2. Mammalian IgG antibodies are commonly used as affinity probes for research and, more and more,
as treatments for diseases. For example, the experimental drug Mzapp recently used to treat two Americans for Ebola virus infection is a combination of three humanized monoclonal antibodies against Ebola proteins. Another example is Avastin, aka Bevacizumab, a humanized monoclonal that targets vascular endothelial growth factor and is used routinely to treat brain, colon, kidney and lung cancers and off-label to treat macular degeneration. The term humanized means they were not originally human antibodies, but have had much of their amino acid sequence changed to the human IgG sequence so they won't be attacked by the human immune system.
Polyclonal IgG is isolated from blood serum of immunized animals; monoclonal IgG typically is isolated from ascites (intraperitoneal fluid) from mice with hybridoma tumors that secrete a single IgG or from hybridoma tissue-culture medium. In the case of Mzapp, the antibodies were isolated from genetically modified tobacco plants.
Concentration and purification of IgG is typically performed by chromatography using Protein A- or Protein G-Sepharose beads, which bind to the Fc region of the antibody.
In a typical protocol, the serum or filtered fluid/medium is passed through a 5 ml Protein A- or Protein G-Sepharose column, which is then washed with phosphate-buffered saline (PBS). IgG is
eluted with 0.05 M Na-acetate, pH 3.0, collecting 2-ml fractions into tubes containing 70 μl 1.0 M Tris base. (For acetic acid, pKa = 4.76; for Tris, pKa = 8.1; for H3PO4, pKa1 = 2.12, pKa2 = 7.21,
pKa3 = 12.32)
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The most common composition of PBS (1X)
Salt Concentration (mmol/L) Concentration (g/L)
NaCl 137 8.0
KCl 2.7 0.2
Na2HPO4 10 1.44
KH2PO4 1.8 0.24
a. What is the pH of this PBS?
b. Why does PBS contain NaCl and KCl, and why is their total amount about 140 mM?
c. What are the concentrations of acetic acid and acetate in the 0.05 M Na-acetate, pH 3.0 used for
elution of the IgG?
d. What is the final pH of the 2-ml fractions containing purified IgG? In making this calculation,
assume any contribution to pH by the IgG is insignificant.
e. Why does 0.05 M Na-acetate, pH 3.0, elute the IgG and why is an aliquot of 1.0 M Tris base put
in the collection tubes?
f. What type of chromatography is this? Explain.