Rat osteocalcin (BGP) ELISA kit instruction manual

Check the content of osteocalcin (BGP) in the liquid sample.

Experimental principle:

This kit uses the double antibody sandwich method to determine the level of rat osteocalcin (BGP) in the specimen. Microporous plates were coated with purified rat osteocalcin (BGP) antibody to make solid-phase antibodies. Osteocalcin (BGP) was added to the monoclonal antibody-coated microwells in turn, followed by HRP-labeled osteocalcin ( BGP) antibody binds to form antibody-antigen-enzyme-labeled antibody complex, and after thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with osteocalcin (BGP) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of rat osteocalcin (BGP) in the sample was calculated by a standard curve.

Kit composition:

1. ELISA plate: one piece (96 wells)

2. Standard product (lyophilized product): 2 bottles, each bottle is diluted with sample diluent to 1ml before use, and its concentration is 100 mIU / mL. After serial dilution, it is diluted to 100 mIU / mL, 50 mIU / mL, 25 mIU / mL, 12.5 mIU / mL, 6.25 mIU / mL, 3.12 mIU / mL, 1.56 mIU / mL, the original solution is directly used as the highest standard concentration, the sample dilution is directly used as the standard concentration 0 pg / mL, pro Prepare within 15 minutes before use.

3. Sample diluent: 1 × 20ml / bottle.

4. Test the diluent A: 1 × 10ml / bottle.

5. Test diluent B: 1 × 10ml / bottle.

6. Test solution A: 1 × 120ul / bottle (1: 100) is diluted with test diluent A1: 100 before use.

7. Detection solution B: 1 × 120ul / bottle (1: 100) is diluted with detection diluent B1: 100 before use.

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).

Sample processing and requirements:

1. Serum: blood coagulates naturally at room temperature for 10-20 minutes, centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully and centrifuge again if a precipitate appears during storage.

2. Plasma: EDTA or sodium citrate should be selected as the anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm) Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.

3. Urine: collected in a sterile tube and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, centrifuge again. Pleural and ascites, cerebrospinal fluid reference implementation.

4. Cell culture supernatant: When detecting secreted components, collect with a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When detecting intracellular components, dilute the cell suspension with PBS (PH7.2-7.4).

The concentration reaches about 1 million / ml. Through repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.

5. Organize the specimen: after cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 ° C. Add a certain amount of PBS (PH7.4) and homogenize the specimen with a manual or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future use.

6. Specimens should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. Experiments should be conducted as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided.

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

Steps

1. Dilution and loading of standard products: 10 standard wells are set on the enzyme-coated plate, 100μl of the standard products are added to the first and second wells respectively, and then the standard products are added to the first and second wells 50μl of diluent, mix well; then take 100μl from the first well and the second well and add them to the third and fourth wells respectively, and then add 50μl of standard diluent to the third and fourth wells respectively, mix well; Then take 50μl each in the third and fourth wells and discard it, then add 50μl each to the fifth and sixth wells, and then add 50ul of the standard dilution solution to the fifth and sixth wells respectively, and mix well; After mixing, take 50μl from the fifth and sixth wells and add them to the seventh and eighth wells respectively. Then add 50μl of the standard dilution solution to the seventh and eighth wells respectively. Take 50μl from the eight wells and add them to the ninth and tenth wells. Then add 50μl of the standard dilution solution to the ninth and tenth wells. After mixing, take 50μl from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50μl, and the concentrations are 900ng / L, 600ng / L, 300ng / L, 150ng / L, 75ng / L).

2. Adding samples: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the steps are the same) and the sample wells to be tested. Add 40μl of sample diluent to the test sample well of the enzyme-coated plate, and then add 10μl of the sample to be tested (the final dilution of the sample is 5 times). 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, shake gently to mix.

3. Incubation: seal the plate with a sealing film and incubate at 37 ° C for 30 minutes.

4. Mixing solution: Dilute 30 times (20 times of 48T) concentrated washing solution with distilled water 30 times (20 times of 48T) and then reserve.

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

6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.

7. Incubation: The operation is the same as 3.

8. Washing: The operation is the same as 5.

9. Color development: Add 50μl of developer A to each well, then add 50μl of developer B, mix gently, and develop at 37 ° C in the dark for 15 minutes.

10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time, the blue color turns to yellow).

11. Determination: Measure the absorbance (OD value) of each well in sequence with blank air conditioner zero and 450nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.

Precautions:

1. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme label coated plate is unopened, the strip should be stored in a sealed bag.

2. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.

3. The sampler should be used at each step of sample addition, and the accuracy should be regularly checked to avoid test errors. 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 a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the test substance in the sample is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please dilute it with a certain multiple (n times) of the sample diluent and then determine it. When calculating, please multiply the total dilution Multiple (× n × 5).

5. The sealing film is limited to one-time use to avoid cross-contamination.

6. Please keep the substrate away from light.

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

8. All samples, washing liquids and various wastes should be treated as infectious agents.

9. The components of different batches of this reagent shall not be mixed. 10. If there is any difference with the English manual, the English manual shall prevail.

Calculation: Taking the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the graph paper, and find out the corresponding concentration from the standard curve according to the OD value of the sample; then multiply by the dilution factor; or use the standard The linear regression equation of the standard curve is calculated by the concentration and OD value of the sample, the OD value of the sample is substituted into the equation, the sample concentration is calculated, and then multiplied by the dilution factor, which is the actual concentration of the sample.

(This picture is for reference only)

Kit performance:

1. The correlation coefficient R between the linear regression of the sample and the expected concentration is more than 0.990.

2. The batch and approval shall be less than 9% and 11% respectively

Detection range: 50ng / L -1000ng / L

Storage conditions and validity period:

1 2-8 ℃.

2 6 months

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