Human vascular endothelial cell adhesion factor (VCAM) ELISA kit instructions - Database & Sql Blog Articles

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Kaixin micro test

Human vascular endothelial cell adhesion factor (VCAM)

ELISA kit instructions

This kit is for research use only.

Experimental principle

The Human Vascular Cell Adhesion Molecule (VCAM) ELISA Kit is designed to measure the concentration of VCAM in biological samples using a double-antibody sandwich immunoassay. The process involves coating a microplate with a specific antibody against VCAM, then adding the sample and a labeled secondary antibody. The enzyme-linked complex forms, and after washing, a substrate is added to produce a color change. The intensity of the color correlates with the amount of VCAM present in the sample. The optical density (OD) is measured at 450 nm, and the concentration is determined by comparing it to a standard curve.

Kit composition

1. 30× Washing solution – 20ml × 1 bottle 2. Stop solution – 6ml × 1 bottle 3. Enzyme reagent – 6ml × 1 bottle 4. Standard (1200μg/L) – 0.5ml × 1 bottle 5. Microplate (12 wells × 8 strips) – 1 plate 6. Standard diluent – 1.5ml × 1 bottle 7. Sample dilution – 6ml × 1 bottle 8. Instruction manual – 1 copy 9. Reagent A – 6ml × 1 bottle 10. Reagent B – 6ml × 1 bottle 11. Sealing film – 2 sheets 12. Sealed bag – 1

Sample requirements

1. Samples should be processed as soon as possible after collection. If not tested immediately, store at -20°C, avoiding repeated freeze-thaw cycles. 2. Avoid using samples containing NaN3, as it may inhibit HRP activity.

Procedure

1. Prepare standards by serial dilution: - 600 μg/L: 150 μl original + 150 μl diluent - 300 μg/L: 150 μl No.5 + 150 μl diluent - 150 μg/L: 150 μl No.4 + 150 μl diluent - 75 μg/L: 150 μl No.3 + 150 μl diluent - 37.5 μg/L: 150 μl No.2 + 150 μl diluent 2. Load 50 μl of standard, 40 μl of sample diluent, and 10 μl of sample into each well. Mix gently. 3. Incubate at 37°C for 30 minutes. 4. Wash 5 times with diluted washing solution. 5. Add 50 μl of enzyme reagent to each well (except blank). 6. Incubate again at 37°C for 30 minutes. 7. Wash again. 8. Add 50 μl of TMB substrate and incubate for 10 minutes. 9. Add 50 μl stop solution to terminate the reaction. 10. Measure OD at 450 nm within 15 minutes.

Calculation

Plot a standard curve using standard concentrations and their corresponding OD values. Determine the sample concentration from the curve or calculate using linear regression. Multiply by the dilution factor to get the actual sample concentration.

Precautions

1. Allow the kit to reach room temperature before use. Store unopened enzyme reagents in a sealed bag. 2. If the washing solution crystallizes, heat it in a water bath before use. 3. Use a pipette for accuracy and avoid contamination. 4. Make a standard curve each time and consider diluting high-concentration samples. 5. Use a new sealing film for each experiment. 6. Keep the substrate away from light. 7. Follow all instructions strictly and rely on microplate reader results. 8. Treat all waste as biohazardous material. 9. Do not mix reagents from different batches.

Storage and shelf life

1. Store the kit at 2–8°C. 2. Shelf life: 6 months.