Instruction manual of human melatonin (MT) ELISA kit

Instruction manual of human melatonin (MT) ELISA kit

This kit is for research use only

Minimum detection limit: 12.5 ng / ml

Specificity: This kit can detect human MT and has no cross reaction with other related substances.

Validity: 6 months

Intended application: ELISA method for quantitative determination of MT content in human serum, plasma 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.

Experimental principle

This kit uses enzyme-linked immunoassay direct competition method to detect MT. The microplate is coated with the antibody MT antibody. Add MT standards or samples, then add biotin-labeled MT. The MT and biotin-labeled MT in the sample competed with the anti-MT antibody on the solid phase plate. Then add horseradish peroxidase-labeled avidin to the microwells to form a solid-phase antibody-biotinylated MT-avidin-HRP complex. After the color development by adding substrate, with the increase of MT concentration in the sample, the color development OD value showed a gradually decreasing linear relationship.

Kit composition and reagent preparation

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

2. Standard: 5 × 1ml / bottle.

Standard

S1

S2

S3

S4

S5

concentration

(Ng / ml)

25

50

100

200

400

3. Biotin-labeled MT: 1 × 6ml / bottle.

4. Horseradish peroxidase labeled avidin (HRP-Avidin): 1 × 6ml / bottle.

5. Substrate A: 1 × 6ml / bottle.

6. Substrate B: 1 × 6ml / bottle.

7. Wash Buffer: 1 × 15ml / bottle, each bottle is diluted 20 times with distilled water.

8. Stop solution (Stop Solution): 1 × 6ml / 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. Ultrasonic cleaner

5. Clean test tubes and Eppendof tubes

6. Series adjustable pipettes and tips. When testing more samples at one time, it is best to use a multi-channel pipette

7. Distilled water, volumetric flask, etc.

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".

Dilution principle of concentrated washing liquid:

Make a 20-fold dilution with distilled water before use. If there is salt precipitation, dilute the water bath to increase the solubility.

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 it first so that the sample conforms to the detection range of the kit.

1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. The blank wells do not add any solution, and the remaining wells are respectively filled with 50ul of standard or sample to be tested. Be careful not to have air bubbles. Add the sample to the bottom of the well of the microtiter plate, then add 50ul of biotin-labeled MT (blank Not added in the hole). Try not to touch the wall of the hole, shake gently to mix. The microplate is covered with a cover or film, and the reaction is performed at 37 ° C for 60 minutes (to ensure the validity of the experimental results, please use a new standard solution for each experiment).

2. After incubation, discard the liquid in the well, spin dry, wash the plate 3-5 times, soak for 10 seconds each time, about 200ul / per well, spin dry.

3. Add 50 ul horseradish peroxidase labeled avidin to all wells. Try not to touch the wall of the hole, shake gently to mix. Encapsulate or cover the microplate, and react at 37 ℃ for 30 minutes

4. Follow step 2 for washing.

5. Add 50ul of substrate solution A and B to each well in sequence, and develop color at 37 ° C in the dark (within 30 minutes, generally within 10-15 minutes. At this time, there is a visible gradient in the back 3-4 wells of the standard Blue, the color of the first 3-4 holes is not obvious, it can be terminated).

6. Add 50ul of stop solution to each well in sequence to stop the reaction (in this case, 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.

7. Measure the optical density (OD value) of each well in sequence at 450nm wavelength using an enzyme-linked instrument. 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.

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. Please keep the substrate away from light.

7. Do not replace the reagents in the kit with reagents from other manufacturers.