Human hypoxia-inducible factor 1a ELISA kit kit detailed explanation - Database & Sql Blog Articles

This reagent is intended for research use only, specifically for serum or plasma samples.

Test Principle:

The HIF-1α ELISA kit operates on a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) method. Known standards and unknown samples are added to microplates for analysis. The process involves incubating the sample with biotinylated antibodies, followed by the addition of avidin-labeled HRP. After washing, substrates A and B are introduced, leading to a color change that correlates with the concentration of HIF-1α in the sample.

Self-contained Materials:

Distilled water, pipettes (5µl, 10µl, 50µl, 100µl, 200µl, 500µl, 1000µl), oscillators, magnetic stirrers, and other necessary equipment are included.

Safety Precautions:

Avoid direct contact with stop solution and substrates A and B. If exposed, 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 Guidelines:

Store all reagents as specified on the label and allow them to reach room temperature before use. Discard diluted standards immediately after use. Keep unused plates sealed to prevent degradation. Avoid mixing reagents from different batches. Always use disposable tips to prevent cross-contamination. Do not use metal-pipette tips when handling stop solution or substrates. Prepare wash solution in clean plastic containers. Ensure all components are well mixed before starting the test. Dry the plate thoroughly after washing, and avoid using absorbent paper directly in the wells. Store samples in small aliquots at -70°C to avoid repeated freezing. Thaw at room temperature only, and avoid heating above 37°C.

Sample Collection & Storage:

For serum: Use endotoxin-free tubes and centrifuge at 1000 x g for 10 minutes to separate serum from red blood cells. For plasma: Use EDTA, citrate, or heparin and centrifuge at 1000 x g for 30 minutes. For cell supernatant: Centrifuge at 1000 x g for 10 minutes to remove particles. Avoid hemolysis or hyperlipidemia, and filter if necessary before testing.

Reagent Preparation:

Prepare standard dilutions fresh for each experiment. Shake the standard before dilution. Dilute the wash buffer 50-fold with distilled water.

Procedure Steps:

Thoroughly mix all reagents before use, avoiding excessive foaming. Determine the number of wells based on samples and standards, with duplicates recommended. Add 50 µl of standard and sample to each well, then add 50 µl of biotinylated antibody. Incubate at 37°C for 1 hour. Wash the plate 3 times with wash buffer, and repeat if using an automated washer. Add 80 µl of streptavidin-HRP and incubate for 30 minutes. Wash again and add 50 µl of substrates A and B. Incubate for 10 minutes in the dark. Stop the reaction by adding 50 µl of stop solution and read OD450 immediately.

Limitations:

Results above the sixth standard may be non-linear and unreliable.

Kit Performance:

1. Sensitivity: Minimum detection below the first standard. Linear dilution range with R > 0.990. 2. Specificity: No cross-reactivity with other cytokines. 3. Repeatability: Coefficient of variation between plates < 10%.

Result Interpretation:

Measure OD450 values using a microplate reader. Plot standard concentrations against OD values to generate a standard curve. Calculate sample HIF-1α levels from the curve. Detection range: 0–800 pg/ml. Sensitivity: 1.0 pg/ml.