Purifying Antibodies

What is Antibody Purification

Antibody purification is a process in which antibodies are extracted from an antiserum (pAb), ascites fluid or cell culture supernatant of a hybridoma cell line (mAb). Once purified, the antibodies can be used in a variety of ways. This process can be performed at labs which offer an antibody purification service, and people can also purify their own, if they have the necessary equipment and tools. This may be done in a lab where people want rapid turnarounds for their research, or when researchers are concerned about protecting the integrity of original research.

One of the major sources of antibodies is antiserum produced in animals. In this case, an animal is repeatedly injected with an antigen until it develops antibodies, and its blood is used to prepare an antiserum which can be purified to access the polyclonal antibodies. Another source of antibodies is a cloned cell which produces antibodies as part of a colony of identical cells; this method is used for mass production of monoclonal antibodies.View more about "Monoclonal Antibody Purification".

How Does Antibody Purification Work

There are several techniques that can be used for antibody purification. The most suitable technique depends on the biological properties of the antibody. Affinity chromatography and protein A/G are both used in the pursuit of antibody purification, for the purpose of isolating antibodies from the rest of the solution. Classically antibody purification relies on using a substance which binds to a specific antibody, holding in place while the rest of the solution is washed away. View more about "Antibody Purification Methods".

Our Capabilities in Antibody Production

Sino Biological has accumulated many years of antibody purification through thousands of monoclonal antibody production projects, including recombinant antibodies from transient transfected HEK293 and CHO cell cultures, mouse, rat and rabbit monoclonal antibodies from hybridoma cultures, and polyclonal antibodies from animal sera. Our experiences cover all sub-types and classes (IgG, IgA, and IgM) of human antibody, mouse antibody, rat antibody, rabbit antibody, canine (dog) antibody, and bovine antibody. Our antibody purification service typically is offered in combination with antibody production and cell culture services. A partial list of our antibody purification is shown below:

• high-quality antibody purification and polishing with monomer purity and endotoxin control
• human, mouse, rat, rabbit, and canine IgG antibody purification services
• human and mouse IgA antibody purification service
• human and mouse IgM antibody purification service
Polyclonal antibody purification with protein A/G affinity resin
• Polyclonal antibody purification with antigen affinity resin

Human antibody purification

• IgG antibody
IgG antibodies are the most abundant antibody class in human and animal blood. IgG antibodies are most widely used as research reagents and also as therapeutic products. Human IgG antibodies are classified into four subtypes: IgG1, IgG2, IgG3, and IgG4. Mouse IgG antibodies are classified as IgG1, IgG2a, IgG2b, and IgG3.

IgG antibodies can be produced from hybridoma cell culture or with transient transfection technology in HEK293 cells or CHO cells. Human IgG antibodies bind well to protein A or protein G, so protein A affinity resin is widely used for human IgG antibody purification, regardless of the sub-class. However, for mouse and rat IgG antibodies, their binding affinity to protein A or G resin varies significantly. As a result, purifying some mouse and rat monoclonal antibodies could be a challenging task. Selection of the right resin and using appropriate purification buffers under optimal process conditions are critical for successful purification of some difficult-to-purify antibodies.

Sino Biological has well trained professional staffs. It has produced and purified thousands of mouse and rat monoclonal antibodies from hybridoma cell culture as well as from transient transfection process. Our platform technology and experience can be of great value to you. By taking advantage of our antibody production and purification services, you can accelerate your antibody discovery and development projects while saving costs at the same time.

• IgA antibody
IgA antibodies are quite different from IgG antibodies. IgA Antibody has two subclasses (IgA1 and IgA2) and can exist in a dimeric form called secretory IgA (sIgA). In its secretory form, IgA is the main immunoglobulin found in mucous secretions, including tears, saliva, sweat, colostrum and secretions from the genitourinary tract, gastrointestinal tract, prostate and respiratory epithelium. It is also found in small amounts in blood.

The dimeric IgA antibody can be produced using recombinant technology. By co-transfecting HEK293 cells with IgA antibody's light chain and heavy chain vectors, monomeric IgA antibodies can be expressed, but homo-dimer IgA antibody formation usually needs co-expression of the J-chain protein, which links the two IgA molecules together.

Sino Biological has accumulated hands-on experience in expression and producing the dimeric form of the IgA antibodies using our cutting-edge transient transfection platform. Our powerful transient transfection platform can offer great services to customers worldwide.

• IgM antibody
IgM antibody usually forms pentamer and hexamer polymers. Monomer IgM antibodies are covalently linked together with disulfide bonds. In pentameric IgM molecules, a J chain is present to hold two IgM molecules together with disulfide bonds. However, in the hexameric form, there is no J chain present.

Due to the large molecule weight and involvement of IgM light chain, heavy chain, and the J chain genes, recombinant IgM expression and production is rarely practiced. IgM antibody purification is particularly challenging because IgM antibody does not bind well to either protein A or protein G, and hence affinity purification is often not an option. In combination of low expression yield, large molecule weight, and poor stability, IgM antibody production seems to be an impossible task for most customers. However, IgM antibody has an advantage to IgG antibodies because of the pentamer structure of the antibody, meaning one IgM molecule could potentially binds to 10 copies of the antigen. As a result, IgM antibody sometimes shows a higher binding capability to IgG antibodies, especially for non-protein type of antigen and epitopes.

Sino Biological has devoted significant resources in developing in-house expertise in recombinant IgM expression, production, and purification. With our advanced mammalian transient transfection platform technology, we have achieved reasonable IgM expression in HEK293 cells. Besides, we have developed highly effective chromatographic purification and polishing steps for IgM recovery and purification. We were able to obtain high purity IgM antibodies. We are starting to offer our expertise in this field to support our customer's recombinant IgM production and purification needs. If you need to produce and purify IgM antibodies, please let an expert like us handle the task for you.

Mouse IgG antibody purification

• Mouse IgG1 Antibody
A very high percentage of Mouse monoclonal antibodies used for research are mouse IgG1 antibodies. Mouse antibody purification from either hybridoma cell culture or transient transfected 293 or CHO cell cultures may not be as straight forward as it looks. Often it can be troublesome due to low binding affinity of some mouse antibodies to protein A and protein G resin, especially for IgG1 and IgG3b antibodies. Precautions need to be taken to avoid antibody flow-through in protein A and protein G affinity purification. Optimization of the loading buffer can increase the binding efficiency of mouse antibodies to protein A and protein G resins, and hence can increase the yield of mouse antibody recovery from cell culture supernatant.

Sino Biological has produced and purified thousands of mouse monoclonal antibodies either in mouse hybridoma cell cultures or transient transfection in HEK 293 cells. Our hybridoma cell culture and transient transfection capacity are virtually unlimited for any scale of mouse IgG antibody production and purification. Our professional mouse IgG1 antibody production team is ready to serve you in meeting your antibody production needs.

• Mouse IgG2a Antibody
Mouse IgG2a antibody is also quite commonly used in life science research. In mouse serum, IgG2a antibody's concentration ranks 2nd among the four subtype antibodies. Therefore, when one immunizes a mouse with an antigen to generate a mouse antibody, the chance of obtaining an IgG2a mouse monoclonal antibody is pretty high. Not surprisingly, it is quite common that one needs to produce and purify mouse IgG2a antibodies.

In the past couple of years, Sino Biological has produced many mouse IgG2a monoclonal antibodies, both for in-house antibody reagent development and for customers worldwide. Our rich experience in mouse IgG2a antibody purification can be of great value to our customers who are interested in using our expertise and capacity to support their antibody production needs.

• Mouse IgG2b Antibody
Mouse IgG2b antibody is the third abundant IgG subtype in mouse serum. It is also quite often that the antibody meeting your application requirement is a mouse IgG2b antibody. Fortunately, mouse IgG2b antibodies bind to protein A and protein G resins quite well, so capturing mouse IgG2b antibody from hybridoma cell culture supernatant is usually straightforward using protein A affinity column.

Sino Biological is highly experienced in producing and purifying mouse IgG2b antibodies. Our experience not only includes mouse IgG2 antibody production and purification from mouse hybridoma cell culture but also includes recombinant mouse IgG2b antibody production and purification from transient transfected HEK293 cell cultures. We've built one of the largest mammalian cell culture facilities with hundreds of cell culture flasks and over 80 stirred-tank bioreactors ranging from 2L to 300 L. Our professional biological service team is ready to support your mouse IgG2b antibody production.

• Mouse IgG3 Antibody
Mouse IgG3 antibody is the least abundant IgG antibody in mouse serum, so it is not very common to find a mouse IgG2 antibody as a research reagent. However, mouse IgG3 antibodies do exist as research reagents and some of them are important tools for research. Mouse IgG3 antibodies bind to protein G better than binds to protein A, so protein G affinity resin is used more often than protein A resins.

Sino Biological has produced plenty of mouse IgG3 antibodies as research reagents in house and hence has accumulated significant experience. We have developed a highly specific mouse antibody subtype detection kits that can be utilized to precisely determine the subtype of mouse antibody. Our experience in mouse IgG3 antibody purification plus our large scale cell culture capacity and facilities allows us to provide reliable and cost-effective mouse IgG3 antibody production and purification services, either from mouse hybridoma cell culture or using our advanced transient transfection technology.

Rat IgG antibody purification

Rat IgG antibodies have four subclasses: IgG1, IgG2a, IgG2b and IgG2c. Each subclass antibody's binding affinity to staphylococcal protein A with the order of: IgG2c > IgG1 > IgG2b > IgG2a (the affinity of IgG2b being very weak, and IgG2a having virtually no binding affinity for protein A). Rat IgG antibodies are often developed against mouse antigen because mouse cannot generate a strong immune response to its own proteins.

Sino Biological offers full services for rat IgG antibody production and purification. Two different strategies are provided: rat hybridoma cell culture or transient transfection in mammalian cells. Transient transfection is offered as an alternative approach if the rat hybridoma cell line is not stable, difficult to grow under serum-free conditions, or it is contaminated. We are experienced in cloning the rat antibody light and heavy chain genes from the hybridoma cells, then use our advanced transient transfection platform to produce the rat antibodies under serum-free conditions at any scale required by customers. Our rat IgG antibody production and purification service is easy to manage, highly reliable, and affordable. It frees up your time and let you focus your expertise on key elements of your research.

Rabbit IgG antibody purification

Rabbit monoclonal antibody has shown great promises in biological research and clinical diagnostic applications. It has a similarly high antigen-binding affinity to rabbit polyclonal antibodies but has much improved batch to batch quality reproducibility. Rabbit monoclonal antibodies also have much higher antigen binding affinities than mouse monoclonal antibodies., sometimes 10 to 100 fold higher. Rabbit monoclonal antibodies also have shown excellent specificity in western blot and immunohistochemistry (IHC) applications.

Sino Biological not only has developed its own proprietary technology for rabbit monoclonal antibody development, but also has developed cutting-edge platform technologies for rabbit monoclonal antibody production and purification. We offer services on rabbit IgG antibody production from rabbit hybridoma cell culture and transient transfected HEK293 cells. Our capacity in large scale cell culture supports both rabbit hybridoma cell culture and HEK293 cell culture, and our expertise and experience in rabbit monoclonal antibody purification allows us to produce high quality rabbit monoclonal antibodies reliably and cost-effectively.

References

1. Low, D., O'Leary, R., & Pujar, N. S. (2007). Future of antibody purification. Journal of Chromatography B, 848(1), 48-63.
2. Hober, S., Nord, K., & Linhult, M. (2007). Protein A chromatography for antibody purification. Journal of Chromatography B, 848(1), 40-47.
3. Gagnon, P. (2012). Technology trends in antibody purification. Journal of chromatography A, 1221, 57-70.
4. Shadle, P. J., Erickson, J. C., Scott, R. G., & Smith, T. M. (1995). U.S. Patent No. 5,429,746. Washington, DC: U.S. Patent and Trademark Office.

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