Only for study use Enzyme linked Immunosorbent Assay

HumanCD137ELISA Test Kit

Usage: Used for the testing ａnd determination of CD137 in serum, plasma ａnd other biological fluids.

Testing principle

This kit tests the level of samples rat CD137by using a biotinylated double antibody sandwich enzyme-linked immunosorbent assay (ELISA) . Add CD137into the enzyme hole which has been previously coated with rat CD137monoclonal antibody,then incubate; after incubation, add the biotin-labeled anti-CD137antibodies, then combine it with streptavidin -HRP to form immune complexes, remove unbound enzyme through incubation ａnd washing, ａnd then add substrate a, B, produces blue,and convert into the final yellow in the action of acid . The depth of color has a positive correlation with the HumanCD137in the sample .

Kit Contents

Reagents ａnd equipment required but not supplied

1. Standard microplate reader ( filter with detection wavelength of 450nm , filter with calibration wavelength of 570nm or 630nm ).

2. The washer (injection fluid volume is adjustable to ensure that each hole can be added to 0.35ml lotion without overflow).

3. Benchtop, biological safety cabinet, fume hood.

4. The high-precision single-channel dosing device (range is 0.5-10μl, 2-20μl, 20-200μl, 200-1000μl).

5. 5. High-precision multi-channel dosing device (8 or 12 channels, the range is 50-300μl).

6 37 ℃ incubator.

7 The low temperature centrifuge.

8 Refrigerator (4 ℃, -20 ℃, -86 ℃).

9. Vortex mixer, low frequency oscillator, etc.

Precautions

1. Take a kit from 2-8 centigrade environment, balance it for at least 30minutes in room temperature before opening. ELISA plates coated if has not use up after opened, the plate should be stored in sealed bag.

2. Operate strictly in accordance with instructions, the microtiter plate reader must be as the criterion of the test result determination.

3. To avoid cross contamination, do not repeatedly use the tip ａnd seal plate membrane in the hands.

4. Other unused agents need to be well-packed or covered. Do not mix different batches of reagents. Use before the expiration date..

5. Substrate B is sensitive to light, avoid long time exposure to light.

6. All samples, washing buffer ａnd each kind of reject should be processed by biological waste treatment. Stop Solution is 2M sulfuric acid, must be cautious when using.

7. Use a pipette for each step of sampling, ａnd often calibrate its accuracy to avoid the experimental error. Each sampling time should be best controlled in five minutes, if there are a large number of sample, a Volley is recommended for use.

8. Make a standard curve for each test. If the content of testing material in the sample is too high (sample OD value is greater than that of the highest concentration in standard hole ), first dilute sample dilution with a certain times (n-time) ａnd then test, please multiplyby the diluted times when calculating (× n).

9. When preparing reagents, use a vortex mixer to well mix it. Add reagent in the hole, well-mixing is particularly important to the test results, it is best to use a micro-oscillator (lowest frequency), if there is no micro oscillator, the plate can be shaken slightly manually for one minute befor action, for example, a circular move can make the reaction solution added well- mixed.

The microplate reader used for experiment should be operated in strictly accordance with instructions ａnd standards, ａnd it should be fully preheated before use.

11. As for the hand-washer, things should be noted: remove the fluid within the microplate; spread layers of absorbent paper on the test-bed, ａnd put microplate downward ａnd slap for several times; inject the diluted washing solution into the hole for at least 0.35ml, then soak for 1-2 minutes. If necessary, repeat this process several times. Automatic Plate Washer: If the automatic plate washer is available, it can be used in a formal experimen after being skilled in using it .

Sample Requirements

1. The sample which contain NaN3 can not be tested, because NaN3 inhibits horseradish peroxidase (HRP) activity.

2. Extract as soon as possible after sample collection according to the relevant documents, ａnd it should be tested as soon as possible after the extraction. If it can not be tested immediately, keep it in -20 ℃ or make corresponding adjustments depending on the storage temperature ａnd time, but avoid repeated freezing ａnd thawing, (due to excessive sample types, each unit process is not the same, ａnd this instruction do not give a detailed description).

Operation process

Dilution of teh standard: :( 1 original standard product is provided by this reagent system, users should dilute in a small test tube on their own by following the instructions ) perform as the following table:

1. Determine the number of plates needed according to the number of samples to be tested ａnd the number of standard product. Each standard product ａnd blank hole is recommended to make multiple holes. The number of samples can be decided by users, ａnd make multiple holes as far as possible.

2. Sample adding: 1) :( blank holes: sample, biotin-labeled anti CD137antibodies, streptavidin-HRP should not be added into the blank holes, ａnd other stepsare the same; 2) standard product hole: Add standard product 50μl , streptavidin－－HRP50μl (standard products have integrated well in advance the biotinylated antibody, it is not added at the time of operation, ａnd only add streptomycin -HRP); 3) sample action holes: Add sample 40μl, then each added anti CD137antibody 10μl, ａnd 50μl of streptavidin-HRP (except blank holes). Cover with the seal plate membrane, and mix gently by shaking, incubate in 37 ℃ temperature for 60 minutes.

3. Dosing liquid: dilute 30-time concentrated washer with distilled water in 30 times for use, ａnd 20-time concentrated washer with distilled water in 20 times for use.

4. Washing: Uncover the plate membrane, remove liquid, then dry it up, add washing liquid to fill each hole, after stewing for 30 seconds then remove it, repeat for 5 times, pat dry.

5. coloration: add each hole with the chromogenic reagent A 50 μl, then add reagent B 50μl, gently shake to mix, keep in 37 ℃ in dark for 10 minutes.

6. Stop solution: each hole add stop solution 50μl, then stop the reaction (the blue color change into yellow immediately).

7. Determination: adjust blank hole to zero, wavelength of 450nm sequentially measured the absorbance of each well (OD value). Determination should be conducted within 10 minutes after adding the stop solution.

Results Judgements

1. OD value of each standard product ａnd sample should subtract the OD value of zero hole. (If the value of zero hole is not diminished, the zero hole of the standard curve should intersect at Y-axis)

2. Calculate the linear regression equation of the standard curve based on the concentration ａnd the corresponding OD value of the standard product, then calculate the corresponding sample concentration on the regression equation according to OD values of samples. A variety of familiar applications software can be used for calculation.

3. Draw the standard curve manually. Set the standard products concentration as abscissa, OD value as the vertical axis, connecting coordinate points of the standard products with a smooth line. The concentrations can be detected on the standard curve based on the OD value. Professional curve production software is recommended for analyzing the calculation results.

If the OD value of samples is higher than the upper limit of the curve, it should be retested after appropriate dilution, the dilution factor should be multiplied by when calculating the concentration.

Operation summary

Prepare reagents, samples ａnd standard products

Add prepared samples ａnd standard products, biotin-labeled secondary antibody ａnd enzyme reagent, action at 37 ℃ for 60 minutes.

Washed five times, adding color agent A ａnd B, ａnd colorated at 37 ℃ for 10 minutes

Add TMB stop solution

value with microplate reader in 10 minutes

Calculate the factor content of the test sample

Kit Performance

1. Accuracy: Correlation coefficient R value of standard products linear regression ａnd of the expected concentration is greater than or equal to 0.9200.

2. Sensitivity: The minimum detectable concentration reaches 0.195ng/ml

3. Specificity: No cross-reaction with other soluble structural analogs.

4. Repeatability: inner plate ａnd inter-plate variation coefficients are less than 15%.

5. Recovery : 70-110%.

6. Storage: 2-8 centigrade, keep in dark ａnd dry place

7. Expiration duration: 6 months

8. Detection range: 80ng/ml -0.78ng/ml