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 samples.

(

CD3

)

Level: Purified mouse CD3 molecule

(

CD3

)

The antibody is coated on a microplate to create a solid-phase antibody. CD3 molecules are then added to the wells coated with monoclonal antibodies. Subsequently, HRP-labeled CD3 antibodies bind to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, TMB substrate is added for color development. Under HRP catalysis, TMB turns blue and eventually becomes yellow when acid is added. The intensity of the color is directly proportional to the concentration of CD3 molecules in the sample. The absorbance at 450 nm (OD value) is measured using a microplate reader to calculate the CD3 concentration in the sample based on a standard curve.

Mouse CD3 molecule

(

CD3

)

ELISA

Kit

Composition

130 times concentrated washing solution 20ml × 1 bottle | 7 stop solution 6ml × 1 bottle

2 enzyme standard reagent 6ml × 1 bottle | 8 standard (48IU/ml) 0.5ml × 1 bottle

3 enzyme label coating plate 12 holes × 8 | 9 standard dilutions 1.5ml × 1 bottle

4 sample dilution 6ml × 1 bottle | 10 instructions 1 copy

5 color developing agent A liquid 6ml × 1 bottle | 11 sealing film 2 sheets

6 color developer B liquid 6ml × 1 bottle | 12 sealed bag 1

Sample requirements

1. Samples should be extracted as soon as possible after collection, following relevant literature. Testing should be done promptly after extraction. If not tested immediately, store at -20°C, avoiding repeated freeze-thaw cycles.

2. Do not use samples containing NaN3, as it inhibits horseradish peroxidase (HRP) activity.

Mouse CD3 molecule

(

CD3

)

ELISA

Kit

Steps

1. Standard Dilution: This kit provides one original standard. Dilute it according to the following chart in a small tube.

Add 150 μl of standard diluent to 150 μl of original standard (24 IU/ml) to make No. 5 standard (12 μl/ml).

Add 1 μl of standard to 150 μl of standard diluent to make No. 4 standard (6 IU/ml).

Add 3 μl of standard to 150 μl of standard diluent to make No. 3 standard (3 μl/ml).

Add 150 μl of No. 2 standard to 150 μl of standard diluent to make No. 1 standard (1.5 IU/ml).

2. Sample Loading: Set up blank wells (no sample or enzyme reagent), standard wells, and sample wells. Add 50 μl of standard, 40 μl of sample diluent, and 10 μl of sample to each well. Final dilution is 5-fold.

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

4. Washing: Use 30x diluted washing solution. Wash 5 times, then pat dry.

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

6. Incubation: Repeat step 3.

7. Washing: Repeat step 4.

8. Color Development: Add 50 μl of developer A and 50 μl of developer B, mix gently, and incubate at 37°C for 10 minutes.

9. Stop Reaction: Add 50 μl of stop solution per well. The color changes from blue to yellow.

10. Measurement: Read absorbance at 450 nm within 15 minutes after adding stop solution.

Calculation

Plot a standard curve using standard concentrations and OD values. Determine the sample concentration from the curve and multiply by the dilution factor. Alternatively, use linear regression to calculate the sample concentration and adjust for dilution.

Mouse CD3 molecule

(

CD3

)

ELISA

Kit

Precautions

1. Allow the kit to reach room temperature (15–30 minutes) before use. Store unsealed enzyme reagents in a sealed bag.

2. Concentrated washing solution may crystallize; heat it in a water bath if needed. It will not affect results.

3. Use a pipette for accurate measurements. Keep loading time under 5 minutes. For large numbers of samples, use a pipetting gun.

4. Prepare a standard curve for each test. Make duplicate wells. If the sample OD is higher than the first standard well, dilute the sample and adjust the final calculation accordingly.

5. Use the sealing film only once to avoid contamination.

6. Protect substrates from light.

7. Follow the manual strictly. Results must be confirmed with a microplate reader.

8. Treat all samples, washes, and waste as biohazardous materials.

9. Do not mix components from different batches.

Storage Conditions and Expiration

1. Store the kit at 2–8°C.

2. Shelf life: 6 months.