Monoclonal antibodies are identical antibodies produced by a single clone of immune cells. In 1964, a method was developed to isolate hybrid cells from two parent cell lines using HAT selection media. In 1975, Kohler and Milstein provided evidence for monoclonal antibodies by fusing normal and malignant cells, for which they received the Nobel Prize. There are three types of monoclonal antibodies: murine, chimeric, and humanized. Murine antibodies come directly from rodents, chimeric combine human and rodent portions, and humanized contain only rodent complementarity determining regions. Monoclonal antibodies are produced by fusing spleen B cells from immunized mice with myeloma cells. Hybridomas are screened to identify those producing the desired antibody

2.  Monoclonal antibodies (mAb) are antibodies that are
identical because they were produced by one type of
immune cell, all clones of a single parent cell. that
specifically bind to that substance; they can serve to
detect or purify. This has become an important tool in
biochemistry, molecular biology and medicine.

3.  1964 Littlefield developed a way to isolate hybrid cells
from 2 parent cell lines using the hypoxanthine-
aminopterin-thymidine (HAT) selection media.
 1975 Kohler and Milstein provided the most
outstanding proof of the clonal selection theory by
fusion of normal and malignant cells. This resulted in
the first monoclonal antibodies, for which they
received the Nobel Prize in 1984

4. Types of Monoclonal
Antibodies
 There are 3 types of monoclonal antibodies.They are
 Murine source monoclonal antibodies
 Chemeric Monoclonal antibodies
 Humanised monoclonal antibodies

5.  Murine source mAbs: rodent mAbs with excellent
affinities and specificities, generated using
conventional Hydrioma technology
 Chimeric mAbs: chimers combine the human constant
regions with the intact rodent variable regions.
 Humanized mAbs: contained only the CDRs of the
rodent variable region grafted onto human variable
region framework

6. Production of Monoclonal
Antibodies
 In order for us to isolate a B lymphocyte producing a
certain antibody, we first have to induce the
production of such a B cell in mice.
 This is typically done in two doses, an initial "priming"
dose and a second "booster" dose 10 days
 A sample of B cells is extracted from the spleen of the
mouse and added to a culture of myeloma
cells (cancer cells).
 The intended result is the formation of hybridomas,
cells formed by the fusion of a B cell and a myeloma
cell. The fusion is done by using polyethylene glycol

7.  HGPRT is hypoxanthine-guanine phosphoribosyl
transferase, an enzyme involved in the synthesis of
nucleotides from hypoxanthine, an amino acid
 . The myeloma cells are HGPRT- and the B cells are
HGPRT.
 The culture is grown in HAT medium, which can
sustain only HGPRT cells
 The B cells that fuse with another B cell or do not fuse
at all die because they do not have the capacity to
divide indefinitely. Only hybridomas between B cells
and myeloma cells survive, being both HGPRTand
cancerous.

8.  The initial collection of B cells used is heterogenous, i.e. they do not
all produce the same antibody.
 A hybridoma cell is initially tetraploid, having been formed by the
fusion of two diploid cells.
 This means that we cannot be certain that the hybridomas will all
produce the desired antibody or even any antibody at all.
 Screening is required to decide which hybridoma cells are actually
producing the desired antibody.

10.  Each hybridoma is cultured and screened after doing SDS-PAGE and
Western blots. The probe used is the epitope of the antibody that is
desired, which may be labeled by radioactivity
 Once we are sure that a certain hybridoma is producing the right
antibody, we can culture that hybridoma indefinitely and harvest
monoclonal antibodies from it.