Presented by:-Jagohool SinghRoll no=1237MSc.Biotechnology 1stMDU ROHTAK

An antibody is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target.

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.

Polyclonal antibodies are antibodies that are derived from different cell lines.

1975, Georges Köhler and Cesar Milstein- awarded Nobel Prize in1984

In 1975, Kohler and Milstein first fused lymphocytes to produce a cell line which was both immortal and a producer of specific antibodies. The two scientists were awarded the Nobel Prize for Medicine in 1984 for the development of this "hybridoma." The value of hybridomas to the field was not truly appreciated until about 1987, when MAbs were regularly produced in rodents for diagnostics.

PRODUCTION OF MONOCLONAL ANTIBODY

Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells

HYBRIDOMA TECHNOLOGY

ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) +

ADJUVANT (emulsification)

Ab titre reached in Serum

Spleen removed

(source of cells)

PRODUCTION OF MONOCLONAL ANTIBODY

Step 2: - Preparation of Myeloma CellsHYBRIDOMA TECHNOLOGY

Myeloma CellsHGPRT-

(Hypoxanthin Guanine

phosphoribosyltransferase)

PRODUCTION OF MONOCLONAL ANTIBODY

Step 3: - Fusion of Myeloma Cells with Immune Spleen Cells &

Selection of Hybridoma Cells

HYBRIDOMA TECHNOLOGY

FUSIONPEG

MYELOMA CELLSSPLEEN CELLS

HYBRIDOMA CELLSELISA PLATE

Feeder CellsGrowth Medium

HAT Medium

1. Plating of Cells in HAT selective Medium

2. Scanning of Viable Hybridomas

Selected by using HAT medium (Hypoxanthine-Aminopterin-Thymidine)

Myeloma cells are unable to grow B cells are able to survive, but can not live for

extended periods

(4) Fusion of myeloma and B cells (using PEG)(5) Separation of cell lines

(6) Screening of suitable cell lines

(7) in vitro (a) or in vivo (b) multiplication(8) Harvesting

Monoclonal antibodies are proving to be very useful as diagnostic, imaging, and therapuetic reagents in clinical medicine.

Many monoclonal antibody diagnostic reagents now available are products for detecting pregnancy, diagnosing numerous pathogenic microorganisms, measuring blood levels of various drugs, and detecting antigens shed by certain tumors.

A pregnant woman has the hormone human chorionic gonadotrophin (HCG) in her urine.

Monoclonal antibodies to HCG have been produced. These have been attached to enzymes which can later interact with a dye molecule and produce a colour change.

The test of HIV infection is based on detecting the presence of HIV antibody in the patient’s blood serum.

a) HIV antigen is attached to the plate.b) Patients serum passed over the plate. Any

HIV antibody in the patients serum will attached to the antigen already on the plate.

c) A second antibody which is specific to the HIV antibody is passed over the plate. This antibody will attach to the concentrated HIV antibody on the plate. This second antibody has an enzyme attached to its structure.

d) Chromagen dye is passed over the complex of concentrated HIV antibody/conjugated antibody.

e) The enzyme will turn the chromagen to a more intense colour. The more intense the colour, the greater the HIV antibody level. This would be the a positive result for a HIV test.

Cancer cells carry specific tumour-associated antigens (TAA) on their plasma membrane.

Monoclonal anti-TAA antibodies have been produced.

Drugs which kill tumour cells or inhibit key proteins in tumour cells are attached to monoclonal anti-TAA antibodies.

Cancer cells are specifically targeted, avoiding damage to healthy host cells.