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10 Hutchens
night. Elute the adsorbed proteins by removal of ammonium sulfate,
that is, elute with 20 mMHEPES buffer containing 0.5MNaCl. Intro
duce 50% ethylene glycol in 20 mM HEPES, pH 8.0 to initiate col-
umn regeneration. Used columns should be washed with 30%
isopropanol and water before reequilibration with column-equilibra-
tion buffer. Collect eluted fractions. Immunoglobulindepleted colosu-al
whey proteins (T-gel column flow-through fractions) and isolated im-
munoglobulins can be concentrated and dialyzed using an Amicon
Model CH2 PBS spiral-cartridge concentrator with a 3000 or 10,000
MW cutoff. An example is provided in Figs. 1 and 2.
3.6. Protein Analyses of Isolated Fractions
and Quantitation of Eluted Immunoglobulins
Monitor the protein elution profile during thiophilic adsorption
chromatography by detection of UV absorbance at 280 nm. Deter-
mine the elution properties of the various immunoglobulins using
ELISA techniques, immuno “dot” blotting procedures (21), or West-
ern (electrophoretic) transfer of the eluted proteins from SDS gels to
nitrocellulose and immune blotting (22). The protein or immunoglobu-
lin concentrations of pooled fractions should be evaluated as described
by Bradford (13) (see vol. 3)or Smith et al. (14) to determine overall
protein and immunoglobulin recoveries; these values should routinely
exceed 90%.
3.6.1. ELBA Methods
for the Detection of Specific Immunoglobulins
Coat 96well microtiter plates (50 pL/well) with afftnity-purified
antibodies (example presented here: rabbit antibodies to bovine im-
munoglobulins obtained from Jackson Immunoresearch Laboratories,
Inc., West Grove, PA) diluted to the appropriate titer with phosphate-
buffered saline (PBS) pH 7.4. The plates can be stored for several days
(at 4OC) before use. Wash the plates 3X in distilled water then add a
100 l.tL aliquot of diluted sample to each well. Human plasma and
unfractionated fetal calf serum (originating from Australia) should be
used at various dilutions as negative and positive controls, respectively.
Dilute purified bovine IgG (Jackson Immunoresearch Laboratories)
with 3% PBS to a final concentration of 0.5 ng/mL. Use this solution
for calibration. Incubate the loaded plates for 1 h at 37OC before wash-
ing (5X) with distilled water containing 0.05% Tween 80. Dilute horse-