Analysis and purification of IgA from Shanghai Xinfan Technology Lecture - Huaqiang

The probe capacitor polarity needle has a head diameter of 5.8 mm. The total length of the 7-pin and 12-pin needles is 5 cm.

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1. DEAE52 Cellulose

2. 0.10 M ZnSO₄ Solution

ZnSO₄·7H₂O: Dissolve 2.88 g in water to make up to 1000.00 ml.

3. Saturated Ammonium Sulfate Solution

4. 10% EDTA-Na Solution

5. Sephadex G-200

6. 0.01 M pH 7.4 PBS Solution

7. 0.10 M pH 6.4 PBS Solution

8. Serum

1. Mix serum with an equal volume of 0.1 M ZnSO₄ solution. Adjust the pH to 7.0, stir at room temperature for 1 hour, then centrifuge to collect the precipitate.

2. In the supernatant, add an equal volume of saturated ammonium sulfate solution. Mix well and let it stand for 30 minutes or overnight in the refrigerator.

3. Centrifuge at 31,000 r/min for 10 minutes, and discard the supernatant.

4. Dissolve the precipitate in 4 ml of 10% EDTA-Na solution.

5. Perform dialysis in normal saline to remove impurities.

6. Pass the sample through a DEAE52 column using 0.01 M pH 7.4 PBS solution as the eluent. Discard the flow-through containing IgG.

7. Elute IgA using 0.1 M pH 6.4 PBS solution.

8. Further purify the IgA by passing it through a Sephadex G-200 column. Use 0.01 M pH 7.4 PBS (0.14 M NaCl) as the eluent. Collect the eluate and measure the OD₂₈₀ value. The first peak corresponds to the purified IgA.

9. Perform dialysis and concentration for final purification and identification.

This detailed procedure ensures high purity of the isolated IgA, suitable for further analysis and application in immunological studies.