• Dog sPDL2 ELISA Kit
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Dog sPDL2 ELISA Kit

Dog sPDL2 ELISA Kit

Catalog Number: abs554432 Application: ELISA Reactivity: Canine Conjugation:
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$381 USD
$381 USD
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Product Details

Product Specification

Usage

Specimen Requirements


1. Specimens should be extracted as soon as possible after collection, following the relevant literature.
Experiments should be performed as soon as possible after extraction.
If experiments cannot be performed immediately, specimens can be stored at -20°C, but repeated freezing and thawing should be avoided.

2. Samples containing NaN3 cannot be assayed, as NaN3 inhibits horseradish peroxidase (HRP) activity.

Procedure


1. Dilution of Standard: This kit provides one standard sample at full strength.
Users can dilute the sample in a small test tube according to the following chart.

4ng/L

32ng/L

Standard No. 5

150μl of the original standard was added to 150μl of the standard diluent

16ng/L

Standard No. 4

150μl of the original standard was added to 150μl of the standard diluent

16ng/L

Standard No. 4 150μl of No. 4 standard was added to 150μl of standard diluent

4ng/L

No. 2 standard

150μl of No. 3 standard was added to 150μl of standard diluent

No. 2 standard

150μl of No. 3 standard was added to 150μl of standard diluent

2ng/L

Add 150 μl of Standard No. 2 to 150 μl of Standard Diluent


2. Sample Addition: Set up blank wells (blank control wells do not contain sample or enzyme-linked reagent; all other steps remain the same), standard wells, and test sample wells.
Accurately add 50 μl of the standard to the enzyme-linked plate.
First, add 40 μl of sample diluent to the test sample wells, followed by 10 μl of the test sample (final sample dilution is 5-fold).
Add the sample to the bottom of the plate well, avoiding contact with the well walls.
Gently shake to mix.


3. Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes.
4. Preparation: Dilute the 30x concentrated wash buffer 30x with distilled water and set aside.
5. Wash: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with wash buffer, let it sit for 30 seconds, then discard.
Repeat this process 5 times and pat dry.
6. Add enzyme: Add 50µl of enzyme-labeled reagent to each well, excluding the blank well.
7. Incubation: Same as in 3.
8. Wash: Same as in 5.
9. Color development: First add 50µl of Color Developer A to each well, then add 50µl of Color Developer B.
Gently shake to mix.
Develop at 37°C in the dark for 10 minutes.
10. Stop: Add 50µl of Stop Buffer to each well to terminate the reaction (the blue color will immediately turn yellow).


11. Measurement: Use the blank well as the zero setting and measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm.
Measurements should be performed within 15 minutes after adding the stop solution.

Calculation


Use the standard concentration as the horizontal axis and the OD value as the vertical axis to draw a standard curve on graph paper.
Based on the sample OD value, find the corresponding concentration from the standard curve; then multiply by the dilution factor.
Alternatively, use the standard concentration and OD value to calculate the linear regression equation for the standard curve.
Substitute the sample OD value into the equation to calculate the sample concentration.
Multiply by the dilution factor to obtain the actual sample concentration.
Species Reactivity Dog
Theory This kit uses a double-antibody sandwich assay to measure canine soluble programmed death ligand-2 (sPDL2) levels in samples. A microplate is coated with purified canine soluble sPDL2 antibody to create a solid-phase antibody. Soluble sPDL2 is then added sequentially to the antibody-coated wells. This antibody then binds to HRP-labeled sPDL2 antibody, forming an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the plate is then developed with the substrate TMB. TMB converts the color to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of soluble sPDL2 in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader, and the concentration of canine soluble sPDL2 in the sample is calculated using a standard curve.
Detection Type Used to determine the content of soluble programmed death ligand-2 (sPDL2) in canine serum, plasma and related fluid samples.
Composition

Serial number

Component name

Specification

Serial number

Component name

Specification

1

30 times concentrated detergent

20ml×1 bottle

6

Developer B solution

6ml×1/bottle

2

Enzyme Labeled Reagent

6ml×1 bottle

7

Stop Solution

6ml×1 bottle

3

Enzyme-coated plate

12 wells×8 strips

8

Standard (64ng/L)

0.5ml×1 bottle

4

Sample diluent

6ml×1 bottle

9

Standard diluent

1.5ml×1 bottle

5

Developer A Liquid

6ml×1 bottle

10

Sealing film

2 photos
General Notes The kit should be removed from the refrigerator and allowed to equilibrate at room temperature for 1 hour before use. If the enzyme-coated plate is opened but not completely used, it should be stored in a sealed bag. Crystals may form in the concentrated wash solution. Warming in a water bath can aid dissolution during dilution. Washing does not affect the results. A pipette should be used for each sample addition step, and its accuracy should be checked frequently to avoid experimental errors. The time for each sample addition should ideally be within 5 minutes. For large numbers of samples, using a dispenser is recommended. A standard curve should be generated with each measurement, preferably in duplicate. If the analyte content in the sample is too high (the OD value of the sample is greater than the OD value of the first standard well), dilute the sample a certain number of times (n times) with sample diluent before measurement. When calculating the total dilution factor, multiply the final dilution by the total number of times (n times 5). Seal the plate with film for single use only to prevent cross-contamination.
  • Store the substrate away from light.
  • Strictly follow the instructions for use. The test results must be determined based on the readings of the microplate reader.
  • All samples, wash solutions, and various wastes should be treated as infectious agents.
  • Components from different batches of this reagent must not be mixed.
    		•
    Storage Temp. If the unopened test kit is sealed and stored at 2-8°C, it is valid for 6 months.
    Test Range 0.5ng/L - 35ng/L