Antibodies are produced by the adaptive immune system in response to invading pathogens. The antibody has specific lock and key recognition for the offending bacteria, virus, or other molecule, which are collectively called antigens. Antibodies are proteins, which are folded polypeptides, or strands of amino acids which have antigen recognition sites that specifically recognize a binding site of its specific antigen. They are produced by B-cells of the adaptive immune system.
The specificity of the antibody-antigen recognition has many uses in biotechnology and sensor development. These applications demand highly pure sources of antibodies. These antibodies are usually conjugated to sensor surfaces in order to detect antigens, or used in combination with nanoparticles or other labeled detection platforms. Purified antibodies with specificity to a particular antigen are commercially available. These antibodies fall into one of two categories depending on how they are generated and harvested: polyclonal or monoclonal. Polyclonal antibodies are capable of recognizing multiple epitopes, or different sites on an antigen. They are derived from different B cell lineages. Monoclonal antibodies recognize only one epitope on an antigen, and are derived from a single B-cell line.
When an antibody is needed against a specific antigen, but it isn’t readily available from a supplier, it is possible to have antibodies made via custom antibody production. A 90% purified sample of antigen is all that is needed to have a custom antibody generated and harvested against that antigen. If a sample of antigen is not available, it is also possible to have custom peptides or proteins synthesized and antibodies created against them. Both polyclonal and monoclonal antibodies can be produced by custom antibody production protocols.
Custom polyclonal antibody production
There are six steps to polyclonal antibody production:
- peptide design
- peptide synthesis
- animal immunization
- serum collection
- titer analysis
- final antibody purification
The first two steps are not necessary if a 90% pure antigen sample is provided by the customer.
Custom polyclonal antibodies can be produced from rabbit, guinea pig, chicken, goat, rat, and mouse, with an average production time of 80 days. Rabbits are used 95% of the time because they respond to most antigens. They have high yield and immune systems that rapidly produce antibodies. It typically takes 70, 90, or 120 days depending on the protocol used. Rat and mouse are only useful for small amounts of antiserum. Goats have the highest yield, but respond slower to antigen (minimum of 120 days). Chicken is really only used if rabbits didn’t react or generate antibodies. Guinea pig antibodies have low background in drosophila research and can be a useful secondary.
Custom monoclonal antibody production
Custom monoclonal antibody production takes 6 months from start to finish. There are 5 steps:
- antigen preparation
- hybridoma development immunizations
- hybridoma development fusions
- subcloning
- monoclonal production and purification.
Antigen preparation: customer can provide highly pure protein or peptide. The company could also design and synthesize a custom peptide or produce a protein.
Immunization: animals are immunized and their serum is tested for antibody production
Fusion: The animals with the best immune response to the antigen are further boosted with the antigen before their spleens are harvested for hybridoma fusion. A hybridoma is a cell line of immortal B cells and myeloma cancer cells. These cells have increased longevity and reproductivity. Cell lines are tested and preserved.
Subcloning: The cell-line is cloned until it is homogeneous and consistently secreting antibody.
Production and purification: 1 liter of cell culture produces 10 to 30 mg of antibody. Purification with protein A or protein G resin.
Custom antibody production is an extremely useful tool when it is necessary to develop an antibody against a rare or unique antigen. The process, especially for monoclonal antibody production is also helpful to others in the scientific realm because once a hybridoma cell line is created to produce an antibody it is possible to preserve that cell line for future use.
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