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.