Bovine beta-casein antibody (Casein-Ab) ELISA kit technical guidance - Database & Sql Blog Articles

Bovine beta-casein antibody

(

Casein-Ab

ELISA

Kit

Technical support

This kit is for research use only.

Popular name:

Bovine beta-casein antibody

(

Casein-Ab

ELISA

Quantitative test kit,

Bovine beta-casein antibody

(

Casein-Ab

)

Enzyme Linked Immunoassay Kit

English abbreviation:

Casein-Ab

ElisaKit

Storage conditions: kit storage 2-8 °C

Validity: 6 months

Specificity: This ELISA test kit can detect both natural and recombinant, and does not cross-react with other related proteins.

Scope of application: This kit is only used for scientific research and is forbidden in clinical practice.

Common methods of ELISA: double antibody sandwich method, indirect method, competition method, and BAS-ELISA.

Experimental principle

The Bovine beta-casein antibody (Casein-Ab) ELISA Kit determines the level of β-casein antibody in the sample using a double antigen sandwich method. The microwell plate is coated with purified antigen to create a solid-phase antigen. The β-casein antibody (Casein-Ab) is then added sequentially, followed by HRP-labeled antigen to form an antigen-antibody-enzyme-labeled complex. After thorough washing, the TMB substrate is added for color development. Under the catalysis of HRP, TMB turns blue and eventually yellow when acid is added. The intensity of the color correlates positively with the concentration of Casein-Ab in the sample. The absorbance (OD value) is measured at 450 nm using a microplate reader, and the sample concentration is calculated based on a standard curve.

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 (240ng/L) – 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

Specimen requirements

1. Specimens should be extracted as soon as possible after collection and according to relevant literature. Perform the experiment as soon as possible after extraction. If testing cannot be done immediately, store the specimen at -20°C but avoid repeated freezing and thawing.

2. Samples containing NaN3 cannot be tested because NaN3 inhibits horseradish peroxidase (HRP) activity.

Kit steps

1. Standard dilution: This kit provides one original standard, which can be diluted in a small tube according to the following chart.

150 ng/L No. 5 standard – 150 μl of original standard + 150 μl of standard diluent

60 ng/L No. 4 standard – 150 μl of No. 5 standard + 150 μl of standard diluent

30 ng/L No. 3 standard – 50 μl of standard diluent + 150 μl of No. 4 standard

15 ng/L No. 2 standard – 150 μl of standard diluent + 150 μl of No. 3 standard

7.5 ng/L No. 1 standard – 150 μl of No. 2 standard + 150 μl of standard diluent

2. Loading: Set up blank wells (no sample or enzyme reagent), standard wells, and sample wells. Add 50 μl of standard to each well, 40 μl of sample diluent, and 10 μl of sample (final dilution 5x). Carefully add samples to the bottom of the well without touching the walls, and gently mix.

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

4. Washing: Dilute 30 times concentrated washing solution with distilled water and use it.

5. Washing: Remove the sealing film, discard the liquid, dry the plate, fill each well with washing solution, let stand for 30 seconds, discard, repeat 5 times, and pat dry.

6. Enzyme addition: Add 50 μl of enzyme labeling reagent to each well except blank wells.

7. Incubation: Repeat step 3.

8. Washing: Repeat step 5.

9. Color development: Add 50 μl of developer A and 50 μl of developer B to each well, mix gently, and incubate at 37°C for 15 minutes.

10. Stop reaction: Add 50 μl of stop solution to each well to terminate the reaction (when the blue color turns yellow).

11. Measurement: Measure OD values at 450 nm using a microplate reader. Perform measurements within 15 minutes after adding the stop solution.

Calculation

Plot a standard curve with standard concentrations on the x-axis and OD values on the y-axis. Determine the sample concentration from the standard curve and multiply by the dilution factor. Alternatively, use linear regression to calculate the equation of the standard curve, input the sample OD value into the equation, and multiply by the dilution factor to get the actual sample concentration.

Precautions

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

2. Concentrated washing solution may crystallize; dissolve by heating in a water bath if needed. It will not affect results.

3. Use a pipette for accurate measurement and avoid errors. Keep loading time under 5 minutes. For large numbers of samples, consider using a pipetting gun.

4. Prepare a standard curve simultaneously, preferably in duplicate. If sample OD exceeds that of the first standard well, dilute the sample first and adjust calculations accordingly.

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

6. Keep the substrate away from light.

7. Follow the manual strictly. All results must be based on microplate reader readings.

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

9. Do not mix components from different batches.

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

Storage conditions and expiration date

1. Storage: 2–8°C.

2. Expiration: 6 months.