7. Ion Exchange Chromatography



Isolation of IgG from Immunoglobulins


A. Principle :


Fractionation of proteins by ion-exchange chromatography exploits differences in their net charges. The immunoglobulin rich fraction prepared by ammonium sulphate  precipitation is applied on DEAE cellulose column at pH 7.2 At this pH DEAE cellulose is positively charged while all the classes of immunoglobulins except IgG carry negative charges. This situation allows only IgG to be eluted out while the other classes of immunoglobulins interact with the matrix and hence are retained in the column.


B. Materials :


a.DEAE-Cellulose (Whatman DE-52).


b.0.01 M SPB, pH 7.2     (Appendix-I)


c.Whatman filter paper (No.1)


d.Chromatography set up.


e.Spectrophotometer or colorimeter.


f.Immunoglobulins (Ig) (g-globulin fraction)




C. Method :


1.Suspend  about 3 g  of DEAE cellulose in 0.01M SPB (pH 7.2) and wash the slurry by changing the buffer 5 or 6 times to remove fine particles.


2.Take  a  glass  column (10 x 2 cm)  with  sintered glass  disc  or glass-wool plug at the bottom and mount it vertically.


3.Inject  about  10 ml of 0.01 M SPB, pH 7.2 through the outlet of column with the help of  syringe and avoid trapping of air bubbles below the sintered glass disc or glass wool plug.


4.Close the outlet and pour thick slurry of DEAE cellulose along the sides of column and allow the buffer to run at about 25 ml/hr. Disturb the settled bed slightly with glass rod if some slurry is to be added.


5.Cover the gel bed with a Whatman 1 filter paper circle (Having diameter slightly lesser than  that of  column) and equilibrate the column by pumping 0.01 M SPB, pH 7.2 at a flow rate of about 25 ml/hr for about 4 hrs.


6.Sample application and elution


(1)Close the outlet and remove the excess buffer above the gel bed.


(2)Carefully  layer on 25 mg of  immunoglobulin fraction in 1 ml volume of 0.01M, SPB pH 7.2 and allow this buffer to enter the gel.


(3)Wash the sample from sides of column with 1 ml of buffer and allow it to enter the gel.


(4)Close the outlet, fill up the column with buffer and connect to the buffer reservoir.


(5)Open the outlet and pump about 30 ml of buffer through the gel at a flow rate of 25 ml/ hr. Collect 2 ml fractions and read their absorbance at 280 nm wave length. Alternatively estimate protein by Lowry's method in a small sample of each fraction.


(6)Test the protein fractions for IgG in Ouchterlony double diffusion using monospecific anti-human IgG. Pool positive fractions, concentrate to 2 ml by ultrafiltration and store at -200C with sodium azide (0.01%) as preservative.




References :


1.Bradwell, A.R. Catty, D. & Houba, V. In: Bench manual of techniques for the preparation of immunological and immunodiagnostic reagents, World Health Organization: Geneva


2.Reddy, M.V.R. Malhotra, A. & Harinath, B.C. Detection of circulating antigen in bancroftian  filariasis by sandwich ELISA using filarial serum IgG. J. Helminthol 58 (1984). 259.