Mouse melatonin (MT) ELISA kit for operation - Database & Sql Blog Articles

Mouse melatonin (MT)

ELISA Test

Kit

Instruction Manual

This kit is for research use only.

Experimental Principle

Mouse melatonin (MT) ELISA Kit is designed to quantitatively determine the level of melatonin in mouse samples using a double-antibody sandwich immunoassay. The microwell plate is pre-coated with purified anti-mouse MT antibodies. After adding the sample, the melatonin binds to the solid-phase antibody. A horseradish peroxidase (HRP)-labeled anti-MT antibody is then added to form an immune complex. Following a wash step, TMB substrate is introduced, and the color changes from blue to yellow under the action of HRP and an acidic stop solution. The intensity of the color is directly proportional to the melatonin concentration in the sample. Absorbance at 450 nm is measured using a microplate reader, and the melatonin 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 Standard Reagent – 6ml × 1 bottle

4. Standard (960pg/ml) – 0.5ml × 1 bottle

5. Enzyme-labeled Coating Plate – 12 wells × 8 strips

6. Standard Dilutions – 1.5ml × 1 bottle

7. Sample Diluent – 6ml × 1 bottle

8. Instruction Manual – 1 copy

9. Reagent A – 6ml × 1 bottle

10. Color Developer 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 and avoid repeated freeze-thaw cycles.

2. Avoid using samples containing NaN3, as it may inhibit HRP activity.

Testing Steps

1. Prepare standards by diluting the original standard according to the provided instructions.

2. Load 50μl of standard and 50μl of sample diluent into the appropriate wells, followed by 10μl of the sample (final dilution 5×).

3. Incubate at 37°C for 30 minutes.

4. Wash the plate 5 times with 30× diluted washing solution.

5. Add 50μl of enzyme-labeled reagent to each well except blank controls.

6. Incubate again for 30 minutes.

7. Wash the plate again.

8. Add 50μl of TMB developer and incubate at 37°C for 15 minutes.

9. Add 50μl of stop solution to terminate the reaction.

10. Measure absorbance at 450nm within 15 minutes.

Calculation

Plot the standard curve using standard concentrations vs. OD values. Use linear regression or direct comparison to calculate the sample concentration, multiplying by the dilution factor.

Precautions

1. Allow the kit to reach room temperature before use. Store unopened enzyme-labeled reagents in a sealed bag.

2. If the washing solution crystallizes, heat gently in a water bath before use.

3. Use accurate pipettes and minimize loading time to ensure precision.

4. Always run a standard curve and consider sample dilution if OD exceeds the first standard well.

5. Use one sealing film per experiment to prevent cross-contamination.

6. Protect the substrate from light.

7. Follow the manual strictly and rely on microplate reader results.

8. Treat all waste as biohazardous material.

9. Do not mix components from different batches.

10. In case of discrepancy, the English manual takes precedence.

Storage Conditions & Expiration Date

1. Store the kit at 2–8°C.

2. Shelf life: 6 months from the date of manufacture.