Sandwich method ELISA for human interferon (α2a, α2b)

Recombinant human α2a / α2b interferon (rHuIFN-α2a, rHuIFN-α2b) is an indispensable part of the production process is the product quality monitoring, usually using HEp-2 / VSV or Wish / VSV system to detect the biological activity of interferon . This system is often not stable due to the influence of VSV or cells, and the detection period is longer. We established a double McAb sandwich ELISA for quantitative detection of rHuIFN-α2a and rHuIFN-α2b with two homemade monoclonal antibodies (McAb) against rHuIFN-α2a and rHuIFN-α2b, with a sensitivity of 60pg / ml. Corresponding to detection. Satisfactory results have been obtained by trial use in the quality control of rHuIFN-α2a production. This kit can also detect natural interferon alpha produced by the human body, and has the potential for basic and clinical disease research.

1. Principle: Two McAbs targeting different epitopes of rHuIFN-α2a and rHuIFN-α2b molecules are selected, one McAb is used as the coating antibody, and the other McAb labeled horseradish peroxidase (HRP) is used as the conjugate to catalyze the bottom Things are developing. Draw a standard curve according to the OD value of the standard product, and find out the content of the sample to be tested on the standard curve.

Second, the kit provides reagents (for testing 22 specimens)

1. Coated antibody: use 0.05M, pH9..6 carbonate buffer for 100-fold dilution during application.

2. Enzyme-labeled antibody: 200-fold dilution with 0.1% BSA-PBS (10-fold dilution before sale).

3. Standard: rHuIFN-α2a (3ng / ml), use 0.1% BSA-PBS for multiple dilution.

4. ABTS substrate: ABTS 1mg + 0.1M, pH5.0 citrate buffer 2ml + 3% H2O24μl (prepared when used).

3. Bring your own reagents and materials

1. Imported 96-well or domestic 40-well polystyrene ELISA plates (sensitivity is slightly better than imported plates).

2. 0.1M, pH7.4 PBS is used with other liquids.

3. Coating buffer: 0.05M, pH9.6 Na2CO3-NaHCO3 buffer.

4. Washing solution: 0.05% Tween 20-PBS.

5. Conjugate dilution: 0.1% BSA-PBS (for diluting specimens, standards and conjugates).

6. Substrate buffer: 0.1M, pH5.0 citric acid-disodium hydrogen phosphate buffer.

7. ELISA reader etc.

4. Operation steps

Add 100μl / well of diluted coating antibody to the ELISA plate

↓ 4 ℃ 24 ～ 48h

Wash 3 times, add 10 times serially diluted specimen to be tested

(Just make 4 dilutions) or multiple dilution standard (6 dilutions) 100μl / well

↓ 37 ℃, 1h

Wash 3 times, add diluted enzyme-labeled antibody 100μl / well

↓ 37 ℃, 45min

Wash 4 times, add 100μl / well of prepared ABTS substrate solution

↓ RT, 30min

410nm OD

↓

Draw a standard curve

Five, matters needing attention:

1. The specimen to be tested must be free of contamination and hemolysis.

2. When adding the ELISA plate after the specimen is diluted, pay attention to add it from low concentration to high concentration in sequence.

3. Do not add sodium azide to the conjugate diluent and substrate buffer.

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