The immune response to an antigen generally involves the activation of multiple B-cells all of which target a specific epitope on that antigen. As a result a large number of antibodies are produced with different specificities and epitope affinities these are known as polyclonal antibodies.
For production purposes these antibodies are generally purified from the serum of immunised animals were the antigen of interest stimulates the B-lymphocytes to produce a diverse range of immunoglobulin's specific to that antigen.
The aim is to produce high titre, high affinity antibodies. Today these polyclonal antibodies are used extensively for research purposes in many areas of biology, such as immunoprecipitation, histochemistry, enzyme linked immunosorbent assays (ELISA), diagnosis of disease, immunoturbidimetric methods, western blots and Biochip technology. Polyclonal antibodies are ideally suited for use in sandwich assays as second stage antigen detectors.
Often polyclonal antibodies will be tagged with reporter molecules such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) so that under specific conditions the antibodies presence can be detected by light or colour changes.
Monoclonal antibodies represent a single B lymphocyte generating antibodies to one specific epitope. B-cells can be isolated easily from the spleen and lymph nodes of immunised animals; however, these cells have a limited life span, and can only divide a limited number of times, coined the 'Hayflick limit'. This prohibits the culture of B-cells directly. For an antibody to be useful in research or industry, it must be readily available in large quantities. Due to the Hayflick limit, this would not be possible using B-cells cultured in vitro as they would eventually stop dividing and the population would die out.
Consequently, in 1975 Kohler and Milstein developed a technology to fuse immortal heteromyleoma cells with lymphocytes, using poly ethylglycol (PEG) to break down cell membranes and allow mixing of the genetic material from both cell types. The resulting cell type is called a hybridoma. This hybridoma takes on the characteristics of both the lymphocyte and heteromyeloma cell, creating an immortal cell with the ability to produce antibody. As the new cell line hybridoma is a product of the fusion of one heteromyeloma cell with one B-cell, the culture only ever has one antibody within the supernatant which, when purified, is called a Monoclonal antibody. This technology allows scientists to extract and purify one antibody from the complex mixture of antibodies present in the in vivopolyclonal response. This cell line, once stabilised via single cell cloning, can be frozen and stored indefinitely under liquid nitrogen, allowing the antibody to be produced in vitro, in large quantities when required.
Monoclonal antibodies can be raised against many targets. Specific antibody characteristics can be identified and selected e.g. sensitivity requirements and cross reactivity levels can be specified and monoclonal antibodies screened to identify any cell lines exhibiting the required characteristics.
Monoclonals can also be generated to cross react with a group of molecules, for example the tricyclic anti-depressants have a similar overall structure with substitutions of differing atoms into the cyclic structure. This is very useful in drug detection when many possible combinations of the drug are to be tested in a patient.
Should I use Monoclonal Antibodies or Polyclonal Antibodies?
Both Polyclonal and Monoclonal antibodies each have their own advantages which make them useful for different applications.
Large quantities of polyclonal antibody are relatively quick and inexpensive to produce compared to monoclonal antibodies. They are non specific in that they are capable of recognising multiple epitopes on any one antigen. This capability provides many advantages:
• Can help to increase the signal produced by the target protein as the antibody will bind to more than one epitope
• Less sensitive to antigen changes than monoclonal antibodies
• Useful when the nature of the antigen is unknown
• More robust detection due to multiple epitopes
• Hybridomas provide an immortal cell line with the ability to produce unlimited quantities of highly specific antibodies
• All batches will be identical and specific to just one epitope a particular advantage when manufacturing procedures must be standardised e.g. clinical tests and therapeutic treatments
• The high specificity of monoclonal antibodies decreases background noise and cross-reactivity, helps provide reproducible results and ensures efficiency in affinity purification