Mouse CD3 molecule (CD3) ELISA kit operating principle - Database & Sql Blog Articles

Mouse CD3 molecule

(

CD3

)

ELISA

Kit

Principle of operation

This kit is for research use only.

Experimental principle

The kit uses a double antibody sandwich method to detect mouse CD3 molecules in the sample. The process involves coating a microplate with purified mouse CD3 antibodies and then adding the sample. HRP-labeled CD3 antibodies are subsequently added, forming an antibody-antigen-enzyme complex. After washing, TMB substrate is added for color development. The intensity of the color is directly proportional to the concentration of CD3 in the sample, which is measured at 450 nm using a microplate reader.

Mouse CD3 molecule

(

CD3

)

ELISA

Kit

Composition

1. 130 times concentrated washing solution – 20ml × 1 bottle

2. Stop solution – 6ml × 1 bottle

3. Enzyme standard reagent – 6ml × 1 bottle

4. Enzyme-labeled coating plate – 12 holes × 8

5. Sample dilution – 6ml × 1 bottle

6. Color developing agent A liquid – 6ml × 1 bottle

7. Color developer B liquid – 6ml × 1 bottle

8. Standard (48IU/ml) – 0.5ml × 1 bottle

9. Standard dilutions – 1.5ml × 1 bottle

10. Instructions – 1 copy

11. Sealing film – 2 sheets

12. Sealed bag – 1

Sample requirements

1. Samples should be extracted as soon as possible after collection and tested promptly. If not used immediately, store at -20°C, avoiding repeated freeze-thaw cycles.

2. Avoid samples containing NaN3, as it may inhibit horseradish peroxidase (HRP) activity.

Mouse CD3 molecule

(

CD3

)

ELISA

Kit

Procedure

1. Standard Dilution: Prepare standards by diluting the original standard according to the provided table.

2. Sample Loading: Add 50μl of standard, 40μl of sample diluent, and 10μl of sample into each well (final dilution 5x).

3. Incubation: Seal the plate and incubate at 37°C for 30 minutes.

4. Washing: Wash the plate 5 times with diluted washing solution.

5. Enzyme Addition: Add 50μl of enzyme-labeled reagent to each well except blank wells.

6. Incubation: Repeat the 37°C incubation for 30 minutes.

7. Color Development: Add 50μl of TMB A and B solutions, incubate at 37°C for 10 minutes.

8. Stop Reaction: Add 50μl of stop solution to each well.

9. Reading: Measure absorbance at 450 nm within 15 minutes of stopping the reaction.

Calculation

Plot a standard curve using standard concentrations and OD values. Use linear regression or interpolation to determine sample concentration, then multiply by the dilution factor.

Mouse CD3 molecule

(

CD3

)

ELISA

Kit

Precautions

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

2. If washing solution crystallizes, warm it gently before use.

3. Use a pipette for accuracy and ensure all steps are completed within 5 minutes.

4. Always run a standard curve and consider diluting high-concentration samples if needed.

5. Do not reuse sealing films to prevent contamination.

6. Keep substrates away from light.

7. Follow the manual strictly and confirm results with a microplate reader.

8. Treat all waste as biohazardous material.

9. Do not mix reagents from different batches.

Storage and Expiry

1. Store the kit at 2–8°C.

2. Shelf life is 6 months.