Monoclonal And Polyclonal Antibody Production

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1. Monoclonal And Polyclonal Antibody Production

2. Antibodies are globular proteins or Immunoglobulins synthesized by a type of white blood cell called plasma cells against the antigen that stimulated its production. The region of antibody that interact with antigen is called Paratope. Antigen antibody interaction is specific. A NTI B O D Y

3. MONOCLONAL ANTIBODY A monoclonal antibody refers to an antibody normally recognizing only a single antigen (e.g. a single protein) and within which only a single common epitope is recognized.

4. Figure: Polyclonal and Monoclonal Antibodies

5. DISCOVERY OF MONOCLONAL ANTIBODIES The idea of ‘magic bullet’ was first proposed by Paul Ehrlich who at beginning of 20 th century postulated that “if a compound could be made that selective targeted a disease causing organism than a toxin for that organism could be delivered along with agent of selectivity”.

6. Figure : Monoclonal antibodies (mAbs) production

7. Step – 1: Immunization of the mice Mice are immunized every 2-3 weeks with an antigen that is prepared for injection

8. Step 2: Screening of Mice for Antibody Production Blood samples are obtained from mice for measurement of serum antibodies whose titer is determined with various techniques, such as enzyme-linked immuno sorbent assay (ELISA) and flow cytometry.

9. Step – 3 : Isolation of antibody producing spleen cells When the antibody titer is high enough, mice are commonly boosted by injecting antigen without adjuvant intra peritoneally or intravenously (via the tail veins) 3 days before fusion but 2 weeks after the previous immunization. If the titer is too low, mice can be boosted until an adequate response is achieved, as determined by repeated blood sampling. Then the mice are euthanized and their spleens removed for in vitro hybridoma cell production.

10. Step 4 : Production of hybridomas A week before cell fusion, myeloma cells are grown in 8-azaguanine (analog of guanine, acts as competitive inhibitor). Myeloma cells lack HPGRT (hypoxanthine phospho ribosyl transferase) enzyme, which is responsible for synthesis of nucleotides. The cells are then screened in HAT (hypoxanthine-aminopterin- thymidine) medium which blocks the pathway for nucleotide synthesis.

11. Step 5 : Screening of hybridomas

12. Step 6 : Culturing hybridoma cells Hybridomas are separated and individually cultured : 1 cell per well These cells are called as clonal culture . Because each cell in the well is derived from singe cell and are therefore identical. After few weeks, when growing cultures can be seen, further screening can be done for desired antibody.

13. Step 7 : Screening for desired antibodies Antigens are immobilized in the wells and the antibodies are transferred (one per well) so that they bind to the complementary antigen. Different antibodies react to different epitopes on the same antigen.

14. Step 8 : Selection and culture of screened antibodies Finally, the desired antibodies are grown in mass culture and are frozen for storage.

15. A polyclonal antibody refers to an antibody normally recognizing only a single antigen but within which a number of different epitopes are recognized . POLYCLONAL ANTIBODY

16. Antibodies used for research and diagnostic purposes are often obtained by injecting a lab animal such as a rabbit with a specific antigen . Within a few weeks, the animal’s immune system will produce high levels of antibodies specific for the antigen . These antibodies can be harvested in an antiserum , which is whole serum collected from an animal following exposure to an antigen. PRODUCING POLYCLONAL ANTIBODIES

17. Because most antigens are complex structures with multiple epitopes, they result in the production of multiple antibodies in the lab animal. This so-called polyclonal antibody response is also typical of the response to infection by the human immune system. Antiserum drawn from an animal will thus contain antibodies from multiple clones of B cells , with each B cell responding to a specific epitope on the antigen. PRODUCING POLYCLONAL ANTIBODIES

18. Antiserum obtained from animals will not only contain antibodies against the antigen artificially introduced in the laboratory, but it will also contain antibodies to any other antigens to which the animal has been exposed during its lifetime. For this reason, antisera must first be “purified ” to remove other antibodies before using the antibodies for research or diagnostic assays. PRODUCING POLYCLONAL ANTIBODIES

19. Fi Figure: The process for harvesting polyclonal antibodies produced in response to an antigen.

20. P OLYCLONAL VS . MONOCLONAL ANTIBODIES . Monoclonal antibodies Polyclonal antibodies Expensive production Inexpensive production Long production time Rapid production More Specificity less Specificity Recognize a single epitope on an antigen Recognize multiple epitopes on an antigen Production is continuous and uniform once the hybridoma is made Different batches vary in composition Few host species options Many host species options A homogenous antibody population. A heterogeneous antibody population.

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