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Custom Immunology Services
Antibody Purification Services |
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Affinity Purification
Protein A, and G purification are excellent techniques for purification of total IgG from serum, ascites, cell culture supernatant and other biological samples. However, the antibody with less cross reactivity and higher specificity requires affinity purification that is specific for a particular antigen. The cross reactants can be inhibitors, interfering IgG or inappropriate buffer solution components. The antibody of interest can be purified away from a complex mixture of biological samples by positive selection or by removing specific contaminants from a sample containing a protein of interest by negative selection.
- The capture of desired antibodies to a specific antigen is generally referred to as positive affinity purification
- The removal of undesired or interfering antibodies of known specificity is generally referred to as negative affinity purification.
The above procedures have several things in common. The antigen is immobilized on some form of support. This is generally a column packing material such as agarose, dextran, polyacrylamide beads, crushed glass or ceramic particles. The biological sample to be purified is exposed to the immobilized antigen on a chromatography column. The immobilized antigen antibody complex is washed free of unbound material (negative selection for undesired or interfering products) and bound antibody is eluted from the column using buffers of varied pH.
Affinity purification is potentially a very powerful tool. Negative affinity purification can generally be safely applied since the desired antibodies remain unbound and are recovered in the pool of the column flow through.
It is generally considered prudent to check the level of recovery by first purifying a small sample, 10 to 20%, of the pool intended for purification by performing a safety net Procedure. In the "SafetyNet" procedure, we first prepare the antigen specific affinity resin(s), and purify 10mL to 20mL of serum to establish an optimized condition for bulk purification and provide opportunity for the client to evaluate purified antibody in their functional assay.
While this increases the overall effort and cost for purification of antigen specific antibody, it does allow the affinity purification strategy to be checked for recovery and the resulting antibody tested for utility before committing the bulk of the sample for purification.
Covance provides a full spectrum of affinity purification services including both positive and negative purifications and can customize procedures to meet your requirements.
Antigen Requirements:
- Approximately 5-10 mg of peptide or protein is required for column preparation. Please call Covance to discuss varying amounts available.
- Fusion proteins can be used for the immunization phase, but the cleaved protein or the protein with an alternate carrier will be needed for screening and purification. Peptides conjugated to a protein carrier can be used for immunization as well, but the unconjugated peptide, linear peptide, or peptide conjugated to an alternate carrier will be needed for screening and purification.
- If you are sending a peptide, we will need the sequence of the peptide. If the sequence is not available, we will need to know if there is a Cysteine present and its location in the sequence.
Note: If you would like us to ship you your column with your purified material, they will be shipped in two separate shipments.
Antigen-specific Affinity Purification vs. Protein A and Protien G
In simple terms, the antigen specific affinity purification isolates antigen specific antibodies, while Protein A and G purifications give total Ig purification from a sample. Purification of an antibody specific for a particular antigen and free of cross reactants from other immunoglobulins is often required. This is accomplished by immobilizing immunization antigen on a column so that only antibodies binding specifically to immunization antigen are isolated.
Efficient antigen specific affinity purification depends on effective presentation of the relevant epitopes on the antigen to binding sites of the antibody. In the case of small peptides, immobilization to a solid support by multiple chemical bonds may cause important epitopes to be blocked, preventing effective antibody binding. Therefore, it is recommended to immobilize antigen using a unique functional group and to use an activated support whose reactive groups occur on spacer arms.
IgG Affinity Purification of Antibodies
The animals immunized with prepared antigens will produce specific antibody against the antigen. Because antibodies have predicable structure, including relatively invariant domains, it has been possible to identify certain protein ligands capable of binding to antibodies. Protein A and Protein G are two bacterial proteins whose antibody-binding properties have been well characterized. These recombinant proteins have been produced and used routinely for affinity purification of antibody types from a variety of species.
Purification by Protein A (from Staphylococcus aureus) and Protein G (from group G streptococci) are based on their ability to bind to the Fc region of IgG.
Protein A and G differ in their ability to bind antibodies of different species and subclasses. For example, Protein A has a strong affinity for rabbit IgG while Protein G is the preferred method for mouse IgG1. The method chosen should be based on these known affinities. Both Protein A and Protein G purifications are rapid, reproducible and amendable to scale-up. |
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| Antibody |
Protein A |
Protein G |
| Rabbit IgG |
s |
s |
| Mouse IgG |
w |
m |
| Mouse IgG2a |
s |
s |
| Mouse IgG2b |
s |
s |
| Mouse IgG3 |
s |
s |
| Rat IgG |
w |
m |
| Goat IgG |
w |
s |
| Sheep IgG |
w |
s |
| Guinea Pig IgG |
s |
w |
| Pig IgG |
s |
w |
| Horse IgG |
w |
s |
| Chicken |
nb |
nb |
w=weak binding, m=medium binding, s=strong binding, nb=no binding
* data represents an assimilation of information from various pieces of literature |
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Ammonium Sulfate Precipitation: Ammonium sulfate is widely used in isolating antibody from a variety of species. A gel-filtration step can be employed to further purify the immunoglobulins.
Aseptic/Low Endotoxin Purification: Purification under aseptic conditions in a dedicated, limited-access facility can generate clean, low-endotoxin antibody preparations. Batch records and extensive SOPs support all activities.
Chicken Antibody Production and IgY Purification
Egg yolks are a rich source of polyclonal antibodies. A single egg contains as much antibody as an average chicken production bleed and a chicken lays an average of 5-7 eggs per week. Many investigators elect to have Covance test the sera by ELISA and then use this information to select the eggs to be purified. IgY purification from eggs presents a non-invasive and cost efficient way to obtain large amounts of antibody. If requested, the product of this purification can be further purified over an antigen coupled affinity column to obtain antigen specific antibody. Secondary chicken antibodies are widely available.
IgY, the predominant serum immunoglobulin of birds is the structural homolog to mammalian IgG, but it is different from mammalian IgG. IgY antibodies are very useful tools in research and have many advantages over conventional antibodies derived from rabbits, mice, rats, and other mammals. Despite the advantages associated with producing antibodies in chickens, the method is not as widely used as might be expected. This could be due to several factors, such as incorrect information about the procedure, a lack of appropriate experience with the technique, or simply a reluctance to use new methods.
In the past decade, R&D organizations have developed significant knowledge to produce immune eggs as effective, economical alternatives to serum-sourced antibodies. Covance adopted this technology very early and developed our own refined procedures for generation and purification of chicken IgY to a variety of antigens. Our chickens are maintained in our custom, animal barrier facility to minimize immune background and all technicians are AALAS trained, validated, and certified in the use of chickens for antibody production. Considering the many benefits of IgY technology in polyclonal antibody production and the utility of these antibodies in medicine, IgY antibodies will have an increasing role in R&D diagnostics and immunotherapy. |
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Mammalian IgG |
Chicken IgY |
| Sampling |
Invasive |
Non-Invasive |
| Amt. of Antibody |
~200 mg IgG per bleed (40 mL of blood) |
~40-100 mg IgY per egg (5-7 eggs per week) |
| Amt. of Antibody/Month |
~200 mg |
~1500 mg |
| Amt. of Specific Antibody |
~5% |
2-10%s |
| Protein A/G Binding |
Yes |
No |
| Cross-Reactivity with Mammalian IgG |
Yes |
No |
| Cross Reactivity with Mammalian Rheumatoid Factor |
Yes |
No |
| Activation of Mammalian Complement System |
Yes |
No |
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Other purification options are available
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Our Shipping Address
Attn: Immunology Services
465 Swampbridge Road
Denver, PA 17517
tel (800) 345-4114
tel (717) 336-4921
fax (717) 336-3481
AAALAC International Accredited, GLP Studies, OLAW Assured, USDA Research Registered
Document Revision Date: 4/5/07 |
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