Mouse free fatty acid (FFA) ELISA test kit instructions - Database & Sql Blog Articles

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Mouse free fatty acid (FFA) ELISA Test Kit

This kit is for research use only. Not for human or animal diagnostic use.

Experimental Principle

The Mouse Free Fatty Acid (FFA) ELISA Kit is based on the double-antibody sandwich method. The microplate is pre-coated with a specific antibody against mouse FFA. After adding the sample, FFA binds to the immobilized antibody. Then, a horseradish peroxidase (HRP)-labeled secondary antibody is added, forming an antibody-antigen-enzyme complex. After washing, TMB substrate is introduced, which turns blue under HRP activity and then yellow when acid is added. The color intensity correlates with the FFA concentration in the sample. The absorbance is measured at 450 nm, and the FFA concentration is determined using a standard curve.

Kit Composition

• 1. 30× Washing Solution – 20ml × 1 bottle
• 2. Enzyme Standard Reagent – 6ml × 1 bottle
• 3. Enzyme-Labeled Coating Plate – 12 wells × 8 strips
• 4. Sample Diluent – 6ml × 1 bottle
• 5. Reagent A – 6ml × 1 bottle
• 6. Color Developer B – 6ml × 1 bottle
• 7. Stop Solution – 6ml × 1 bottle
• 8. Standard (1600μmol/L) – 0.5ml × 1 bottle
• 9. Standard Dilutions – 1.5ml × 1 bottle
• 10. Instruction Manual – 1 copy
• 11. Sealing Film – 2 sheets
• 12. Sealed Bag – 1

Sample Requirements

1. Samples should be processed immediately after collection. If not tested right away, store at -20°C. Avoid repeated freeze-thaw cycles.

2. Do not use samples containing NaN3, as it may inhibit HRP activity and affect results.

Step-by-Step Procedure

1. Standard Dilution: Prepare standards according to the provided dilution chart.
2. Loading: Add 50μL of standard or 40μL of sample diluent plus 10μL of sample to each well (final dilution: 5x).
3. Incubation: Seal the plate and incubate at 37°C for 30 minutes.
4. Washing: Wash 5 times with diluted washing solution.
5. Add Enzyme: Add 50μL of enzyme-labeled reagent to each well (except blank wells).
6. Incubation: Repeat incubation at 37°C for 30 minutes.
7. Color Development: Add 50μL of TMB developer and incubate at 37°C for 15 minutes.
8. Stop Reaction: Add 50μL of stop solution to terminate the reaction.
9. Measurement: Read OD at 450nm within 15 minutes after stopping the reaction.

Calculation

Plot the standard curve using OD values vs. concentrations. Use linear regression or interpolate from the curve. Multiply by the dilution factor to get the actual sample concentration.

Precautions

• Allow the kit to reach room temperature before use.
• Dilute concentrated washing solution with distilled water before use.
• Use a pipette for accurate measurements; avoid cross-contamination.
• Always include a standard curve and run duplicates for accuracy.
• Store unused enzyme-labeled plates in a sealed bag.
• Keep substrates away from light.
• Follow the manual strictly; results must be confirmed by a microplate reader.
• Treat all waste as biohazardous material.
• Do not mix reagents from different batches.
• In case of discrepancy, the English manual takes precedence.

Storage Conditions & Expiration

• Storage: 2–8°C
• Expiration: 6 months from date of manufacture