PF4 ELISA Kit

Catalog No. ABIN456191

Product Details PF4 ELISA Kit

Target: PF4 (Platelet Factor 4 (PF4))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0.312-20 ng/mL

Minimum Detection Limit: 0.312 ng/mL

Application: ELISA

Purpose: This immunoassay kit allows for the use in vitro quantitative determination of human Platelet Factor 4, PF-4 concentrations in plasma.

Sample Type: Plasma

Analytical Method: Quantitative

Specificity: This assay recognizes recombinant and natural human PF-4.

Cross-Reactivity (Details): No significant cross-reactivity or interference was observed.

Sensitivity: < 0.78 ng/mL
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.

Characteristics: Homo sapiens,Human,Platelet factor 4,PF-4,C-X-C motif chemokine 4,Iroplact,Oncostatin-A,PF4,CXCL4,SCYB4

Components: Reagent (Quantity): Assay plate (1), Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay DiluentB 1 x 10ml Detection Reagent A (1x120µl), Detection Reagent B (1x120µl), Wash Buffer 1 x 30ml 2 (25 x concentrate) Substrate (1x10ml), Stop Solution (1x10ml), Plate sealer for 96 wells (1x5)

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol: The microtiter plate provided in this kit has been pre-coated with an antibody specific to PF-4. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for PF-4 and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3'5, 5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain PF-4, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of PF-4 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reagent Preparation:

Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 2,000 ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. Please firstly dilute the stock solution to 200 ng/mL and the diluted standard serves as the high standard (200 ng/mL). The Sample Diluent serves as the zero standard (0 ng/mL). 3 ng/mL 2,000 200 100 50 25 12.5 6.25 3.12 0 Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.

Sample Collection: Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8 °C within 30 minutes of collection. Store samples at -20 °C or -80 °C. Avoid repeated freeze-thaw cycles. Note: Plasma samples to be used within 7 days may be stored at 2-8C, otherwise samples must stored at -20 °C (≤ 3 months) or -80 °C (≤ 6 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature. It is recommended that all samples be assayed in duplicate.

Assay Procedure:

Allow all reagents to reach room temperature. All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Arrange and label required number of strips. Prepare all reagents, working standards and samples as directed in the previous sections.
1. Add 100 uL of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37 °C.
2. Remove the liquid of each well, don’t wash.
3. Add 100 uL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37°C. Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
5. Add 100 uL of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hours at 37 °C.
6. Repeat the aspiration/wash as in step
4. 7. Add 90 uL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 30 minutes at 37°C. Protect from light.
8. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well at once, using a microplate reader set to 450 nm.
Important Note:
1. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards can be used only once.
2. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
3. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
4. Duplication of all standards and specimens, although not required, is recommended.
5. When mixing or reconstituting protein solutions, always avoid foaming.
6. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
7. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
8. Do not substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.

Calculation of Results:

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the PF-4 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Restrictions: For Research Use only

Handling

Handling Advice: 1. The kit should not be used beyond the expiration date on the kit label.
2. Do not mix or substitute reagents with those from other lots or sources.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

Storage: 4 °C/-20 °C

Storage Comment: The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.

Target Details for PF4

Target: PF4 (Platelet Factor 4 (PF4))

Alternative Name: PF4 (PF4 Products)

Synonyms: CXCL4 ELISA Kit, PF-4 ELISA Kit, SCYB4 ELISA Kit, Cxcl4 ELISA Kit, Scyb4 ELISA Kit, Pf4a ELISA Kit, RATPF4A ELISA Kit, PF4A ELISA Kit, CXCL4L1 ELISA Kit, CXCL4V1 ELISA Kit, PF4-ALT ELISA Kit, SCYB4V1 ELISA Kit, PF4 ELISA Kit, platelet factor 4 ELISA Kit, platelet factor 4 variant 1 ELISA Kit, PF4 ELISA Kit, Pf4 ELISA Kit, LOC703451 ELISA Kit, PF4V1 ELISA Kit, LOC100524373 ELISA Kit, LOC102181854 ELISA Kit, LOC100630489 ELISA Kit

Background: Platelet Factor (PF4) is a 70 amino acid, 30,000 D molecular ratio protein. PF4 is released from activated platelet alpha granules in a tetrameric form complexed with platelet proteoglycan. Upon release, the half-life of PF4 is very short, less than 5 minutes, as it quickly binds to endothelial cell glycosaminoglycans where it is stored. PF4 possesses a powerful anti-heparin activity as it binds to heparin, forming a stochiometric complex, where 1 mg of PF4 will inhibit 27 IU of heparin.