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Mouse Hyaluronidase ELISA Kit

SKU:
MOFI00879
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q91ZJ9
Sensitivity:
2.344ng/ml
Range:
3.906-250ng/ml
ELISA Type:
Sandwich
Synonyms:
HAase
Reactivity:
Mouse
$779
Frequently bought together:

Description

Kit Name

Hyaluronidase is an enzyme that plays a vital role in tissue remodeling, wound healing, and various physiological processes. The Mouse Hyaluronidase ELISA Kit empowers researchers to study the dynamics of hyaluronidase levels, providing insights into its involvement in tissue homeostasis, disease models, and therapeutic interventions.

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:

Mouse Hyaluronidase ELISA Kit

Product Code:

MOFI00879

Size:

96 Assays

Alias:

HAase

Detection Method:

Sandwich ELISA

Application:

This immunoassay kit allows for the in vitro quantitative determination of Mouse HAase concentrations in serum plasma and other biological fluids.

Sensitivity:

2.344ng/ml

Range:

3.906-250ng/ml

Storage:

4°C for 6 months

Note:

For Research Use Only

Additional Information

Recovery

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

88-104

Matrix

Recovery Range (%)

Average (%)

serum (n=5)

88-98

93

EDTA plasma (n=5)

88-104

94

UFH plasma (n=5)

89-95

93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse HAase 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)

85-104%

86-103%

96-104%

EDTA plasma (n=5)

88-96%

87-100%

84-101%

UFH Plasma (n=5)

80-94%

82-90%

80-100%

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 exact instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. 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-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!

2.

Aliquot 0.1ml standard solutions into the standard wells.

3.

Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.

4.

Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.

5.

Seal the plate with a cover and incubate at 37 °C for 90 min.

6.

Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.

7.

Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.

8.

Seal the plate with a cover and incubate at 37°C for 60 min.

9.

Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.

10.

Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.

11.

Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.

12.

Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.

13.

Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.

14.

Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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.

Hyaluronidase Background

Hyaluronidase

Hyaluronidase is an enzyme that plays a crucial role in breaking down hyaluronic acid, a naturally occurring polysaccharide found in various connective tissues, synovial fluid, and the extracellular matrix of animals, including humans. Hyaluronic acid is responsible for maintaining tissue hydration and lubrication, as well as contributing to the structural integrity of tissues.

Hyaluronidase Structure

Hyaluronidase enzymes are glycosidases, which means they catalyze the hydrolysis of glycosidic bonds in complex carbohydrates. These enzymes are often present in various organisms, including bacteria, animals, and some venomous creatures. The structure of hyaluronidase can vary among species, but it generally consists of an active site responsible for the cleavage of glycosidic bonds in hyaluronic acid molecules.

Hyaluronidase Function

The primary function of hyaluronidase is to degrade hyaluronic acid and other glycosaminoglycans, like chondroitin sulfate, into smaller fragments. This enzymatic degradation is essential for various physiological processes, including tissue remodeling, wound healing, and the spread of substances within tissues. By breaking down the large molecules into smaller components, hyaluronidase promotes tissue permeability and facilitates the movement of cells and fluids.

Hyaluronidase Mechanism of Action

Hyaluronidase catalyzes the hydrolysis of the glycosidic bonds that link the sugar units in hyaluronic acid and other glycosaminoglycans. This cleavage results in the fragmentation of the large polysaccharide chains into smaller fragments. The enzyme achieves this by attacking specific chemical bonds within the glycosidic structure, leading to the breakdown of the molecule into simpler sugars.

Hyaluronidase Clinical Significance

Hyaluronidase has several clinical applications. It is commonly used as an adjuvant in medical procedures and treatments where enhanced tissue permeability is desirable. For instance, hyaluronidase is employed to aid the dispersion and absorption of fluids, such as local anesthetics, in subcutaneous injections. It also finds use in intravenous therapy to improve the distribution of injected fluids in cases of emergency or certain medical conditions.

Hyaluronidase Inhibitors

Researchers have explored the development of hyaluronidase inhibitors for various purposes, including potential therapeutic applications. These inhibitors could help regulate the enzymatic activity of hyaluronidase and prevent excessive tissue degradation, which might be beneficial in certain disease conditions or during specific medical interventions.

Mouse Hyaluronidase ELISA Kit FAQs

Q: What is the purpose of the Mouse Hyaluronidase ELISA Kit?

The Mouse Hyaluronidase ELISA Kit is specifically designed to quantify the concentration of hyaluronidase in mouse samples. Hyaluronidase is an enzyme that plays a vital role in tissue remodeling, wound healing, and various physiological processes. This kit empowers researchers to study the dynamics of hyaluronidase levels, providing insights into its involvement in tissue homeostasis, disease models, and therapeutic interventions.

Q: What type of samples can be used with this kit?

This ELISA kit is versatile and can be used with a wide range of sample types, including cell culture supernatants, serum, plasma, and other biological fluids. It accommodates diverse research applications, allowing you to explore hyaluronidase dynamics in various biological contexts.

Q: Is the Mouse Hyaluronidase ELISA kit compatible with other species besides mouse?

This Mouse Hyaluronidase ELISA kit has been developed specifically for mouse samples. While it may cross-react with HYAL protein from other species, it is recommended to consult the product manual or contact technical support for information regarding cross-reactivity.

Q: Where can I find additional technical support or assistance with the kit?

For any technical inquiries or assistance regarding the kit, you can reach out to our team. They will be available to answer your questions and provide the necessary guidance to ensure a successful experiment.

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