While simple in principle, antibodies are deceivingly complex in structure and production. Do you know the advantages and disadvantages of monoclonal, polyclonal and recombinant antibody production?
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Polyclonal → Monoclonal → Recombinant
Polyclonal antibodies are characterized by a pool of immunoglobulins containing antibodies specific to multiple epitopes on a given antigen. Production of polyclonal antibodies begins by immunizing a host with an antigen of interest and harvesting the resulting antibodies through bleeds. Separating leukocytes and erythrocytes from the whole blood yields antibody containing serum – which can be used directly in assays as antiserum. Antibodies can be further purified from antiserum through isolation of immunoglobulins by protein A or G or antigen specific immunoglobulins through immunoaffinity purification.
Polyclonal antibodies are relatively inexpensive to produce, can be produced quickly, and have high affinity binding. This makes them ideal for laboratory tests requiring precipitation, such as CHIP and IP, along with other experiments including Western Blots and FACS.
Monoclonal antibodies are characterized by a pool of immunoglobulin containing antibodies specific to one single epitope on a given antigen. The mouse is the common source of monoclonal antibodies; mice are immunized with an antigen of interest and the resulting immune cells are then harvested from the spleen and fused to an immortal myeloma cell line to create a hybridoma. The hybridomas are cultured and serve as a consistent, long term source of antibodies with lot-to-lot consistency.
Monoclonal antibodies are characterized by their responsiveness to a single epitope, high specificity, and reproducibility. They are particularly useful for projects requiring large amounts of antibody.
Through the development and use of antibody engineering technology, many types of recombinant antibodies have become available. Fab, Fab’, and F(ab)’2 represent only a small selection of the possibilities, each with their own advantages and disadvantages. Here, for simplicity, we will outline one of many types of recombinant antibody currently available: Single Domain Antibody (aka VHH antibody).
The Single Domain Antibody, or VHH antibody, is a recombinant monoclonal antibody derived from camelids (llama, alpaca, camel, etc.). After immunization, peripheral blood mononuclear cells are collected and RNA is from them. The collected RNA is then used to construct a cDNA phage display library containing sequences of antigen specific binding fragments. The library is screened to select the high affinity binding antibody fragments, these fragments are then sequenced and expressed recombinantly.