Antibody Purification Methods
Antibody purification can be divided into two groups: precipitation methods and
chromatographic methods. The latter is grouped further into non-affinity and affinity
chromatography.
The choice of purification methods available include:
- Polyethylene Glycol (PEG) and /or Ammonium Sulphate Precipitation
- Immunoaffinity purification
- Protein A affinity Chromatography
- Protein G Affinity Chromatography
- Protein A/G affinity chromatography
- Ig Antibody Affinity Purification
- Antibody Purification by Ion exchange chromatography
- Hydrophobic interaction chromatography
- Size exclusion chromatography
- Ammonium sulfate precipitation
- HPLC purification
- Procedures and methodology used for the purification and a
Quality Control report (including analysis of yield, purity and immunoreactivity)
are provided with each purified product
Antibody purification using Protein A, G, or A/G
Protein A/G is a recombinant or genetically engineered protein that combines both
Protein A and Protein G binding properties. Protein A/G is designed to contain four
Fc binding domains from Protein A and two from Protein G. Protein A or Protein G
purification removes the IgG fraction based on the specificity of these proteins
for the Fc portion of the IgG. Protein A is produced from Staphylococcus aureus.
It has the capacity to bind at least two molecules of IgG. The binding is specific
to the Fc portion and does not affect the antigen binding sites. Protein G is isolated
from Group G streptococci and binds the Fc region of the IgG in a similar manner
to Protein A. Protein A and G have differing binding efficiencies for IgG from different
species. It is important to check this before deciding which method to use. For
example, Protein G works well with sheep Ig, but Protein A does not. Neither Protein
A nor Protein G will bind to chicken Ig. Care should be taken when eluting the antibody
from the column to avoid denaturation of the antibody.
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Type of Sample
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Isotype
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Average Concentration
|
|
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Serum
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lgG
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Serum: 1 to 3 mg/ml
|
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Ascitic Fluid
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Ascitic fluid: 1 to 5 mg/ml
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Cell culture supematant
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Cell Culture supematant: 5 to 20 ug/ml
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Service Includes:
- Preparation of protein A or G affinity column, loading an delution
of bound antibodies
- SDS-PAGE analysis of purified antibodies
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|
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Peptide Immunoaffinity Purification
The most commonly used method to purify antigen-specific antibodies from crude sera
is immunoaffinity purification. Unlike Protein A or G, the non-specific Ig fraction
is not retained. In this procedure, peptide antigen is bound covalently to a solid
support. The antibodies within the polyclonal sample that are specific for the peptide
antigen bind to the support column. The unbound antibodies are removed from the
column by washing and the specific antibodies are eluted from the column. The product
of immunoaffinity purification is highly specific antibodies. Immunoaffinity purification
can occasionally cause denaturation of the antibody due to the conditions used to
elute the bound antibody from the column. It is important to compare the response
generated by the purified sample against the response generated by the crude sera.
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Type of Sample
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Min Peptide Quantity
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Average Concentration
|
|
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Serum
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5 to 7 mgs
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50 to 100 ug of immuno-purified antibodies/mL
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Service Includes:
- Peptide immobilization on sepharose beads
- Loading of antiserum on the affinity column and elution of
bound antibodies
- LSIA testing on crude serum and immunopurified antibodies
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|
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IgY Purification from Egg Yolk
Chicken antibodies IgY are the equivalent to mammalian IgG antibodies and can be
isolated from serum or egg yolk. The egg yolks contain at least the same concentration
of IgY antibodies than found in chicken or rabbit serum. IgY can be used in most
biochemical and cellular applications, including western blots. As the Fc region
of chicken IgY is sufficiently different from mammalian IgG chicken antibodies do
not cross-react with mammalian proteins, do not bind with mammalian rheumatoid factors
or Fc-receptors and with proteins A or G. Therefore the background signal or false
response in certain immunochemical assays is reduced.
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Type of Sample
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Yield of Purification
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|
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Eggs, or egg yolks
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~10 to 15 mg of lgY / egg yolk
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Service Includes:
- Preparation of the egg yolk extract by two-step precipitation
- Loading of the egg yolk extract on the ion exchange column
and elution of bound antibodies
- ELSIA testing on starting material and immunopurified antibodies
- SDS-PAGE analysis of purified antibodies
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|
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Polyethylene Glycol and /or Ammonium Sulphate Precipitation
This method is useful for concentration and partial purification of antibodies from
most sources and all species including purification of IgY's from chicken egg yolks.
Although, on its own the yields of antibodies are impure, it is the method of choice
when combined with ion exchange for large volumes of antisera and or followed by
Antigen Affinity purification to enrich for specific antibodies. It is not recommended
for purification of tissue culture supernatant.
Ig Antibody Affinity Purification
This method can be used to isolate monoclonal antibodies from supernatants which
contain high levels of serum Ig. It is the choice for secondary antibody production.
Size Exclusion/Gel Filtration Chromatography
A single gel matrix or a sequence of size exclusion columns can be used for purification
of IgM and IgG antibodies. The purification strategy employed is dependent on the
immunoglobulin class and the source of the material.
Antibody Purification by Ion Exchange Chromatography
This is usually the methods of choice when purifying antibodies from ascites, as
the monoclonal can be resolved from the host immunoglobulins. It has been used in
adjunct to ammonium sulfate precipitation for purifying antibodies from ascites
fluid or where protein A and protein G elution conditions may damage the antibody.
It is also applicable to all IgG isotypes.
Aseptic/Low Endotoxin Removal, Purification and Testing
Purification under aseptic conditions in a dedicated, limited access facility to
generate clean, low-endotoxin antibody preparations.
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