Human Hydroxyproline (Hyp) ELISA Kit

Instruction Manual of Human Hydroxyproline (Hyp) ELISA Kit

Human Hydroxyproline (Hyp) ELISA Kit is for research use only

Detection range: 156 ng / ml-10000 ng / ml

Minimum detection limit: 39 ng / ml

Intended application: ELISA method for quantitative determination of Hyp content in human serum, plasma or other related biological fluids.

Overview: hydroxyproline (hydroxyproline) is one of the imino acids, usually with a hydroxyl group at the fourth position, but sometimes also at the third position. Since there are two asymmetric carbon atoms, there are 4 kinds of stereoisomers. There is no D-hydroxyproline in nature. L-hydroxyproline is contained in animal gum and collagen. L-Allohydroxyproline is a component of the toxic polypeptide phalloidine obtained from Amanita Phalloides. This amino acid does not exist in general proteins. Among the methyl derivatives of hydroxyproline in nature are L-stachydine, D-stachydine, and γ-hydroxypallic acid. In vivo, hydroxyproline is synthesized from proline, but not in a free state, but is hydroxylated in the peptide chain. Its decomposition is similar to proline, but it is carried in a state with hydroxyl groups, so it becomes 4-hydroxyglutamic acid.

Experimental principle: The microtiter plate is coated with purified antibody to make a solid phase carrier, and the specimen or standard, biotinylated anti-Hyp antibody, and HRP-labeled avidin are sequentially added to the microwell coated with anti-Hyp antibody , After thorough washing, develop color with substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The color depth is positively correlated with Hyp 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.

Human hydroxyproline (Hyp) ELISA kit composition and reagent preparation:

1. Assay plate: one piece (96 wells).

2. Standard product (Standard): 2 bottles (lyophilized product).

3. Sample Diluent: 1 × 20ml / bottle.

4. Biotin-antibody Diluent: 1 × 10ml / bottle.

5. Horseradish peroxidase-labeled avidin dilution (HRP-avidin Diluent): 1 × 10ml / bottle.

6. Biotin-antibody: 1 × 120μl / vial (1: 100)

7. Horseradish peroxidase labeled avidin (HRP-avidin): 1 × 120μl / vial (1: 100)

8. Substrate solution (TMB Substrate): 1 × 10 ml / bottle.

9. Wash Buffer: 1 × 20ml / bottle, each bottle is diluted 25 times with distilled water.

10. Stop Solution (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 multi-channel pipettes

6. Distilled water, volumetric flask, etc.

Collection and preservation of specimens:

1. Serum: Please leave whole blood samples at room temperature for 2 hours or overnight at 4 ° C and centrifuge at 1000 xg for 20 minutes. Take the supernatant for testing, or store the samples at -20 ° C or -80 ° C, but avoid repeated Freeze and thaw.

2. Plasma: EDTA or heparin can be used as anticoagulant. Centrifuge the sample at 2-8 ° C 1000 xg for 15 minutes within 30 minutes after collection, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing. melt.

3. Cell culture supernatant or other biological specimens: centrifuge at 1000 xg for 20 minutes, take the supernatant for detection, or store the specimen at -20 ℃ or -80 ℃, 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 the specimen: firstly, the approximate content of the sample to be tested is determined through literature search, and the appropriate dilution factor is determined. Only when it is diluted to the range of the standard curve, the test result is accurate. Detailed records should be made during the dilution process. When calculating the concentration at the end, it was diluted "N" times, and the concentration of the specimen should be multiplied by "N".

Standard dilution principle: 2 bottles, each bottle is diluted with sample diluent to 1ml before use, and then left to stand for more than 10 minutes after capping, and then repeatedly inverted / rubbed to help dissolve, its concentration is 10000 ng / ml, do After serial dilutions, dilute 10000 ng / ml, 5000 ng / ml, 2500 ng / ml, 1250 ng / ml, 625 ng / ml, 312 ng / ml, 156 ng / ml, and the sample dilution is directly used as the standard concentration 0 ng / ml, prepared within 15 minutes before use.

For example, to prepare a 5000 ng / ml standard: take 0.5ml (not less than 0.5ml) of the above standard at 10000 ng / ml and add it to an Eppendorf tube containing 0.5ml of sample diluent, mix well. The rest of the concentration can be deduced by analogy.

Dilution principle of biotin-labeled antibody: before use, dilute with biotin-labeled antibody diluent. Before dilution, prepare according to the pre-calculated total amount required for each experiment (100μl per well). 0.1- 0.2ml. For example, 10 μl of biotin-labeled antibody plus 990 μl of biotin-labeled antibody dilution is prepared, mixed gently, and prepared within one hour before use.

Dilution principle of horseradish peroxidase-labeled avidin: before use, dilute with horseradish peroxidase-labeled avidin diluent, and prepare according to the pre-calculated total amount required for each experiment before dilution (Each 100μl), 0.1-0.2ml should be prepared in actual preparation. For example, 10 μl horseradish peroxidase-labeled avidin plus 990 μl horseradish peroxidase-labeled avidin dilution is prepared, mix gently, and prepare within one hour before use.

Operation steps: Before starting the experiment, please configure all the reagents in advance. When the reagents or samples are diluted, they should be mixed evenly. Try to avoid foaming when mixing. A standard curve should be made for each test. 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 100μl of sample diluent to blank wells, and add 100μl of standard or test sample to the remaining wells. Be careful not to have air bubbles. Add samples to the bottom of the wells of the microtiter plate. Try not to touch the walls of the wells. The target plate is covered with a cover or film 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 100 μl of biotinylated antibody working solution to each well (the ratio of 1 μl of biotinylated antibody plus 99 μl of biotinylated antibody dilution is prepared, mix gently and prepare within one hour before use), 37 ° C, 60 minutes.

3. After incubating for 60 minutes, discard the liquid in the wells, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, 350μl / well, spin dry.

4. Add 100 μl of horseradish peroxidase-labeled avidin working solution (with biotin-labeled antibody working solution) to each well at 37 ° C for 60 minutes.

5. After incubating for 60 minutes, discard the liquid in the well, spin dry, wash the plate 5 times, soak for 1-2 minutes each time, 350μl / well, spin dry.

6. Add 90μl of substrate solution to each well in sequence, and develop color at 37 ° C in the dark (within 30 minutes, at this time, the first 3-4 wells of the standard product have a visible blue gradient before the 3-4 wells, and the gradient of the back 3-4 wells is not obvious. , You can terminate).

7. Add 50μl 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 the same as that of the substrate solution. 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, only the substrate solution and 2N H2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring.

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. Standards, biotin-labeled antibody working solution, horseradish peroxidase-labeled avidin working solution, please use according to the required amount. Do not reuse diluted standard, biotin-labeled antibody working solution, or horseradish peroxidase-labeled avidin working solution.

5. It is recommended to set a double-hole test when testing samples to ensure the accuracy of the test results.

Washing method:

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

2. Manual plate washing method: suck up (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, and force the enzyme plate down to pat several times; the recommended washing buffer Inject at least 0.3ml into the hole and soak for 1-2 minutes. Repeat this process several times as needed.

Calculation: Taking the concentration of the standard as the abscissa (logarithmic coordinate) and the OD value as the ordinate (ordinary coordinate), draw a standard curve on semi-logarithmic coordinate paper, and find the corresponding curve from the standard curve according to the OD value of the sample Concentration; multiply by the dilution factor; or calculate the linear regression equation of the standard curve using the concentration of the standard and the OD value, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor, which is the actual sample concentration.

Explanation:

1. Store the kit: -20 ℃ (when not in use for a long time); 2-8 ℃ (when used frequently).

2. When the concentrated washing liquid is stored at low temperature, salt will precipitate out. When diluted, it can be heated and dissolved in a water bath.

3. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions.

4. The well of the enzyme-linked plate just opened may contain a little water-like substance. This is normal and will not have any impact on the experimental results.

Precautions:

1. When mixing protein solutions, try to be as gentle as possible to avoid foaming.

2. The washing process is very important, inadequate washing is easy to 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 a standard curve at the same time of each measurement, it is best to make a double hole.

5. If the content of the substance to be tested 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.

Specificity: This kit can detect natural or recombinant human Hyp at the same time, and does not cross-react with other related proteins.

Validity: 6 months