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What is the difference between monoclonal and polyclonal antibodies?

Posted on: September 24, 2020, by Jason
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What are monoclonal antibodies?

1. Definition

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.

2. History

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.

3. Monoclonal Antibody Production

  1. Immunize animals
    Immunization of animals is the process of immunizing mice with the target antigen to make the mice produce sensitized B lymphocytes. Generally, female BALB/c mice aged 6-8 weeks are selected and injected according to the pre-established immunization protocol. The antigen enters the peripheral immune organs through the blood circulation or lymphatic circulation, stimulates the corresponding B lymphocyte clones, activates, proliferates, and differentiates into sensitized B lymphocytes.
  2. Cell Fusion
    The mice are killed with carbon dioxide, and the spleen is taken out aseptically. Then squeeze and ground it in a petri dish to make a spleen cell suspension. The prepared syngeneic myeloma cells and mouse spleen cells are mixed in a certain proportion, and add the fusion promoter polyethylene glycol. Under the role of polyethylene glycol, various lymphocytes can fuse with myeloma cells to form hybridoma cells.
  3. Selective cultivation
    The purpose of selective culture is to screen fused hybridoma cells, and HAT selective medium is generally used. In the HAT medium, the unfused myeloma cells die due to lack of hypoxanthine-guanine-phosphoribosyltransferase and unavailable salvage pathway to synthesize DNA.

    Although unfused lymphocytes have hypoxanthine-guanine-phosphoribosyltransferase, they cannot survive for long time in vitro.

    Only the fused hybridoma cells can survive and proliferate in HAT medium because they have obtained hypoxanthine-guanine-phosphoribosyltransferase from spleen cells and the ability of unceasing proliferation from myeloma cells.
  4. Screening and cloning of hybridoma positive clones
    Only a few of the hybridoma cells grown in HAT medium are cells that secrete predetermined specific monoclonal antibodies. Therefore, screening and cloning are necessary. The limiting dilution method is usually used for clonal culture of hybridoma cells. Using sensitive, rapid, and specific immunological methods, the positive hybridoma cells that can produce the required monoclonal antibodies are screened out and cloned and expanded. After a comprehensive identification of the immunoglobulin type, subclass, specificity, affinity, epitope of the recognized antigen and its molecular weight of the monoclonal antibody secreted by it, it should be frozen in time.
  5. Mass production of monoclonal antibodies
    The large-scale preparation of monoclonal antibodies mainly adopts animal in vivo induction and in vitro culture methods.

4. Cloning Method

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.

monoclonal-and-polyclonal-antibodies
monoclonal-and-polyclonal-antibodies

5. Application

Since the advent of monoclonal antibodies, they have been applied in many fields of medicine due to their unique characteristics.

  1. Laboratory medicine diagnostic reagents
    As a diagnostic reagent in laboratory medicine laboratories, monoclonal antibodies are widely used in technologies such as enzyme-linked immunosorbent assay, radioimmunoassay, immunohistochemistry and flow cytometry due to their strong specificity, high purity, and good uniformity. And the application of monoclonal antibodies has greatly promoted the development of commercial kits.

    Commercial kits made of monoclonal antibodies are widely used in:
    1. Detection of pathogenic microorganism antigens and antibodies;
    2. Detection of tumor antigens;
    3. Detection of immune cells and their subgroups;
    4. Hormone determination;
    5. Determination of cytokines.

      The recognition of antigens by monoclonal antibodies is very different from that of polyclonal antibodies. Different kits use different monoclonal antibodies and have different antigen recognition sites, leading to certain differences in test results. Therefore, the standardization issue needs further study.
  2. Purification of protein
    Monoclonal antibodies are important ligands in affinity chromatography. The monoclonal antibody is adsorbed on an inert solid phase matrix (such as Spehrose 2B, 4B, 6B, etc.) and prepared into a chromatography column. When the sample flows through the chromatographic column, the antigen to be separated can specifically bind to the solid phase monoclonal antibody, and the remaining components cannot bind to it. After the chromatographic column is fully eluted, the ionic strength or pH of the eluate is changed, the antigen to be separated is dissociated from the antibody, and the antigen to be purified can be obtained by collecting the eluate.
  3. Tumor-oriented therapy and radioimmunoimaging technology
    The monoclonal antibody directed against a certain tumor antigen is connected with a chemotherapy drug or radiotherapy substance, and the targeting effect of the monoclonal antibody is used to carry the drug or radiotherapy substance to the target organ and directly kill the target cell, which is called tumor-directed therapy.

    In addition, radioimmunoimaging can be performed by connecting radioactive markers with monoclonal antibodies and injecting them into patients to assist in tumor diagnosis. Monoclonal antibodies are mainly murine antibodies, and the serum of heterogeneous animals can cause allergic reactions in humans. Therefore, the preparation of human-human monoclonal antibodies or humanized antibodies is more important, but no significant progress has been made in this regard.

What is polyclonal antibody? 

1. What does polyclonal antibody mean?

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.

2. Structure

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.

3. Classification

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.

4. Application

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.

Monoclonal vs Polyclonal antibodies

1. Concept

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.

2. Production

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.

3. Cons and Pros

A. Monoclonal antibody

Advantages:

  • Highly specific recognition of an epitope of the antigen
  • Immortal hybridoma cell lines have the ability to produce unlimited amounts of antibodies
  • Immortal hybridoma cell lines have the ability to produce unlimited amounts of antibodies
  • Minimal background noise and cross-reaction
  • Excellent affinity and purification

Excellent affinity and purification:

  • Development of monoclonal antibodies takes time and requires high technical skills.
  • They can produce numerous specific antibodies, but may not be detected in multiple species.
  • Susceptible to changes in epitopes. Even minor changes in conformation may result in greatly reduced binding capacity.

B. Polyclonal antibody

Advantages:

  • The price is cheap and the production is relatively fast.
  • Due to the recognition of multiple epitopes, the overall antibody affinity to the antigen is higher.
  • High sensitivity for detecting low amounts of protein.
  • Strong ability to capture target protein (recommended as capture antibody in sandwich ELISA).
  • Antibody affinity can lead to faster binding to the target antigen (recommended for analysis that requires rapid capture of proteins; such as IP or ChIP).
  • Advanced reagents for detecting natural proteins.
  • Easy to couple with antibody markers, it is unlikely to affect the binding ability.

Disadvantages:

  • Differences between batches of different animals at different times.
  • Since it recognizes multiple epitopes (affiliative purified antibodies show minimal cross-reactivity), the possibility of cross-reactivity is high.

4. Other differences

 CharacteristicsMonoclonal AntibodyPolyclonal Antibody
1SpecificityStrongWeak
2StabilityRelatively poor, sensitive to physical and chemical conditionsGood
3Requirements for immunogensImpure immunogens can also produce highly purified antibodiesHigh immunogen purity is required
4StandardizationEasy to standardize,
little difference between batches
Difficult,
big differencebetween batches
5RecognitionOnly one epitope on the antigen is detectedCan recognize multiple epitopes
6Cross Reactionnot easy to cross react with other proteinscommon and difficult to avoid nonspecific reactions
7Precipitation and agglutinationMost don'tCommon
8Production CostHighLow
9Preparation PeriodLongShort
10PriceHighLow

Conclusion

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.

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