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Section 2.3.3
Purification and
Conjugation of Antiserum
Alternative Purification
7* Prepare a column of DEAE Sephacel of 3.5 ml, total volume and
equilibrate with dialyzing buffer.
8* Wash the column several times with dialyzing buffer which is also
used as an elution buffer.
9* Apply the dialyzed fraction and collect fractions of about 0.5 ml:
measure the concentration of the fractions at 280 nm.
After stabilizing the gel for the particular conditions of use, the gas should
be removed both from the eluting buffer and the stabilized gel to avoid
air-bubble formation. This can be done by shaking the mixture slowly and
using an air pump.
Mounting the column is easy: Take the required volume; verify the height
the column will reach according to the specificity of each case; mark
where necessary, taking care that the column is exactly perpendicular to
the surface of the table to ensure that the sample flows evenly. Fill the
column by closing the lower outlet and pouring the gel, which has been
previously stabilized and degassed to prevent bubbles.
The speed of the flow is specific for each case. Generally, a slow flow
prevents the column from overpacking or from losing its optimum
characteristics. Normally, a flow of 10–20 ml per hour is enough. Once
the column is packed, it is washed several times with eluting buffer to
ensure that the preservative has been completely removed.
To insert the sample, temporarily stop the flow and insert a sample that is
5% of the volume of the column.
6. Pour the contents of the dialysis bag into a vial (previously treated
with silicone and rinsed in distilled water).
8. Store at 4 C.
2. CIP-Gugerli Method
a) When purifying the IgG, Gugerli proposes skipping the step of passing
through the DEAE-Sephacel column after dialyzing and before
conjugation.
b) Unlike the CIP method for enzymatic conjugation, Gugerli does not
dialyze, so the process takes less time (3 hours).
6. Add 50 g/l of sucrose, place the mixture on ice, mix well and pour
into a chromatography column in Sephacryl S-300 gel (25 mm x
200 mm, previously buffered) plus Tris/HCl, pH 8.0.
7. Elute with the same Tris/HCl solution, collect 2-ml fractions, and
measure the concentration of protein at 280 nm. The antibody
conjugated with the enzyme will elute as the first fraction, then the
elution of unreacted antibodies and enzymes could be observed.
9. Store at 4 C.
Immunoglobulin Titration
For the distribution of the dilutions in the ELISA plates, see the diagram.
Note: The wells of the edges that are not used should be filled with the
appropriate buffer solution in each step of the test (to prevent nonspecific
reactions).
APPENDIX
0.2 g KH2PO4
2.9 g Na2HPO4 12 H20
8.0 g NaCl (137 mM)
0.2 g KCl (2.7 mM)
0.2 g NaN3 (3 mM)*
0.1 g KH2PO4
1.45 g Na2HPO4 12 H20
4.0 g NaCl (69 mM)
0.1 g KCl (1.4 mM)
0.1 g NaN3 (1.5 mM)*
IMPORTANT:
Depending on the batch and the provider, the enzyme’s label could
provide the following information (for example):
X = 12,648 units/ml
X = 26,691 units/ml
X= 46.83 µl
2. Reagents