Instruction Manual of Rat Hydroxyproline (Hyp) ELISA Kit

Instruction Manual of Rat Hydroxyproline (Hyp) ELISA Kit

This kit is for research use only

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

Minimum detection limit: 39 ng / ml

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

Validity: 6 months

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

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

Hydroxyproline (hydroxyproline), is one of the imino acids, usually with a hydroxyl group in the fourth position, but sometimes also in 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

Coat the microplate with purified antibody to make a solid-phase carrier. Add samples or standards, biotinylated anti-Hyp antibody, and HRP-labeled avidin to the anti-Hyp antibody-coated microwells. After washing, color was developed with the 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.

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. HRP-avidin Diluent: 1 × 10ml / bottle.

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

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

8. Substrate solution (TMB Substrate): 1 × 10ml / 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 heating thermostat 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: Whole blood specimens should be left at room temperature for 2 hours or overnight at 4 ° C and centrifuged at 1000 xg for 20 minutes. Supernatant can be taken for detection, or the specimens should be stored 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 ° C or -80 ° C, 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 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 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 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.

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

Steps

Before starting the experiment, please configure all reagents in advance. When the reagents or samples are diluted, they should be mixed well. 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 well walls. 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 100μl of biotinylated antibody working solution to each well (proportion of 1μl of biotinylated antibody plus 99μl of biotinylated antibody dilution), 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, 350μl / per 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 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 microplate; place a few layers of absorbent paper on the experimental table, and force the microplate down several times; pat the recommended wash buffer Inject at least 0.3ml of solution into the hole 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.

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 concentration from the standard curve according to the OD value of the sample; Multiply by the dilution factor; or use the standard concentration and OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor to obtain 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 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.