There is a big difference between monoclonal and polyclonal antibodies. Monoclonal antibodies are highly uniform antibodies that are produced by a single B cell clone and target only a specific epitope. Hybridoma antibody technology is based on cell fusion technology, which combines sensitized B cells and myeloma cells with immortal reproduction capacity into B cell hybrids tumor.
A single hybridoma cell with this characteristic is used to cultivate a cell population to prepare a specific antibody against an epitope, that is, a monoclonal antibody.
The development of monoclonal antibodies has gone through four stages, namely:
Murine monoclonal antibodies, chimeric antibody, humanized monoclonal antibody and fully human monoclonal antibodies.
Fully human monoclonal antibodies: The variable region and constant region of the antibody are of human origin, eliminating immunogenicity and toxic side effects.
The related technologies for the preparation of fully human antibodies mainly include:
Human hybridoma technology, EBV transformation B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology and single B cell antibody preparation technology.
Humanized and fully humanized antibodies drugs prepared from humanized and fully humanized antibodies overcome various shortcomings of animal-derived antibodies and chimeric antibodies because of their high affinity, high specificity, and low toxic and side effects. It has become an inevitable trend in the development of therapeutic antibody drugs.
The positive wells that have been tested for antibodies can be cultured and cloned to obtain monoclonal antibodies secreted by the offspring of a single cell. Generally speaking, the earlier the cloning time, the better. Because during this period, various hybridoma cells grow vigorously at the same time, competing for nutrition and space, and the cells producing the specified antibodies may be submerged and eliminated.
However, the cloning time should not be too early, otherwise the cell traits are unstable, and the number of cells is small and easy to lose. The cloned positive hybridoma cells, after a period of culture, will also lose the ability to produce antibodies due to cell mutations or loss of specific chromosomes, so they need to be cloned again and again. The number of cloning is determined by the secretion ability and the immunity of the antigens. Generally speaking, the number of clones of a strong immune antigen can be less, but at least 3 to 5 clones can make it stable. There are many methods of cloning, including limiting dilution method, micromanipulation method, soft agar plate method and fluorescence activation separation method.
Since the advent of monoclonal antibodies, they have been applied in many fields of medicine due to their unique characteristics.
Antigens are usually composed of multiple antigenic determinants. One type of antigenic determinant stimulates the body, and the antibody produced by a B lymphocyte receiving the antigen stimulation is called Monoclonal Antibody. The body is stimulated by multiple antigenic determinants, and various monoclonal antibodies are produced accordingly. These monoclonal antibodies are mixed together to form polyclonal antibody.
The molecular structure on the antigen that can cause the body to produce antibodies is called epitope. There can be several different antigenic determinants on an antigen, so that the body produces several different antibodies, and the final antibodies are plasma cells. A plasma cell population that only acts on one antigenic determinant is a clone. The specific antibody produced by a clone is called a monoclonal antibody. Monoclonal antibodies can specifically bind to a single specific antigenic determinant, just as a missile accurately hits the target. On the other hand, even if it is the same antigenic determinant, several clones can produce antibodies in the body, forming a mixture of several monoclonal antibodies, called polyclonal antibodies.
Antigens are usually composed of multiple epitopes. One type of epitope stimulates the body. The antibody produced by a B lymphocyte receiving the antigen is called a monoclonal antibody. The body is stimulated by a variety of antigenic determinants, and a variety of monoclonal antibodies are produced accordingly. These monoclonal antibodies are mixed together to form polyclonal antibodies. Except for diverse antigenic determinants, the same type of epitope can also stimulate the body to produce five types of antibodies: IgG, IgM, IgA, IgE and IgD.
Polyclonal antibodies are a group of immunoglobulins secreted by the body's plasma cells that stimulated by heterologous antigens (macromolecular antigens, hapten conjugates). Polyclonal antibodies are widely used in research and diagnosis due to their ability to recognize multiple epitopes and cause precipitation reactions with short preparation time and low cost.
A good polyclonal antiserum contains multiple antibodies against different epitopes of a certain antigen. Since polyclonal antiserum usually contains antibodies against different epitopes of a certain antigen, including denatured-resistant epitopes, it will also have an effect in deep-fixed samples. In the staining of paraffin-embedded tissue sections, Polyclonal antibodies are often used. According to the different needs of the experiment, polyclonal antibodies are used to label the corresponding antigen.
In addition, in agricultural production, polyclonal antibody is used for on-site monitoring of pesticide residues; in clinical applications, polyclonal antibodies are mainly used for pathogen detection, disease diagnosis and treatment, such as protein immunosuppressants for transplantation response and treatment of autoimmune diseases.
Cloning: refers to asexually reproduced cell line, which is a cluster of cell lines formed by the division and reproduction of a single progenitor cell. In all members of this family, if no mutation occurs, the genes are exactly the same.
Polyclonal antibody (pAb): Immunize animals with an antigen containing multiple epitopes, which can stimulate multiple B cell clones to produce different antibodies against multiple epitopes. The obtained immune serum is actually a mixture of multiple antibodies.
Monoclonal antibodies (mAb): Homologous antibodies produced by a clone of B cells that recognize an epitope. Highly uniform, strong specificity, high titer, little or no cross-reactivity.
Differences from monoclonal antibody preparation:
B lymphocytes treated with specific antigens and myeloma cells are fused to obtain hybridoma cells. After screening by HAT medium and detecting the titer by ELISA, a positive clone is obtained, and then do a cell culture or inject the cells into the abdominal cavity of animals (usually balb/c mice) and culture them with ascites, collect the supernatant/ascites and purify them to obtain monoclonal antibodies.
The preparation of polyclonal antibody is not as cumbersome as monoclonal antibodies. You only need to inject the antigen (the higher the purity, the better) directly into the animal body for immunization. After 3 to 4 immunizations, the titer is qualified by ELISA, and the blood is centrifuged. The supernatant can be purified to obtain polyclonal antibodies. Therefore, the preparation cycle of polyclonal antibodies is shorter than that of monoclonal antibodies, and the initial preparation price is lower than that of monoclonal antibodies.
A. Monoclonal antibody
Excellent affinity and purification:
B. Polyclonal antibody
|Characteristics||Monoclonal Antibody||Polyclonal Antibody|
|2||Stability||Relatively poor, sensitive to physical and chemical conditions||Good|
|3||Requirements for immunogens||Impure immunogens can also produce highly purified antibodies||High immunogen purity is required|
|4||Standardization||Easy to standardize, |
little difference between batches
big differencebetween batches
|5||Recognition||Only one epitope on the antigen is detected||Can recognize multiple epitopes|
|6||Cross Reaction||not easy to cross react with other proteins||common and difficult to avoid nonspecific reactions|
|7||Precipitation and agglutination||Most don't||Common|
The gap between monoclonal and polyclonal antibodies is enormous, and these differences make them useful in different fields. Monoclonal antibodies occupy a vital place in medicine, while polyclonal antibodies shine in the fields of agricultural production and disease detection and treatment.