This reagent is intended for research use only and is suitable for the analysis of serum or plasma samples.
Test Principle:
The HA ELISA Kit is based on a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). Known concentrations of HA standards and unknown samples are added to microplates for detection. The sample and biotinylated antibodies are incubated together, followed by washing. Avidin-labeled HRP is then added, and after further incubation and washing, the unbound enzyme conjugate is removed. Substrates A and B are then introduced simultaneously, resulting in a color change. The intensity of the color is directly proportional to the concentration of HA in the sample, allowing for quantitative analysis.
Self-Contained Materials:
Distilled water, micropipette tips (5 µl, 10 µl, 50 µl, 100 µl, 200 µl, 500 µl, 1000 µl), oscillators, magnetic stirrers, and other necessary equipment are included.
Safety Precautions:
Avoid direct contact with the stop solution and substrates A and B. If exposure occurs, rinse thoroughly with water immediately. Do not eat, drink, smoke, or apply cosmetics during the experiment. Never use your mouth to pipette any reagents from the kit. Always wear gloves and lab coats when handling chemicals.
Operational Precautions:
Reagents should be stored as per the label instructions and allowed to reach room temperature before use. Standards after dilution must be discarded and not reused. Unused microplate wells should be returned to the sealed bag promptly to prevent contamination. Other reagents should be properly covered and not mixed across batches. Use the kit before the expiration date. Always use disposable pipette tips to avoid cross-contamination. Avoid using metal-tipped pipettes when handling the stop solution and substrates. Prepare wash buffer in clean plastic containers and 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 keep substrate B away from light. Read OD values immediately after completing the reaction. Maintain consistent reagent addition order to ensure uniform incubation times. Follow the manufacturer's instructions precisely for incubation duration and volume.
Sample Collection, Processing, and Storage:
For serum: Avoid any cell irritation during collection. Use pyrogen-free and endotoxin-free tubes. After blood collection, separate serum from red blood cells via centrifugation at 1000 x g for 10 minutes. For plasma: EDTA, citrate, or heparin anticoagulated blood 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 particles and polymers. Storage: If not used immediately, divide samples into small aliquots and store at -70°C to avoid repeated freezing. Avoid hemolyzed or lipemic samples. If there are visible particles, centrifuge or filter the sample before testing. Thaw samples at room temperature, not above 37°C, and ensure even thawing.
Reagent Preparation:
Standards should be prepared freshly during the experiment and not stored. Shake the standard solution well before dilution. The wash buffer should be diluted 50-fold with distilled water.
Procedure:
Before starting, mix all reagents thoroughly to avoid foaming, which could introduce air bubbles and 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. For each sample, it is advisable to run duplicates to improve reliability. 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 for 1 hour at 37°C. Remove the liquid, wash each well with wash buffer for 30 seconds, and pat dry with absorbent paper. Repeat this process three times. If using an automated washer, increase the number of washes by one. Add 80 µl of avidin-HRP conjugate to each well, gently mix, and incubate for 30 minutes at 37°C. Wash again three times, and add 50 µl of substrates A and B. Incubate for 10 minutes at 37°C in the dark. Afterward, add 50 µl of stop solution to each well and measure the OD at 450 nm immediately.
Limitations:
Results beyond the sixth standard may be non-linear and cannot provide accurate quantification.
Kit Performance:
1. Sensitivity: The minimum detectable concentration is lower than that of the first standard. The dilution linearity is excellent, with a correlation coefficient (R) of over 0.990. 2. Specificity: No cross-reactivity with other human cytokines. 3. Repeatability: Inter-assay variation is less than 10%.
Result Interpretation:
1. Measure the OD value of each well 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 determine the HA concentration in the sample. 3. Detection range: 0–80 ng/mL. 4. Sensitivity: 0.1 ng/mL.
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