Dog sPDL1 ELISA Kit

Catalog Number: abs554431 Application: ELISA Reactivity: Canine Conjugation:

Price:

$381 USD

Size: 96T

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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 freeze-thaw cycles should be avoided.
2. Samples containing sodium nitrate (NaN3) cannot be assayed, as NaN3 inhibits horseradish peroxidase (HRP) activity.

Procedure

1. Dilution of the 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.

5ng/L

40ng/L	Standard No. 5	150μl of original standard was added to 150μl of standard diluent
20ng/L	Standard No. 4	150μl of standard No. 5 was added to 150μl of standard diluent
5ng/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
2.5ng/L		To 150 μl of Standard No. 2, add 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. To the test sample wells, first add 40 μl of sample diluent, then add 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 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. Washing: 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 five 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 step 3.
8. Washing: Same as in step 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 Solution to each well to terminate the reaction (the blue color will immediately turn yellow). 11. Measurement: Use the blank well as a zero setting and measure the absorbance (OD) of each well sequentially at a wavelength of 450 nm. Measurements should be performed within 15 minutes after adding the stop solution. Plot a standard curve on graph paper, with the standard concentration as the horizontal axis and the OD value as the vertical axis. Based on the sample OD value, determine the corresponding concentration from the standard curve. Multiply this value by the dilution factor. Alternatively, calculate the linear regression equation for the standard curve using the standard concentration and OD value. Substitute the sample OD value into the equation to calculate the sample concentration. Multiply this by the dilution factor to obtain the actual sample concentration.

Standard Curve

Species Reactivity

Dog

Theory

This kit uses a double-antibody sandwich assay to measure canine soluble programmed death ligand-1 (sPDL1) levels in samples. A microplate is coated with purified canine soluble sPDL1 antibody to create a solid-phase antibody. Soluble sPDL1 is then added sequentially to the antibody-coated wells. This antibody then binds to HRP-labeled soluble sPDL1 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 sPDL1 in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader, and the concentration of canine soluble sPDL1 in the sample is calculated using a standard curve.

Detection Type

Used to determine the content of soluble programmed death ligand-1 (sPDL1) 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 (80ng/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

1. After removing the kit from the refrigerator, allow it to equilibrate at room temperature for 1 hour before use. If the enzyme-coated plate is opened but not completely used, store it in a sealed bag.
2. Crystals may form in the concentrated wash solution. Warming in a water bath during dilution can aid dissolution. This will not affect the results during washing.
3. Use a pipette for each sample addition step and frequently calibrate its accuracy to avoid experimental error. The time for each addition should ideally be within 5 minutes. For large numbers of samples, using a dispenser is recommended.
4. Develop a standard curve with each measurement, preferably in duplicate. If the analyte concentration 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) with sample diluent before measurement. When calculating the total dilution factor (×n×5), multiply the final dilution factor.
5. Plate sealing film is for single use only to avoid cross-contamination.
6. Protect the substrate from light. 7. Strictly follow the instructions for use. Test results must be based on the readings of the microplate reader. 8. All samples, wash solutions, and waste should be treated as infectious agents. 9. Components from different batches of this reagent must not be mixed.

Storage Temp.

Unopened test kit, sealed and stored at 2-8℃, valid for 6 months

Test Range

1ng/L - 45ng/L

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Dog sPDL1 ELISA Kit