Plant polyclonal antibody production is an important biotechnology that produces antibodies that are highly specific to specific plant antigens by immunizing animals, usually mice or rabbits. The general process of plant polyclonal antibody production and its key steps are described below.
1.Antigen preparation.
Select antigen: Determine the plant antigen that needs to be used to prepare antibodies, which can be plant tissue extracts, purified proteins, organelles, or expressed recombinant proteins.
Extraction and purification: Antigen of interest is extracted from plant samples using appropriate methods and purified to ensure the purity and activity of the antigen.
2.Immunization animal selection and immunization.
Animal selection: Appropriate immunized animals are selected, commonly including mice and rabbits. Factors such as the size of the animal, the characteristics of the antigen, and the needs are considered.
Immunization program: After the antigen is mixed with an immune adjuvant, it is injected into the immunized animal, and the immune response is observed and recorded.
3.Serum collection and antibody testing.
Serum collection: At the end of the immunization cycle, serum samples are collected from immunized animals to obtain antibodies from them.
Antibody detection: Detect antibody levels and specificity in serum using appropriate methods (e.g., ELISA, Western blotting, etc.) to screen for highly effective positive samples.
4.Cell fusion and hybridoma preparation.
Cell fusion: B cells from immune mice or rabbits are fused with tumor cells, such as myeloma cells, to produce hybridoma cells.
Hybridoma screening: In a Petri dish containing the appropriate medium, hybridoma cells are screened and cultured to screen for hybridomas that produce antibodies of interest.
5.Monoclonal antibody screening and amplification.
Dilution limiting: Hybridoma cells are progressively diluted to the single-cell level so that each cell grows in a Petri dish.
Screening and amplification: Monoclonal hybridomas that produce specific antibodies are screened out by ELISA and other methods, and these monoclonal cells are expanded.
6.Antibody purification and identification.
Purification: Purify antibodies using appropriate methods (e.g., protein ag affinity chromatography, ion exchange chromatography, etc.) to remove impurities and improve purity.
Identification: Characterize the function and properties of purified antibodies, including evaluation of parameters such as specificity, affinity, and potency.
7.Antibody application and preservation.
Application: The prepared plant polyclonal antibody is used in specific experiments or research projects, such as western blotting, immunohistochemistry, immunoprecipitation, etc.
Preservation: Preserve the remaining antibodies in an appropriate manner (e.g., cryopreservation) for future use.
Through the above process, polyclonal antibodies with high specificity and affinity for specific plant antigens can be prepared, providing important experimental tools and support for plant biology research and applications.