TECO® Hyaluronic Acid (HA) PLUS ELISA

Reagents and Materials Supplied

Materials Required and not Supplied

• Pipettes 10 µl – 1000 µl
• Multichannel pipettes for 50 µl –100 µl
• Graduated cylinders for reconstituting or diluting reagents
• Manual Aspiration System or Automatic washer for ELISA plates
• Aqua dest
• Vortex mixer
• ELISA plate reader suitable for 96 well formats and capable of measuring at 450 nm (Reference: 590-650 nm)
• ELISA plate shaker (500 rpm) (orbital shaker)
• Software package for data generation and analysis

The TECO® assay kit for hyaluronic acid is a sensitive sandwich assay using a microtiter plate coated with HA binding protein (HABP) and HRP conjugated HABP for detection. This HRP conjugated HABP binds to sample HA and is followed by a substrate reaction. The color development is catalyzed quantitatively dependent on HA levels of the samples.

Assay Principle

The TECO® assay kit for hyaluronic acid is a sensitive sandwich assay using a microtiter plate coated with HA binding protein (HABP) and HRP conjugated HABP for detection. This HRP conjugated HABP binds to sample HA and is followed by a substrate reaction. The color development is catalyzed quantitatively dependent on HA levels of the samples.

Assay Procedure

All determinations (standards, controls and samples) should be assayed in duplicate. When performing the assay, the standards, controls and samples should be pipetted as fast as possible (<15 minutes). To avoid distortions due to differences in incubation times, HABP-HRP conjugate, substrate solution and stop solution should be added to the plate in the same order and with the same time interval as the samples. A multichannel pipette is essential.

Allow all reagents to stand at room temperature (20–25 °C) for at least 30 minutes. During all incubation steps, plates should be sealed with the adhesive foil or a plastic cover. For light protection, incubate in a dark chamber or cover plate with aluminium foil.

1. Allocate the wells of the Microtiterplate  1  for standards, controls and samples.
2. Dilute standards ( A  till  F ), controls ( C1  and  C2 ) and samples 1:50 with Sample Diluent  3 .
3. Pipette 100 µl of each diluted standards ( A  till  F ), controls ( C1  and  C2 ) and samples into the corresponding wells.
4. Cover the wells with a plastic cover and incubate the plate for 2h ± 5 min at room temperature (20–25 °C) on a shaker (500 rpm).
5. After incubation, aspirate the wells by using a plate washer or manually decant by inverting the plate. Wash the wells 3 times with 350 µl diluted wash buffer per well. After the last wash cycle tap the inverted wells on a dry absorbent surface to remove excess wash solution. The use of an automatic plate washer is recommended.
6. Following the last washing step, pipette 100 µl of the HABP-HRP conjugate  6  in each well (multichannel pipette).
7. Cover the wells with a plastic cover and incubate the plate for 30 ± 5 min at room temperature (20–25 °C) on a shaker (500 rpm).
8. After incubation wash the wells 5 times with wash buffer as described in step 5.
9. Pipette 100 µl of the TMB substrate solution  7  in each well (multichannel pipette).
10. Incubate the plate for 30 min, in the dark, at room temperature (20–25 °C) on a shaker (500 rpm).
11. Stop the reaction by adding 100 µl of stop solution  8  (multichannel pipette).
    Measure the color reaction within 10 minutes at 450 nm (reference filter between 590–650 nm). If the extinction of the std  F  exceeds 3.0, the measurement should be repeated at 405 nm.

Protocols for the different automatic ELISA systems are available.

Research Use

Hyaluronic acid (HA), also known as hyaluronan or hyaluronate is a large linear non-sulfated glycosaminogly-can (GAG) with a molecular weight between 106 and 107 Da. It is a major component of connective tissues and thus distributed ubiquitously in the organism. About one-half of the body’s entire hyaluronan is found in the skin and about one fourth in the skeleton and its supporting structures like ligaments and joints. Hyaluronan is synthesized by fibroblasts and other specialized connective tissue cells.

Hyaluronan is especially important for the structure and organization of extracellular matrices. The hyaluro-nan network acts as an osmotic buffer and is reponsible for water homeostasis as well as it regulates protein distribution via the formation of flow and diffusion barriers. Additionally, hyaluronan interacts with proteins and cell surfaces and thus has a strong influence on cell proliferation, differentiation and tissue repair.

Turnover and catabolism

The tissue half-life of hyaluronan differs between species and varies from about one to several days. A certain amount of hyaluronan is degraded locally, but the much larger part is removed and degraded by the lympha-tic system. The remainder enters the blood circulation where it is removed primarily by liver endothelial cells. A minor portion is metabolized by the kidneys and the spleen.

Adult cartilage is avascular and depends upon the synovial fluid to provide nutrition; as well as, the disposal of metabolic wastes. Thus, hyaluronan resulting from cartilage degradation is first released into the synovial fluid where it enters the blood and lymph stream, respectively, through the highly vascularized synovial membrane.

The serological half-life of hyaluronan is about 2–5 minutes. The normal adult human serological level of hya-luronan varies between 10 and 100 μg/l and the total hyaluronan turnover in serum is estimated to be in the range of 10–100 mg/24 h. Serum hyaluronan is influenced by various factors including age, sex and ethnicity as well as food intake and the level of physical activity.