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

Mouse Melatonin (MT) ELISA Test Kit

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

Experimental Principle

The Mouse Melatonin (MT) ELISA Kit is based on the double-antibody sandwich immunoassay technique. The microtiter plate is pre-coated with a specific monoclonal antibody against melatonin. After adding the sample, the melatonin in the sample binds to the coated antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming an immune complex. Following washing steps, TMB substrate is added, and the color develops in proportion to the amount of melatonin present. The reaction is stopped with a stop solution, and the absorbance is measured at 450 nm using a microplate reader. The concentration of melatonin in the sample is determined by comparing the OD values to a standard curve.

Kit Components

• 30× Washing Solution – 20ml × 1 bottle
• Stop Solution – 6ml × 1 bottle
• Enzyme-labeled Reagent – 6ml × 1 bottle
• Standard (960pg/ml) – 0.5ml × 1 bottle
• Enzyme-labeled Plate – 12 wells × 8 strips
• Standard Dilutions – 1.5ml × 1 bottle
• Sample Diluent – 6ml × 1 bottle
• Instruction Manual – 1 copy
• Reagent A – 6ml × 1 bottle
• Reagent B – 6ml × 1 bottle
• Sealing Film – 2 sheets
• 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 and interfere with results.

Testing Procedure

1. Standard Dilution: Prepare a series of standards by diluting the original standard according to the provided chart.
2. Loading: Add 50μl of standard and 50μl of sample (diluted 5x) to respective wells. Mix gently without touching the well walls.
3. Incubation: Seal the plate and incubate at 37°C for 30 minutes.
4. Washing: Wash the plate 5 times with diluted washing solution, then pat dry.
5. Add Enzyme: Add 50μl of enzyme-labeled reagent to each well except blank controls.
6. Incubation (again): Incubate for another 30 minutes at 37°C.
7. Color Development: Add 50μl of TMB developer to each well and incubate for 15 minutes at 37°C.
8. Stop Reaction: Add 50μl of stop solution to each well to terminate the reaction.
9. Measurement: Read the OD value at 450nm within 15 minutes of stopping the reaction.

Calculation

Plot the OD values of the standards against their concentrations to create a standard curve. Use this curve to determine the melatonin concentration in the samples. Multiply the calculated value by the dilution factor to obtain the actual sample concentration.

Precautions

• Allow the kit to reach room temperature before use. Store unopened enzyme reagents in a sealed bag.
• If the washing solution crystallizes, warm it in a water bath before use.
• Use accurate pipettes and ensure consistent timing during sample loading.
• Always run a standard curve and consider diluting high-concentration samples if necessary.
• Use a new sealing film for each experiment to prevent cross-contamination.
• Keep the substrate away from light.
• Strictly follow the instructions. Results must be confirmed with a microplate reader.
• Treat all waste materials as biohazardous.
• Do not mix components from different batches.
• In case of discrepancies, the English manual takes precedence.

Storage Conditions & Expiration

• Store the kit at 2–8°C.
• Shelf life: 6 months from the date of manufacture.