Protocols
Please note that the protocols below apply to affinity purified rabbit polyclonal Genomic Antibody Technology™ antibodies.
Western Blot Protocol
Immunohistochemistry Protocol
Hybridoma Development FAQ
Q: How many animals are used in the immunization phase?
A: On a standard hybridoma project, we immunizes 15 mice (5 BALB/c and 10 Swiss Websters).
Q: How many clones will I get?
A: In a standard hybridoma project, a maximum of 5 clones are selected.
Monoclonal Antibody FAQ
Q: What is the antibody yield from ascites fluid?
A: The yield of monoclonal antibody in ascites fluid depends on two major factors. First, the inherent ability of the hybridoma cell line to synthesize and secrete monoclonal antibody. This inherent monoclonal antibody production rate may be improved with molecular biology intervention. Second, the health, clonality, and environment in which the hybridoma cell line is synthesizing and secreting monoclonal antibody contribute to the productivity of the cell line. The full inherent potential of the cell line to produce monoclonal antibody may be compromised if the culture viability is poor, if the cell line is allowed to exhaust critical media components or if the clonality of the cell line is low.
SDIX's mean antibody yield from ascites production using hundreds of cell lines over the last 2 years is 18.9 mgs of antibody per mouse. The mean has a wide variance due to inherent different among cell lines to synthesize and secrete monoclonal antibody. Some cell lines have produced less than 1 mg of antibody per mouse while others have produced over 50 mgs of antibody per mouse.
Q: What is the antibody yield from roller bottle supernatant?
A: The yield of monoclonal antibody also depends on the inherent ability of the cell line to synthesize and secrete monoclonal antibody and the health, clonality and environment in which the hybridoma cells are synthesizing and secreting monoclonal antibody. Changes in media formulation may improve the antibody yield. SDIX means antibody yield from roller bottle supernatant is 35 mgs per liter. This mean has a large variance due to inherent differences among cell lines to synthesize and secrete monoclonal antibody and due to the improvements in yield realized by manipulating the media formulation. Some cell lines produce less than 10 mgs of antibody per liter while others produce over 50 mgs per liter.
Q: Which method should I use for monoclonal antibody production, ascites or roller bottles?
A: Three major factors are used to determine the production mode: economics, ethical considerations, and the desire to define the environment in which the hybridoma cell line produces antibody.
Economics: For most cell lines, the cost of antibody produced in ascites is less than the cost of antibody produced using roller bottles. This difference becomes very important when tens or hundreds of grams of monoclonal antibody are produced.
Ethical considerations: Many individuals and many organizations choose to avoid the use of animals when a suitable alternative to the use of animals is available. All of SDIX's animal facilities are AAALAC-accredited. Click here for more information about SDIX's accreditations, registrations and licenses.
Defining the environment in which the hybridoma cell line produce antibody: Ascites fluid is a complex mixture of many mouse proteins and other biomolecules. These molecules are present in the ascites product. The identities and concentrations of all of these proteins and biochemicals is not known. These molecules are usually removed during purification of monoclonal antibody from ascites fluid. To remove the uncertainty of the influence of the these proteins and biomolecules on the outcome of antibody purification, production of monoclonal antibody is roller bottles is available. The environment in which the hybridoma cell line makes antibody (the media formulation) is easily defined, especially when serum-free media formulations are used.
Q: Why use SCID mice for ascites production?
A: SCID is an acronym for Severe Combined ImmunoDeficient mice. These mice lack the ability to synthesize and secrete endogenous antibodies. When SCID mice are used for ascites production, the only antibody present in the ascites fluid is the monoclonal antibody produced by the hybridoma cell line used for ascites production.
Q: What do you need to get started?
A: To produce antibody in ascites fluid or in roller bottles, we require frozen cryovials of the hybridoma cell line and a completed Project Submission Form. This form captures your antibody production experience with the cell line (if any) and information required for successful antibody production (such as the media formulation).
Polyclonal Antibody FAQ
Q: Which species should I use for polyclonal antibody production?
A: Several factors are considered when determining the best species for polyclonal antibody production. If a protein antigen is homologous to a protein found in the species being immunized, a different species should be considered. For example, chickens frequently generate antibodies to conserved mammalian proteins. The amount of antibody required also determines the appropriate host species. Small amounts of sera containing antibody are obtained from mice, rats and guinea pigs. Larger amounts of sera are obtained from rabbits, the most popular species at SDIX for immunization. The largest volumes of sera (or plasma) are obtained from sheep and goats. Also, consider the time required to obtain a fully-matured antibody response. Rabbits, chickens, guinea pigs, rats, and mice typically require 4 immunizations over approximately 70 days to reach optimal immune response. Goats and sheep require more immunizations over 200 days to reach full immune maturity.
Q: What type of antigens can be used for polyclonal antibody production?
A: Nearly any molecule can be injected into animals, but some molecules are much better immunogens than others. Full-length proteins, either expressed recombinantly or purified from natural sources, are the best immunogens. Peptides may be easier and cheaper to produce, but anti-peptide antibodies frequently fail to recognize the full-length protein from which they were derived. Small molecules such as vitamins, antibiotics, drugs, dyes, and metabolites are not immunogenic due to their small size. This class of antigen is known as haptens. Haptens must be conjugated to carrier proteins before they can be recognized by the immune system. Other molecules such as carbohydrates, lipoproteins, and phospholipids are poor immunogens, either generating undesirable IgM responses only or failing to generate any response.
Q: To make large volumes of antisera, should I start additional animals or should I use extended immunization protocols on fewer animals?
A: Consider your timeline and consider the amount of antibody you require in a single lot of antibody product. If large amounts of antibody are required in a short period of time, more animals are started on an immunization protocol. If a single large lot of antibody product is desired, more animals are started on an immunization protocol. A single large lot of antibody minimizes the amount of qualification testing required before use in your laboratory or manufacturing operation. If you are uncertain of the antibody response that will be elicited by your antigen, extended immunization protocols may be preferable. In this approach, several animals might be started on a standard immunization protocol. At the conclusion of the standard protocol, the animals with the best antibody response are continued on extended protocols.
Contact Technical Support
Still have questions? Please call (800) 544-8881 or email antibodysupport@sdix.com