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Canine HS (Heparan Sulfate) ELISA Kit (CNFI00073)

SKU:
CNFI00073
Product Type:
ELISA Kit
Size:
96 Assays
Reactivity:
Canine
Sensitivity:
<18.75ng/ml
Range:
31.25-2000ng/ml
€649
Frequently bought together:

Description

Canine HS (Heparan Sulfate) ELISA Kit (CNFI00073)

system_update_alt Datasheet system_update_alt MSDS

Key Features

Save Time Pre-coated 96 well plate
Quick Start Kit includes all necessary reagents
Publication Ready Reproducible and reliable results

Overview

Product Name:

Canine HS (Heparan Sulfate) ELISA Kit

Product Code:

CNFI00073

Size:

96 Assays

Detection Method:

Competitive ELISA, Coated with Antigen

Application:

HS ELISA Kit allows for the in vitro quantitative determination of Heparan Sulfate concentrations in serum, plasma, tissue homogenates and other biological fluids.

Sensitivity:

<18.75ng/ml

Range:

31.25-2000ng/ml

Storage:

2-8°C for 6 months

Note:

For Research Use Only

Additional Information

Recovery

Matrices listed below were spiked with certain level of Heparan Sulfate and the recovery rates were calculated by comparing the measured value to the expected amount of Heparan Sulfate in samples.

Matrix

Recovery Range (%)

Average (%)

serum(n=5)

89-104

98

EDTA plasma(n=5)

87-101

95

heparin plasma(n=5)

90-104

98

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Heparan Sulfate and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample

1:2

1:4

1:8

serum(n=5)

85-103%

87-105%

87-104%

EDTA plasma(n=5)

82-100%

91-98%

84-101%

heparin plasma(n=5)

83-99%

85-100%

88-100%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Kit Components

Component Quantity Storage

ELISA Microplate (Dismountable)

8x12 strips

4°C for 6 months

Lyophilized Standard

2

4°C/ -20°C

Sample/Standard Dlution Buffer

20ml

4°C

Biotin-labeled Antibody (Concentrated)

120ul

4°C (Protection from light)

Antibody Dilution Buffer

10ml

4°C

HRP-Streptavidin Conjugate (SABC)

120ul

4°C (Protect from light)

SABC Dilution Buffer

10ml

4°C

TMB Substrate

10ml

4°C (Protection from light)

Stop Solution

10ml

4°C

Wash Buffer (25X)

30ml

4°C

Plate Sealer

5

-

Other materials required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir

Protocol

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Equilibrate the TMB substrate for at least 30 min at 37°C beforeuse. When diluting samples and reagents, they must be mixed completely andevenly. It is recommended to plot a standard curve for each test.

Step Procedure

1.

Set standard, test sample and control (zero) wells on the pre-coatedplate respectively, and then, record their positions. It isrecommended to measure each standard and sample in duplicate. Washplate 2 times before adding standard, sample and control (zero) wells!

2.

Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank or Sample per well. The blankwell is added with Sample Dilution Buffer. Immediately add 50 µL of biotin-labelled antibody workingsolution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thoroughmixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro-ELISA platewell, avoid touching plate walls and foaming).

3.

Wash: Aspirate each well and wash, repeating the process three timesWash by filling each well with Wash Buffer (approximately 350µL)using a squirt bottle, multi-channel pipette, manifold dispenser orautomated washer. Complete removal of liquid at each step is essentialto good performance. After the last wash, remove any remaining WashBuffer by aspirating or decanting. Invert the plate and pat it againstthick clean absorbent paper.

4.

HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC workingsolution to each well. Cover with a new Plate sealer. Incubate for30minutes at 37°C.

5.

Wash: Repeat the aspiration/wash process for five times.

6.

TMB Substrate: Add 90µL of TMB Substrate to each well. Coverwith a new Plate sealer. Incubate for about 10-20 minutes at 37°C.Protect from light. The reaction time can be shortened or extendedaccording to the actual color change, but not more than 30minutes.When apparent gradient appeared in standard wells, you can terminatethe reaction.

7.

Stop: Add 50µL of Stop Solution to each well. Color turn toyellow immediately. The adding order of stop solution should be as thesame as the substrate solution.

8.

OD Measurement: Determine the optical density (OD Value) of each wellat once, using a microplate reader set to 450 nm. You should open themicroplate reader ahead, preheat the instrument, and set the testing parameters.

Sample Preparation

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type Protocol

Serum

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

Plasma

Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.

Urine & Cerebrospinal Fluid

Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.

Cell culture supernatant

Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.

Cell lysates

Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Tissue homogenates

The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.

Tissue lysates

Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C

Breast Milk

Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

Canine HS Background

Heparan sulfate is a complex carbohydrate molecule that belongs to the class of glycosaminoglycans (GAGs). It is composed of repeating units of disaccharides, consisting of glucuronic acid (or its derivative iduronic acid) and N-acetylglucosamine. Heparan sulfate is found on the cell surface and in the extracellular matrix of various tissues throughout the body.

Cell Signaling and Regulation

Heparan sulfate plays a crucial role in cell signaling by interacting with a variety of growth factors, cytokines, and morphogens. It acts as a modulator, regulating signaling pathways involved in embryonic development, tissue repair, and homeostasis. Key interactions with fibroblast growth factors (FGFs), transforming growth factor-beta (TGF-β), and Hedgehog proteins highlight the importance of heparan sulfate in orchestrating these complex processes.

Cell Adhesion and Migration

Contribution to cell adhesion and migration is another significant function of heparan sulfate. Acting as a binding site for cell adhesion molecules, it facilitates these essential processes during tissue development, inflammation, and wound healing. By assisting cellular movements and promoting tissue organization, heparan sulfate plays a crucial role in guiding the dynamic behavior of cells.

Extracellular Matrix Organization and Anticoagulant Activity

Heparan sulfate also plays a role in the organization of the extracellular matrix. Through interactions with collagens, elastin, and other components, it contributes to the structure and integrity of tissues. Additionally, heparan sulfate exhibits anticoagulant activity by acting as a cofactor for antithrombin III, preventing excessive blood clotting. These multifunctional properties of heparan sulfate underline its significance in maintaining tissue architecture and preventing thrombotic events.

Canine HS FAQs

What is the Canine HS ELISA Kit?

The Canine HS ELISA Kit (CNFI00073) is an enzyme-linked immunosorbent assay designed to measure Canine Heparan Sulfate (HS) levels in biological samples

What are the benefits of using the Canine HS ELISA Kit?

The Canine HS ELISA Kit provides accurate quantification of Canine HS levels in various biological samples, such as serum, plasma, and tissue extracts. Its high sensitivity ensures the detection of even low HS levels. By delivering reliable and precise measurements, the kit facilitates detailed investigations into the role of HS in canine health and disease.

Where can I find more information about the Canine HS ELISA Kit?

For more detailed information about the Canine HS ELISA Kit, including technical specifications, performance characteristics, and ordering details, please refer to the product brochure or contact our customer support team. We are here to assist you with any inquiries you may have.