Hybridoma Technology--Cell Fusion & Production of Antibodies

ZOOLOGY Topic : HYBRIDOMA TECHNOLOGY--CELL FUSION & PRODUCTION OF ANTIBODIES ------

OBJECTIVES: To familiarise the term ‘Monoclonal antibodies’ and Understand the technique involved in”Hybridoma Technology”

INTRODUCTION:

Monoclonal antibodies(mAbs) are a series of identical antibodies produced by one type of immune cell. They are highly specific and recognize unique epitopes, or binding sites, on a single antigen. Monoclonal antibodies possess two important characteristics,namely high specificity and high reproducibility. Because of these unique properties, they are widely used in several applications. They play a key role in immunotherapy(1) because each type of monoclonal antibody will target a specific targeted antigen in the body .The discovery and development of hybridoma technology created the foundation for monoclonal antibodies.

ANTIBODIES :

Antibodies are specific immunoglobulin molecules produced by the body in response to an antigenic stimulus as an adaptive immune response to fight against infection.They are present in the blood and tissue fluids .The only cells that make antibodies are B lymphocytes.

TYPES OF ANTIBODIES:

POLYCLONAL ANTIBODIES: Antibodies that are derived from different B lymphocyte cell lines. Arise from multiple cell clones.They are used in diagnostic assays particularly to detect antigens.

MONOCLONAL ANTIBODIES :

Monoclonal antibodies are a series of identical antibodies produced by a single clone of B cell. In other words, monoclonal antibodies (mAbs) are a mixture of homogeneous molecules with high affinity towards a specific antigen.Due to their high specificity and high reproducibility, they are widely used in diagnostic, clinical and therapeutic applications. With more than 20 molecules in clinical use, monoclonal antibodies have emerged as important therapeutic molecules today. mAbs are used in the treatment of tumours, cancer, transplantation of organs, autoimmune diseases and cardiovascular diseases.

HYBRIDOMA TECHNOLOGY:

In 1975, monoclonal antibodies(mAbs) were first generated by Köhler and Milstein.They were awarded a share of the Nobel Prize in the year, 1984 in physiology and medicine. The method for production of monoclonal antibodies is called “Hybridoma technology”. This is a well-established method to produce monoclonal antibodies (mAbs) specific to antigens of interest. The production of antibodies by ‘Hybridoma technology’ was a major breakthrough in biotechnology. Hybridoma technology is a technique for the production of a large number of identical antibodies. Hybridomas are cells produced by the fusion of a short lived antibody producing B lymphocytes with an immortal myeloma cell. Each hybridoma multiply indefinitely and expresses only one type of specific monoclonal antibody. Hybridoma Technology Involved in Hybridoma Technology Steps Involved in Hybridoma Technology

STEPS INVOLVED IN HYBRIDOMA TECHNOLOGYved in H

A)CELL FUSION TECHNIQUE:

1.The hybridoma process starts by injecting a mouse with a desired antigen. As part of immune response, the B lymphocytes are produced in the mouse.The B cells secrete antibodies that are specific to the antigen.

2.Then B cells(splenocytes2) are isolated from the mouse spleen.

3.These splenocytes are fused with myeloma cells in the presence of Polyethylene glycol(PEG)which causes cell membranes to fuse ultimately producing a hybrid cell called a hybridoma, which has both the antibody-producing ability of the B-cell and the indefinite growth potential of the myeloma cell.

B)HYBRIDOMA SCREENING AND GENERATION OF ANTIBODIES:

4. The hybridomas can be grown and expanded in culture . Fused cells or hybridomas are selected by incubating in HAT medium (hypoxanthine-aminopterin thymidine) for roughly 10 to 14 days.Myeloma cells do not synthesize nucleotides by de-novo synthesis as aminopterin present in HAT medium blocks de-novo synthesis. Myeloma cells have a defective HGPRT enzyme(hypoxanthine-guanine phosphoribosyl transferase), blocking their ability to use the salvage pathway(3) also. Thus,unfused myeloma cells die in HAT medium.Unfused B-lymphocytes die because of a short life span. The purpose of using HAT medium is to eliminate the unfused myeloma cells.

5. Only the hybridomas have the ability to divide and proliferate on the HAT medium because genome from the B-lymphocyte makes them HGPRT positive.only the B cell-myeloma hybrids survive, since the HGPRT gene coming from the B cells is functional.These cells produce

antibodies (a property of B cells) and are immortal (a property of myeloma cells).

6. The generated hybridomas are subject to multiple rounds of screening by Enzyme linked immunosorbent assay (ELISA)5 and selection in order to identify the hybridomas that produce the best monoclonal antibodies.

7. Hybridoma cell lines are cryopreserved(4) for future use.

PRODUCTION OF ANTIBODIES in vivo and invitro :

In vivo approach :

Hybridoma cells are injected into the peritoneal cavity. They grow in a constant manner and continuously secrete antibodies in the ascitic fluid. Ascites are collected and purified .Ascitic fluid contains high concentration of antibodies.This method is quicker,easier and economical.

In vitro: ● Hollow-fiber bioreactors: Cell culturing system based on hollow fibers, small capillary membranes arranged in parallel array and bundled in a tubular shell . The culture medium is pumped and nutrients are supplied via hollow fiber membranes.The cells grow to a high density on the large interior surface area of the capillary fibers and are capable of secreting high concentrations of mAbs.

FIG: PRODUCTION OF MONOCLONAL ANTIBODIES

KEY POINTS TO REMEMBER:

● Monoclonal antibodies are a series of identical antibodies produced by a single clone of B cell.hey possess high affinity towards a particular antigen. ● mAbs are widely used in diagnostic,clinical and therapeutic applications.

● KOHLER & MILSTEIN(1975) discovered the principle for production of monoclonal antibodies." ● The method for production of monoclonal antibodies is called “Hybridoma technology”. ● “ Hybridoma technology” is a technique for the production of a large number of identical antibodies. ● Hybridoma technique: a) Cell fusion : Hybrid cells are produced by fusing B lymphocytes with Myeloma cells. ● Fused cells are incubated in HAT medium b)Production of antibodies: Hybrid cells are cloned to produce a large number of cells. ● Unfused myeloma cells die in the HAT medium and unfused B lymphocytes die because of their short life span. ● Only fused cells survive inHAT medium ● Fused cells are screened for ELISA ● Each positive hybrid that produces specific antibodies are cloned and recloned to get a large number of desired antibodies. ● Hybridomas are then cryopreserved for future use.

KEY WORDS: 1. Immunotherapy : Treatment of disease by activating or suppressing the immune system (or) a type of cancer treatment.

2. Splenocytes : white blood cells in the spleen

3. Cryopreservation: The process of cooling and storing of cells at very low temperatures to maintain their viability. 4. Salvage pathway: Refers to a short sequence of enzyme-catalyzed reactions in the purine and pyrimidine metabolism which uses pre formed purine or pyrimidine bases to form nucleotides.

5. ELISA : Enzyme-linked immunosorbent assay is a test that detects and measures antibodies in a sample.

WEB LINKS:

1.sciencemag.org 2.jamanetwork.com 3.cancer.org 4.timesnownews.com 5.future-science.com 6.Monoclonal antibodies: a tool in clinical research: Indian Journal Of Clinical Medicine 2013:4 9-21 7.ncbi.nlm,nih.gov 8. britannica.com

Dr. Krishna kumar vepakomma Professor & Principal(Retd.) LMS content generator [email protected] Mobile : 9849562910