The immune system produces antibodies as a specific response to an immunogen challenge—an animal immune system acts via two principal mechanisms of humoral type responses and cell-mediated responses. Antigens or immunogens include the molecules inducing specific immune responses.
Consideration should be provided at the outset for procuring the commercially produced antibodies in antibody production service from the compliant sources with CCAC guidelines. Suppliers of mAbs produced in vitro have been listed by the Focus on Alternative Group, UK. This article discusses the rules and regulations governing antibody production.
Each attempt needs to be made to obtain an already available material or use an in vitro production method. Any production of monoclonal antibodies through the ascites method needs justification by the investigator to the animal care committee.
The commercially produced monoclonal antibody production must be sourced from the sources in compliance with CCAC guidelines. Another provision is the fact that they need accreditation from AAALAC.
The monoclonal antibody production in mice through the ascites method creates a lot of issues of concern in regards to the potential for unnecessary and severe pain for animals. Recognizing that problem, several in vitro replacements for the method of rodent ascites in the antibody production process have been developed. Many countries have banned routine in vivo production. It is noteworthy that CCAC Ethics of Animal Investigation bears a rule of following the “Three Rs” of Russell & Burch (1959): Replacement, Reduction, and Refinement.
Ideally, a replacement in vitro system needs to be less expensive, produce high monoclonal antibody production concentrations, not need culture conditions, and need a reasonable short period for producing pure and adequate quantities of monoclonal antibody production.
Clearly Defined Close Monitoring and Endpoints of the animal’s condition are needed for minimizing the potential for distress and pain.
According to CCAC guidelines on choosing appropriate endpoints in experiments through animals for teaching, research, and testing, there should be a development of clear endpoints to minimize the distress and pain of animals. There should also be a clear schedule for the reportage and monitoring. SOPs should also be established for every new adjuvant or immunogen combination.
In selecting strain and species of animals for mAb production, the primary consideration needs to minimize distress or pain.
They used rats or mice to be of similar strain for immunization that produces hybridoma clones and for the production of mAb so that the used tissue is histocompatible. The animal of choice is ordinarily BALB/c mice because many of the used parental myeloma cells in the fusion process are from BALB/c mice. Retired breeders may provide advantages for the production of ascites because of the previously stretched abdominal musculature. You should note that SCID mice need barrier facilities for extensive procedures and appropriate housing for care.
Immunization SOPS needs to be developed based on the guidelines for adjuvant choice, injection route, volume, and several injection sites. The procedure of immunization does not require an adjuvant. It can be performed in short periods. The procedures seem to lead to less stress for mice and need many boosts in a short time frame, eliminating the adjuvant requirement.
The regulation for priming is that Freund’s Incomplete Adjuvant or the less invasive adjuvant needs to be used as the agent for intraperitoneal priming bearing a 0.3ml maximum volume administered in a single injection. There needs to be just the institutional animal care committee in the proposal for Pristane use. The volume needs to be of a limit of 0.2ml.
Hybridomas need testing for the presence of adventitious virus and mycoplasma agents before using it in vivo monoclonal antibodies’ propagation. The viral contaminations are common in inoculated murine specimens with a marine-derived hybridoma. This contamination type typically leads to infection of a host animal and the spreading of diseases throughout the facility of an animal. Additionally, the virus called lymphocytic choriomeningitis, leading to diseases in humans, has been isolated from the lines of murine tumor cells. Further, the mouse hepatitis virus also can influence the subsequent production of mAbs.
Up to 3 * 10^6 hybridoma cells with a maximum volume of 0.1 ml could be injected into the primed mouse peritoneal cavity. The tumor cell lines that are prone to solid tumors create less ascitic fluid, which can be optimized for the production of ascites through the performance of serial passages of tumor cells that are non-attached. Selecting hybridoma cell lines not adhering to the tissue culture flask has been proven to cause a reduction in the likelihood of forming solid tumors. Most 3*10^6 cells have been observed to cause the top necessity for providing good mAbs development and ascitic fluid.
Regulation of antibody production is usually a complex process involving the activities of a variety of cytokines. Several significant differences exist between regulating murine B and human lymphocytes. That is especially with regards to IL-2 action. IL-2 in humans plays a critical role in regulating the activation of cell B.
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