The antibody shift: From ascites to culture

Antibodies are the body’s ultimate weapon, produced by B lymphocytes to circulate in the blood and other body fluids and defend against invading pathogens. These proteins can quickly recognize unique antigens and either tag the offending cell for attack by other members of the immune system or neutralize the target directly. Given this innate ability to bind and tag any designated antigen, the antibody’s utility in research, diagnostics and, more recently, therapeutics is constantly expanding as novel uses continue to be discovered.

Antibodies have become an essential part of a variety of research disciplines, integral to western blots, enzyme-linked immunosorbent assays (ELISA), immunohistochemistry (IHC), flow cytometry, and much more. In diagnostics and therapeutics, they provide accurate antigen recognition and selective binding to disease-specific cell types. With over 300 companies that manufacture antibodies and thousands of antibodies to choose from, whether monoclonal antibodies generated from a single clone of B lymphocytes or polyclonal antibodies produced by several B lymphocyte clones, selecting the highest quality antibody specific to a particular application is a critical part of experiment planning and execution.

However, because it can be difficult to identify the right antibody for a given study, dissatisfaction is common amongst researchers with respect to antibody validation and experimental reproducibility. While there is quite a bit that researchers can do to ensure success, meticulous antibody production is critical to study performance. Understanding the methods for the manufacture of antibodies and how each is more robust for certain applications can help not only in antibody choice but in authentication of experiments.

Ensuring quality in monoclonal antibody production

Quality monoclonal antibody production generally relies on two well-known methods. In vivo production using ascites, developed in 1975 by Kohler and Milstein, can produce high concentrations of high affinity monoclonal antibodies and has the ability to include post-translational modifications such as glycosylation within the animal model. This method has transformed immunology, leading to the creation of thousands of different antibodies for both research and therapeutic use. Nevertheless, there are limiting factors associated with ascites, particularly ethical considerations for the mouse models involved.  As a result, in vitro methods have more recently become a standard for successful antibody production.

Most antibody production methods begin with injection of an antigen into mice or rats. Their extracted lymphoid cells can be fused with myeloma cells to create cloned hybridomas. The hybridoma cell lines that produce the desired antibody are isolated and cloned to become antibody production houses. In vivo production requires a second animal model, where hybridoma cells are injected into the mouse and expand as ascites tumors, secreting high concentrations of the target antibody into the surrounding fluid. These antibodies can then be collected and purified.

While the in vivo method is a confirmed approach for antibody production, concern surrounds the use of animal models in such a way. Laws and guidelines limit animal use in favor of newer in vitro methods, highlighting animal welfare yet still recognizing the utility that in vivo production provides. In some cases, only ascites models can be used to make antibodies depending on the application and type of antibody needed. Cognizant of this issue, the National Institutes of Health (NIH) lists circumstances where the ascites method is permitted, including when hybridoma cell lines cannot adapt to in vitro conditions, for higher antibody production needs, or to solve issues with decreased antibody activity following purification from cell culture.

In vitro methods circumvent ethical issues with animal use by growing hybridoma cells in culture, where the cells secrete antibodies directly into the culture medium. This approach generates quality antibodies with the advantages of easy purification and large-scale production potential but cannot be used if antibodies require post-translational modification or in cases where high yields are required.

What to look for in a good antibody

Maintaining antibody structure and reactivity along with preventing system contamination are two primary issues seen during antibody production that can seriously impair study outcomes. Stringent purification procedures might alter the native structure of an antibody and result in a loss of reactivity or binding ability. Denaturation can also occur, enhancing immunogenicity and causing decreased in vivo antibody retention time. Contamination from the presence of other immunoglobulins in the animal model could hinder antibody collection in in vivo methods and impact immunoreactivity, whereas issues with potential endotoxins, yeast, fungus, or mycoplasma in cell culture can affect the quantity and quality of antibodies produced in vitro. Ensuring that a vendor produces antibodies void of these issues and provides validated antibodies is crucial for their overall performance in the lab.

One of the most important properties of an antibody is its ability to bind selectively to an antigen. One must first have a defined specification of the target analyte to help determine which antibody is right for what application. Once set, reviewing in-depth validation data by ELISA testing, western blots and IHC using biologically relevant sample types and tissues can provide an accurate reflection of how the antibody will function.

Whether used for screening, diagnosis, or even treatment, sensitivity, specificity and reproducibility are all essential factors to consider in quality antibodies. From a vendor’s perspective, care must be taken in the production process to ensure successful manufacture, including initial immunization to correctly trigger B lymphocyte differentiation into more mature configurations and selective fusion of the antigen-sensitized B lymphocytes with myeloma cells to generate effective hybridoma cell lines. Superior hybridoma technology can support the generation of monoclonal antibodies with high affinity and specificity, contributing to positive validation and experimental reproducibility.

Cell Sciences supplies both in vitro and in vivo produced monoclonal, polyclonal and functional biologically active antibodies, depending on which method best generates the quality, specificity and reactivity needed for successful and reproducible results. As the number of applications enabled by antibody-based technologies continually expands, Cell Sciences is working to supplement the large arsenal of currently available antibodies and meet the increased demand for the development of antibodies for new targets of interest.

Cell Sciences offers an extensive selection of monoclonal antibodies produced in cell culture and preservative free.

Here are examples of our top sellers:

CDM265 Mouse Anti-Human IL-6  (clone B-E8)

CDM005 Mouse Anti-Human IL-2R alpha (clone B-G3)

CMV015 (Mouse Anti-Human VEGF (clone 3 6D3)

CDM049 Mouse Anti-Human E-Selectin/CD62E (clone B-P7)

CDM288 Mouse Anti-Human IL-17F (clone B-F60)  

CDM012 Mouse Anti-Human IL-6ST (clone B-R3)

CDM271 Mouse Anti-Human IL-12 p35 + p70 (clone B-T21)

CMV012 (Mouse Anti-Human VEGFR-2/KDR (clone 3 4H3)