This reagent is intended for research use only and is suitable for the detection of hyaluronic acid (HA) in serum or plasma samples.
Test Principle:
The HA ELISA Kit operates on the principle of a solid-phase sandwich enzyme-linked immunosorbent assay. Known concentrations of HA standards and unknown samples are added to microtiter plates. The sample and biotinylated antibodies are incubated together, followed by washing. Avidin-conjugated HRP is then added, and after another incubation and washing step, the unbound enzyme conjugate is removed. Substrates A and B are then added simultaneously, leading to a color development that correlates with the HA concentration in the sample. The intensity of the color is directly proportional to the amount of HA present.
Self-contained Materials:
Distilled water, pipette tips (5 µl, 10 µl, 50 µl, 100 µl, 200 µl, 500 µl, 1000 µl), oscillators, magnetic stirrers, and other necessary equipment are included in the kit.
Safety Information:
Avoid direct contact with the stop solution and substrates A and B. In case of exposure, rinse thoroughly with water. Do not eat, drink, smoke, or apply cosmetics during the experiment. Never use your mouth to handle any reagents from the kit.
Operational Precautions:
Store reagents as instructed on the label and allow them to reach room temperature before use. Standards should be prepared fresh and discarded after use. Unused microtiter strips should be returned to the sealed bag immediately to prevent degradation. Keep all unused reagents properly covered and avoid mixing reagents from different batches. Use the kit before the expiration date. Always use disposable tips to prevent cross-contamination. Avoid using pipettes with metal parts when handling the stop solution and substrates. Prepare wash solutions in clean plastic containers. Mix all reagents and samples thoroughly before use. Ensure the microplate is fully dried after washing, and do not place absorbent paper directly into the wells. Store substrate A in a sealed container to prevent evaporation, and protect substrate B from light. Avoid contact with skin and wear appropriate protective gear. Read OD values immediately after the reaction is stopped. Maintain consistency in reagent addition order and incubation times as specified in the protocol.
Sample Collection, Processing, and Storage:
For serum: Avoid any cell damage during collection. Use endotoxin-free tubes. After blood collection, separate serum from red blood cells by centrifuging at 1000 x g for 10 minutes. For plasma: EDTA, citrate, or heparin anticoagulated plasma can be used. Centrifuge at 1000 x g for 30 minutes to remove the pellet. For cell supernatant: Centrifuge at 1000 x g for 10 minutes to remove debris. For storage: If not used immediately, divide the sample into small aliquots and store at -70°C to avoid repeated freezing. Avoid using hemolyzed or lipemic samples. If there are visible particles in the serum, centrifuge or filter the sample before testing. Thaw samples at room temperature only; avoid heating above 37°C.
Reagent Preparation:
Standards should be freshly prepared and not stored. Shake the standard before dilution.
Dilution of Wash Buffer (50x): Dilute with distilled water to a 50-fold concentration.
Steps:
Before starting, mix all reagents thoroughly. Avoid creating excessive foam to prevent air bubbles during loading, which may affect accuracy. Determine the number of wells needed based on the number of samples and standards. It is recommended to include each standard and blank well. Each sample should be tested in duplicate if possible.
Add 50 µl of diluted standard and 50 µl of sample to each well. Immediately add 50 µl of biotinylated antibody, cover the plate, gently shake, and incubate at 37°C for 1 hour.
Remove the liquid, fill each well with wash buffer, shake for 30 seconds, and discard. Repeat this process three times. If using an automated washer, increase the number of washes by one.
Add 80 µl of streptavidin-HRP to each well, gently shake, and incubate at 37°C for 30 minutes.
Repeat the washing steps as described above. Add 50 µl of substrates A and B to each well, mix gently, and incubate at 37°C for 10 minutes in the dark.
Add 50 µl of stop solution to each well immediately after the reaction. Read the OD values at 450 nm as soon as possible.
Limitations:
Results above the sixth standard are non-linear and cannot be accurately quantified using the standard curve.
Kit Performance:
1. Sensitivity: The minimum detectable concentration is below that of the first standard. The dilution linearity is excellent, with a correlation coefficient (R) of 0.990. 2. Specificity: No cross-reactivity with other human cytokines. 3. Repeatability: Inter-assay coefficient of variation is less than 10%.
Result Interpretation:
1. Measure the OD value at 450 nm using a microplate reader. 2. Plot the OD values (Y-axis) against the corresponding HA concentrations (X-axis) to generate a standard curve. Use this curve to calculate the HA concentration in the sample. 3. Detection range: 0–80 ng/mL. 4. Sensitivity: 0.1 ng/mL.
Figure 8 Fiber Optic Cable,Communication Cable,Outdoor Optical Cable,Aerial Cable
Guangzhou Jiqian Fiber Optic Cable Co.,ltd , https://www.jqopticcable.com