Plant Growth Hormone (GH) ELISA Test Kit Instruction Manual

This kit can only be used for scientific research and should not be used for medical diagnosis.

Plant Growth Hormone (GH) ELISA Test Kit Instruction Manual

Detection principle

The kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). To the coated microwells pre-coated with growth hormone (GH) antibody, specimens, standards, and HRP-labeled detection antibodies were sequentially added, incubated and thoroughly washed. Using the substrate TMB to develop color, TMB is converted to blue under the catalysis of peroxidase and converted to the final yellow color by the action of an acid. The color depth is positively correlated with growth hormone (GH) in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader to calculate the sample content.

Sample collection, processing and storage methods

1. The sample should not contain sodium azide (NaN3) because sodium azide (NaN3) is an inhibitor of horseradish peroxidase (HRP).

2. Extract the specimen as soon as possible after the collection, and extract it according to the relevant literature.

3. Plant extract or other related samples: centrifuge at 1000 xg for 20 minutes and take the supernatant for testing.

4. Storage: If the sample is not detected in time, please fill it once and freeze it at -20 °C to avoid repeated freezing and thawing, thaw at room temperature and ensure that the sample is fully thawed evenly.

Bring your own items

1. Microplate reader (450nm)
2. High-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL
3. 37 ° C incubator

Operational precautions

1. The kit was stored at 2-8 ° C and equilibrated for 20 minutes at room temperature before use. The concentrated washing liquid taken out from the refrigerator will crystallize, which is a normal phenomenon, and the water bath is heated to completely dissolve the crystals before use.
2. The slats not used in the experiment should be immediately put back into the ziplock bag and sealed (low temperature dry) for storage.
3. The S0 standard with a concentration of 0 can be regarded as a negative control or blank; the sample has been diluted 5 times according to the instructions, and the final result multiplied by 5 is the actual concentration of the sample.
4. Incubation is carried out in strict accordance with the time indicated in the instructions, the amount of liquid added and the order.
5. Shake well all liquid components before use.

Kit composition

Note: The concentration of standard (S0-S5) is: 0, 5, 10, 20, 40, 80 pg/mL

Reagent preparation

Dilution of 20× Wash Buffer: Distilled water was diluted 1:20, ie 1 part of 20× Wash Buffer plus 19 parts of distilled water.

Washing method

1. Manually wash the plate: Drain the liquid in the hole, fill each hole with the washing liquid, let stand for 1 min, then drain the liquid in the hole, pat dry on the absorbent paper, and wash the plate 5 times.

2. Automatic washing machine: Inject 350μL of washing solution into each hole, soak for 1min, and wash the plate 5 times.

Steps

1. The required slats were taken out from the aluminum foil pouch after equilibrating for 20 min at room temperature, and the remaining slats were sealed back to 4 ° C with a ziplock bag.
2. Set standard and sample wells, standard wells with different concentrations of standard 50μL;
3. The sample well was first added with 10 μL of the sample to be tested, and then the sample dilution was 40 μL; the blank well was not added.
4. In addition to the blank wells, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each well of the standard well and the sample well, and the reaction well was sealed with a sealing plate, and incubated at 37 ° C in a water bath or incubator for 60 min.
5. Discard the liquid, pat dry on the absorbent paper, fill each well with the washing solution, let stand for 1 min, remove the washing solution, pat dry on the absorbent paper, and repeat the washing 5 times (can also be washed with a washing machine).
6. 50 μL of each of the substrates A and B was added to each well, and incubated at 37 ° C for 15 min in the dark.
7. 50 μL of the stop solution was added to each well, and the OD value of each well was measured at a wavelength of 450 nm within 15 min.

Result judgment

Draw a standard curve: In the Excel worksheet, the standard concentration is used as the abscissa, and the OD value is plotted as the ordinate. The linear regression curve of the standard is drawn, and the concentration values ​​of each sample are calculated according to the curve equation.

Kit performance

1. Accuracy: The linear regression coefficient of the standard and the expected concentration correlation coefficient R value, greater than or equal to 0.9900.
2. Sensitivity: The minimum detection concentration is less than 1.0 pg/mL.
3. Specificity: Does not cross-react with other soluble structural analogs.
4. Repeatability: The coefficient of variation between the plates and the plates is less than 15%.
5. Storage: 2-8 ° C, protected from light and moisture.
6. Validity: 6 months

Disclaimer

1. The kit is for research use only and should not be used for clinical trials or human experiments. Otherwise, the consequences will be borne by the experimenter and the company will not be responsible.
2. In strict accordance with the instructions, the experimenter violates the instructions, and the consequences are borne by the experimenter.

FOR RESEARCH USE ONLY.

Plant Grow Hormone (GH) ELISA Kit instruction

Intended use

The GH ELISA kit is intended Laboratory for Research use only and is not for use in the therapeutic procedures. The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the The concentration of GH in the sample, the GH ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density versus GH concentration. The concentration of GH In the samples is then determined by comparing the OD of the samples to the standard curve.

Sample collection and storages

1. Can't detect the samples which contain NaN3, because NaN3 inhibits HRP activity of the horseradish peroxidase.

2. Extract as soon as possible after Specimen collection, Extracted according to the relevant literature.

Cell culture supernates and plant exact fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20°C or -80°C. Avoid repeated freeze-thaw.

Materials required but not supplied

1. Standard microplate reader (450nm)

2. Precision pipettes and Disposable pipette tips.

3. 37 °C incubator

Precautions

1. Do not substitute reagents from one kit to another. Standard, conjugate and microplates are matched for optimal performance. Use only the reagents supplied by manufacturer.

2. Do not remove microplate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.

3. Mix all reagents before using.

Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25°C)

Supplies supplied

Note: Standard (S0 → S5) concentration was followed by: 0,5,10,20,40,80 U/mL

Reagent preparation

20×wash solution:Dilute with Distilled or deionized water 1:20.

Assay procedure

1. Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.

2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.

3. Add Sample: Add testing sample 10μl then add Sample Diluent 40μl to testing sample well; Blank well doesn't add anyting.

4. Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C.

5. Aspirate each well and wash, repeating the process four times for a total of five washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential After the last wash, remove any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper towels.

6. Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.

7. Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not

Appearance uniform, gently tap the plate to ensure thorough mixing.

8. Read the Optical Density (OD) at 450 nm using a microtiter plate reader within 15 minutes.

Calculation of results

1. The standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average OD (450 nm) obtained for each of the six standard concentrations on the vertical (Y) axis versus the corresponding concentration on the horizontal (X) axis.
2. First, calculate the mean OD value for each standard and sample. All OD values, are subtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software.
3. To determine the amount in each sample, first locate the OD value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration.
4. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve.
5. The sensitivity by this assay is 1.0 U/mL
6. Standard curve

Storage: 2-8 ° C.

Validity: six months.

FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!

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