Polyclonal antibody purification assay

Summary

The octanoic acid extraction method can be used to isolate and extract Ig from human, rabbit and mouse serum and McAb from hybridoma-induced ascites and culture supernatants.

Operation method

Octanoic acid extraction

Materials and Instruments

Animal serum samples
Octanoic acid Acetic acid buffer PBS
Dialysis bags High-speed cryogenic centrifuge

Move

I. Materials and reagents
1. Octanoic acid
2. 60 mmol/L, pH 4.0 acetate buffer, PBS
3. Dialysis bags
4. High-speed cryogenic centrifuge
II. Operational steps
1. The sample to be extracted with 60 mmol / L, pH 4.0 acetate buffer diluted 3 to 4 times, adjust the pH to 4.5, the specimen containing high lipid can be used silica powder or glass fiber adsorption method to remove lipids.
2. Slowly add n-octanoic acid while stirring at room temperature, 25 μl per ml of sample, or 30 μl per ml of sample if the sample volume is less than 5 ml, and stir for 30 min.
3. 10 000 × g centrifugation for 30 min, take the supernatant through the qualitative filter paper filtration, loaded with dialysis bag, with 20 times the volume of PBS, 4 ℃ dialysis over the liquid, the middle of the liquid change 3 ~ 4 times.
4. Then add 0.27 g of ammonium sulfate per mL of mixture and stir for 30 min.
5. Centrifuge at 5,000 g for 15 min, collect the precipitate, dissolve it in a small amount of PBS, and then centrifuge at 15,000 × g for 20 min. The supernatant is the extracted Ig, most of which is IgG, accounting for about 90%, and a small amount of IgA and IgM, with a recovery rate of >80%. The whole operation can be completed within 24 h.

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