Monoclonal and Polyclonal Antibodies
Monoclonal and Polyclonal Primary Antibodies
Antibodies, also known as immunoglobulins, are proteins produced by B cells in response to the presence of an antigen. Antigens are recognised as anything that triggers an immune response and the subsequent production of antibodies. Antigens can be displayed on the surface of bacteria, viruses and fungi as well as being small molecules such as toxins and foreign particles. Antibodies bind to the antigen helping to prevent harm. They also act as markers, alerting the body to the presence of an antigen and causing the correct antibodies to be produced in response. Antibodies belong to a large Ig superfamily each made up of basic structural units, consisting of 2 heavy chains and 2 light chains. Antibodies are grouped according to their heavy chain, with 5 different classes currently recognised IgA, IgD, IgE IgG and IgM. These classes have different roles within the immune response.
When an immune response is triggered by an antigen a large number of antibodies are produced. The antibodies produced can have a range of different epitope affinities and specificities, binding to different regions of the antigen and with differing binding strengths. The antibodies produced from this response are called
polyclonal. Polyclonal antibodies therefore represent the total number of Ig produced by an animal in response to an antigen.
Antibodies which recognise a specific region on the antigen are generated from a single line of B cells and are called
monoclonal. These antibodies are clones of one another and do not vary in their specificity.
Due to antibodies having high specificity and selectivity they can be utilised in a number of ways including research, diagnosis and therapy. Both polyclonal and monoclonal antibodies have valuable characteristics that can be applied to different areas in scientific research. The advantages of their use is discussed below.
The major benefit for the production of polyclonal antibodies is the ease at which they can be made. Large quantities of polyclonal antibodies can be made quickly and inexpensively when an antigen is injected into an animal initiating an immune response. These antibodies may then be collected from the animal's serum.
Other benefits of polyclonal antibodies include:
- Their ability to recognise multiple epitopes on any one antigen
- Broader recognition means they can be used for a large number of applications, advantageous when the nature of the antigen is unknown
- Less sensitive to antigen changes, for example, antigen changes across species
Monoclonal antibodies on the other hand require a more labour intensive and time consuming production process and are therefore more expensive to produce. These antibodies bind to a specific epitope on the target antigen and represent only one line of antibodies produced by a specific B lymphocyte following an immune response. B cells have a limited life span so cannot be isolated for the continual production of monoclonal antibodies, instead these lymphocytes need to be fused with immortal cells. The resulting cell type is called a hybridoma, taking on the antibody producing characteristics of the B lymphocyte and the immortality of the immortal cell. This secures the production of the monoclonal antibody for as long as the antibody is required. The antibody that this cell produces will be identical and highly specific. Furthermore, after a specific hybridoma cell clone is selected, it can be frozen and stored, and thawed and allowed to grow in vitro when more antibody is required.
Other benefits of Monoclonal antibodies include:
- Continuous source of consistently identical antibodies
- Ability to produce highly specific and identical antibodies beneficial when used within standardised procedures, clinical tests, therapeutic treatments
- Reduction in background noise when used as a detection method, reduced cross reactivity, helps provide reproducible results when used within experimental research
- Monoclonals can also be used to detect groups of molecules which have a similar structure but only vary with substitutions of differing atoms. This makes them particularly useful during drug development and detection when many combinations of the drug are to be tested in a patient.
Considerations when developing monoclonal antibodies is the high technology required along side greater expertise and training to produce them.
Monoclonal or Polyclonal
When deciding upon which antibodies to use it is crucial to consider their properties. Because antibodies recognise a relatively small region of an antigen they can cross react with similar regions on other antigens but this tends to be with less affinity. The specificity of an antibody refers to its ability to recognise a certain epitope when compared to other epitopes. A highly specific antibody will show less cross reactivity. Furthermore the binding affinity of an antibody is also influenced by the conformational shape of the antibody and the antigen.
The shape of the antibody or antigen can be manipulated by factors such as association with other proteins, post translational modifications, temperature, pH and salt concentration. Polyclonal antibodies are particularly useful as a detection method providing a more robust detection signal. They can help amplify the signal through their ability to bind to more than one epitope which means that more than one detection antibody may bind to the target protein. Conformational change to an antigen or antibody will have less effect in respect to polyclonal antibodies but can have significant effect on the binding ability of a monoclonal antibody.