THEORY: When foreign organisms enter a host system the body has particular mechanisms that

allow it to destroy and remove this invading organism, this is known as immunity and it can be
sub-divided into two types/classes; innate or adaptive. Innate immunity is a natural non-specific,
general immunity that has a fast and equal response to all invading organisms. When this is not
enough the other type of immunity is activated, the adaptive immunity is specific for particular
antigen and is not as rapid as innate. In this experiment we would try to isolate the
immunoglobulin IgG from whole serum using precipitation, dialysis and ion exchange
chromatography, but in order to fully understand the mechanisms for isolation and quantification
we must first have and understand of what IgG is.
Immunoglobulins are glycoprotein molecules produced by plasma cells and are also known as
antibodies. They bind to its corresponding antigen to form a antigen-antibody complex which are
large insoluble compounds the precipitate out of solution. Antibodies consist of 2 identical heavy
chains (50 70kD) and 2 identical light chains (23kD) which arrange to form a Y shaped
structure held together by both inter and intra chain disulphide bonds. The light chain
structures contain one constant domain (110 amino acids) and one variable domain (110 amino
acids), while the heavy chain consist of three constant domains (330 - 440 amino acids) and one
variable domains (110 amino acids) with the two variable domains interacting to form the
antigen binding site of the antibody (Figure 1).

Figure 3: Structure of Antibody. ( Lehneger, Principles of Biochemistry, 2008)

These immunoglobulins can then be divided into five different types; IgG, IgA, IgE, IgM and
IgD based on differences in amino acid sequence of the constant domains in the heavy chains.

IgG is the most abundant immunoglobulin, making up 75 80% of the serum antibodies and is
one of the two antibodies synthesized in response to an infection. This antibody is the only one
capable of crossing the membranes of small blood vessels to reach extracellular antigen and
cross the placenta membrane. IgA is the second most common antibody present and is most
abundant in secreations such as mucus, saliva, tears, etc. There are two very different types of
IgA, one that is secretory and the other is not, they have to separate shape and places of
synthesis. Secrertory IgA ( IgA2) is synthezied in the B cells and is dimer with a polypeptide
attached to it (J). Non secretory IgA (IgA1) is synthesized in the bone marrow and is
monomeric. The third most common antibody is IgM and it is the largest antibody present
consisting of 5 Y structures joined together by bonds between their heavy constant regions and
an additional polypeptide chain. It is activated/synthesized in response to primary infections and
is the first antibody produced by babies. IgM is also very good at activating the compliment
system. Another type of antibody is the IgE which as a very low concentration in serum, this is
due to the fact almost all produced IgE is bound within a tissue cell like mast cell and once it
comes into contact with antigen it recruits the suitable molecules for the removal of this invading
pathogen. The last antibody is the IgD which is found on the surface of B cells and the with
monomeric IgM acts as receptors for B cell activation

Figure 2: Different types of Ig (The Microbial World, 2006)

As IgG is the most abundant antibody in serum this is the one we would attempt to isolate and
quantify, this is done by first adding a salt, which in this case is sodium sulphate. As the salt is
added to the solution it would cause a change in the environment in which the IgG is in (change
in pH) which as increase addition of salt would cause the IgG to precipitate out of solution. This
is based on the idea that the solubility of the IgG depends on the salt concentration of the
solution, as an increase concentration would decrease the concentration of water in solution that
would interact with the IgG. Hydration circles would form around the ions of the salt and not the
IgG. By centrifuging at low speeds would cause it precipitate to settle at the bottom and be able
to be extracted.
The second method uses was dialysis, in this, the extracted IgG was placed in a semi-permeable
bag/sack and the left in a buffer solution for a measured period of time. The sack would allow for
the movement of the salt ions to move from the sack into the buffer solution but would prevent
the IgG from following. This would help purify the extracted sample. This IgG that would be
removed from the sack after allotted time would not completely pure and so must undergo some
sort of further purification, this can be facilitated by ion exchange chromatography which
separates molecules based on their charge. The resin that will be used is DEAE cellulose which
is a positively charge resin that binds negatively charged particles, as IgG is positively charged it
would be expected to be eluted first. The absorbance of the elutes are then taken at 260nm and
280nm with an expected reading to be above 1.00.
DISCUSSION:
In this experiment we first had to isolate and then quantify the amount of IgG present in the
serum. The first part of the experiment dealt with the precipitation of the IgG from serum by
addition of sodium sulphate. This is added slowly to the solution while mixing to ensure it
completely dissolves because precipitation of IgG will only occur when salt is in excess in
solution in order to truly compete for the water molecules present in solution. The addition of
sodium sulphate occurs twice at different w/v percentages. This is because not all IgG would
precipitate during the first addition, because even though the ions in the salt have a higher
affinity for the water molecules some hydration circles would still form around the IgG so by

repeating this process at a different saturation level guarantees the highest level of precipitation
that can occur. The precipitation of IgG can be a result of three types of interactions;
electrostatic, hydrophobic and Van der Waals forces. In electrostatic interactions, like forces
would repel each other and so molecules within solution would re-arrange themselves in a way
to reduce the amount of interaction between like charges. It is not as important in the attraction
of the IgG molecules but it is important is the prevention of them separating. A next type of
interaction, which is by far the most important interaction, is hydrophobic interactions. IgG is a
type of protein and like all proteins consist of both a hydrophilic and hydrophobic region and as
all proteins when in solution the arrange themselves to prevent high levels of interactions
between water and the hydrophobic region. When the salt is added to the solution and starts to
compete for water molecules, ions of salt have a higher affinity, and the hydration spheres around
IgG begins to deteriorate and move towards the salt ions, the IgG must now re-arrange and
clump together to prevent the hydrophobic interactions.
After the IgG was isolated and purified it was placed in a UV spectrometer and its absobance
read at 260nm and 280nm. This is done because proteins show a strong absorbance at this region
in the UV spectra due to the presence of aromatic rings. As IgG has the highest concentration in
serum it would expect to show a high absorption reading. This was not observed however with
the maximum reading being 0.207 at 280nm. This may due to the fact that the extract of
precipitation was left for two weeks to dialyze in a fridge and as IgG is a protein it would
eventually become denatured and not absorb any light within the UV region
We also did gels with the elutes that showed the highest absorption, and it was seen the higher
the absorption in the 280nm region the more distinct the bands fromed.