Separation of circulating filarial
antigen (CFA-2) from Plasma
A. Principle :
Gel
filtration is a separation technique useful for the purification of thousands of
enzymes, polysaccharides, nucleic acids, proteins and other biological
macromolecules. Gel filtration permits
the preparative separation of molecules by their native molecular weight due to
the differential penetration of the gel matrix by molecules of different size.
Very large molecules which never enter the gel matrix, move through the
chromatographic bed fastest. Smaller molecules which can enter the gel pores,
move more slowly through the column, since they spend a portion of their time
in the stationary phase (gel). Molecules are therefore, eluted in order of
decreasing molecular size.
B.Materials
:
a.Pre-swollen
Ultrogel ACA 34.
b.0.05 M
SPB, pH 7.2 (Appendix- I)
c.Chromatography
set up.
d.Circulating
filarial antigen (CFA). (Appendix- II)
e.Gel
filtration molecular weight markers.
C. Method
:
1.Transfer about
100 ml volume of pre-swollen
ultrogel ACA 34
to a vacuum flask containing 50
ml of 0.05 M SPB, pH 7.2.
2.Mix with
a glass rod and deaerate the gel suspension with the help of vacuum pump until
the air bubbles stop coming out.
3.Pack the
chromatography column (LKB, Sweden) with bed dimension 1.6x 50 cm
at 40C using either a peristaltic pump at
a flow rate of 25 ml/hr or at a constant ydrostatic pressure of 70-100 cm H2O.
4.Equilibrate
the column with deaerated 0.05 M SPB, pH 7.2 for 24 hr with a flow rate of 15
ml/hr.
5.Apply 1
ml of CFA (20 mg/ml) without injecting air bubbles to the gel and elute with
0.05 M SPB at a flow rate of 8 ml/hr.
6.Collect
2 ml fractions and read their absorbance at 280 nm wave length and record with
automatic one channel recorder.
7.Pool
fractions in each protein peak separately and concentrate by ultra filtration.
8.Check
antigenic activity in pooled fractions by CIEP using FSIgG.
9.The
second protein fraction showing filarial antigens is designated as CFA2.
Determination
of molecular weight
1.Equilibrate
the column with 0.05 M SPB pH 7.2.
2.Apply a
mixture of gel filtration molecular weight marker proteins.
3.Collect
fractions and determine elution volumes (Ve) of each marker protein from the
recorded peaks.
4.Determine
void volume (Vo) by elution volume of Blue Dextran 2000.
5.Determine
gel bed volume (Vt).
6.For each
protein determine the value of Kav.
7.Draw
standard graph by plotting Kav against log molecular weight.
Kav =
Ve-Vo / Vt-Vo
8.Calculate
Kav for each protein peak of CFA Viz, CFA1, CFA2, CFA3 and CFA4.
9.From the
standard graph estimate the approximate molecular weight for each peak.
References
:
1.Reddy, M.V.R., Prasad, G. B. K. S. and Harinath, B. C. Isolation and evaluation of
antigens from microfilaraemic plasma and immunecomplexes for Diagnosis of bancroftian
filariasis. Indian J. Pathol, Microbiol. 29 (1986), 179.
2.Gel
Filtration Theory and Practice. Pharmacia Fine Chemicals, 1981.