per well, spin dry.
6. Add 90ul of substrate solution to each well in sequence, and avoid color development at 37 ° C (within 30 minutes, at this time, the first 3-4 wells of the standard product are clearly visible to the naked eye)

Gradient blue, the gradient after 3-4 holes is not obvious, you can terminate).
7. Add 50ul of stop solution to each well in sequence to stop the reaction (at this time, the blue color turns to yellow). The order of adding the stop solution should be as much as possible with the substrate solution

The order of joining is the same. In order to ensure the accuracy of the experimental results, the termination solution should be added as soon as possible after the substrate reaction time expires.
8. Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. Test within 15 minutes after adding stop solution.
Note:
1. When using the reagent kit for the first time, the user should centrifuge various reagent tubes for several minutes so that the reagents are concentrated to the bottom of the tube.
2. Leave one well for each experiment as a blank zero-adjusting well. No reagents are added to this well, but only the substrate solution and 2NH2SO4 are added last. Use first when measuring

This hole adjusts the OD value to zero.
3. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test, and add a cover or film to the enzyme label plate.
4. Store unused microplates or reagents at 2-8 ° C. Standard products, testing solution A working fluid, testing solution B working fluid

The required amount is configured for use. Do not reuse the diluted standard, test solution A working solution or test solution B working solution.
5. It is recommended to set double-hole measurement when testing samples to ensure the accuracy of the test results.
Plate washing method Manual plate washing method: suck (do not touch the wall) or shake off the liquid in the enzyme plate; place a few layers of absorbent paper on the experimental table with the enzyme plate facing down

Pat hard several times; inject at least 0.3ml of the recommended wash buffer into the well and soak for 1-2 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 skillfully.
Calculate the concentration of the standard as the abscissa (logarithmic coordinate), the OD value is the ordinate (normal coordinate), draw a standard curve on semi-logarithmic coordinate paper,

According to the OD value of the sample, find the corresponding concentration from the standard curve; multiply by the dilution factor; or calculate the standard curve using the concentration and OD value of the standard

Linear regression equation, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor, which is the actual concentration of the sample

.
Precautions
1. When mixing protein solutions, try to be as gentle as possible to avoid foaming.
2. The washing process is very important. Insufficient washing can easily cause false positives.
3. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.
4. Please make the standard curve at the same time of each measurement, it is better to make the complex hole.
5. If the content of IGF-I in the specimen is too high, please dilute it and then determine it. When calculating, please multiply by the dilution factor.
6. When preparing standard products and testing solution working fluid, please prepare with corresponding diluent, not to be confused.
7. Please keep the substrate away from light.
8. Do not replace the reagents in the kit with reagents from other manufacturers.

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