Monoclonal antibodies are produced by clones of single B cells that are directed against a single antigen or epitope. Polyclonal antibodies represent a mixture produced by various B cell clones, recognizing multiple epitopes. The hybridoma technology developed by Milstein and Kohler in 1975 involves fusing antibody-producing B cells with myeloma cells to form immortal hybridomas. This allows for the mass production of monoclonal antibodies through selection in HAT medium, where hybridomas survive due to inheriting genes from both parent cells. Monoclonal antibodies have various applications including disease diagnosis, antigen purification, and studying the immune system.

1. MONOCLONAL ANTIBODIES:
 Are monospecific antibodies these antibodies are produced form
clones of single lymphocyte directed aganist a single antigenic
determinant or epitope
 Monoclonalantibodies are produced by single B cell clones of a
single parent
 POLYCLONAL ANTIBODIES:
 Represents a mixture of antibodies synthesized by variety of
such B lymphocyte clones
 The resulting polyclonal antibodies in the antiserum are
heterogenous comprising a mixture of antibodies each specific
for one epitope
 They recognize multiple epitopes making them more tolerant of
small changes in the nature of the antign
 Preferred choice for detection of
 Hybridoma technology:is a method of formig hybrid cell
lines ( hybridomas) by fusing a specific antibody produding B
cell with a myeloma cell
 Invented by milstein and kohler in 1975 they shared the nobel
prize in 1984
 Hybridomas are somatic cell hybrids produced by fusing
antibodies forming spleen cells with myeloma .
 PRODUCTION OF MONOCLONAL ANTIBODIES:
 Antibody B cells normally die after several weeks in cell culture
in vitro
 Myelomas are capable of dividing indefinitely so called
immortal cell lines
 Hybridoma cell lines share the properties of both fusion patners
 IMMUNISATION:
 The very first step in hybridoma technology is to immunize an
animal (usually a mouse), with appropriate antigen.

2.  After several weeks of injections, mouse responds by producing
antibodies against the injected antigen
 Many of the B cells producing these antibodies will reside in the
mouse spleen which is surgically removed as a source of these
cells
 CELL FUSION:
 After the antibodies production the plasma cells which are
removed are to be fused with myeloma cells
 Fusion of cells can be achieved by using polyethylene
glycol(reagent for fusing cell membrane
 Selection of Hybridomas:
 When the cells are cultured in HAT medium, only the hybridoma
cells grow, while the rest will slowly disappear.
 Myeloma cells is deficient in an enzyme important to recycle
purine nucleotides
 Aminopterin is a synthetic derivative of pterin , acts as a
competitive inhibitor for enzyme dihydrofolatereductase which
catalyze the reduction of dihydrofolate
 Addition of aminopterin inhibits the denovo nucleotide synthesis
pathway
 Normal cells survive in the medium as they are able to use the
salvage pathway for nucleic acid synthesis and die after a short
period of time
 Myeloma cells are deficient in enzyme HGPRT so they are
unable to utilize the salvage pathway and die in the aminopterin
containing medium
 Hybridoma cells will survive as they inherit HGPRT from the
lymphocyte parent
 Application of hybridoma:
 Hybridoma cells produce blood group antibodies on a large
scale

3.  The phenotypic difference between Blymphocytes and T
lymphocytes can be identified
 The origin of tumour cells can be traced
 Hybridoma technology helps to locate cancerous tisssues
 Function of the immune system can be studied
 Antigen can be purified
 Antigenic determiants on various infectious agents can be
identified
 Diagnosis of infections disease such as gonorrhoea