The production of antibodies relies on the in vivo humoral response. Simple immunizations of many foreign molecules, viruses or cells can elicit a strong antibody response whilst some substances fail to induce a strong response. The immune system can be manipulated to increase the response by modifying either the antigen or the host. Until recently these modifications were empirically discovered but now increased understanding of the immune system allows a more rational approach.
Proteins, peptides, carbohydrates, nucleic acids, lipids and many other naturally occurring or synthetic compounds can act as successful immunogens. Peptides and non protein antigens usually need to be conjugated to a carrier protein (bovine serum albumin or keyhole limpet hemocyanin) to become good immunogens. This is due to their small size and the carrier is needed to provide the class II T receptor binding sites. Additionally these may need to be administered with an adjuvant to ensure a high quality/quantity response. Adjuvants are non-specific stimulators of the immune response. They allow smaller doses of antigen to be used and the antibody response is more persistent.
Hybridoma cell culture at Abcam
Polyclonal antibodies are made by immunizing with antigen X. Repeated immunizations of the same antigen at intervals of several weeks, stimulates specific B cells to produce large amounts of the anti X antibodies in the blood. Because many different B cells are stimulated by antigen X, the blood will contain a variety of anti X antibodies, each binding X in a slightly different way. The immune-sera can be used in its crude form where high levels of specific antibodies are present, or the specific antibodies can be isolated from sera components by affinity purification.
To produce monoclonals the same immunization protocol is used and all antibody forming cells (e.g. mature B cells/B cells) are removed. These are fused with immortal tumor cells to become hybridomas, which are screened for antibody production. The hybridomas that produce antibodies are given clone names, which are uniquely assigned to permit identification. The antibody producing hybridoma cells are cloned by isolation and cultivated using tissue culture or within mice to produce ascites fluid. Unlike polyclonal antibodies these are homogeneous antibodies with defined specificity. The antibody secreted by the cells into the culture media can be harvested. The tissue culture supernatant can be used in its crude form, or it can be further purified by affinity purification.