Monoclonal Antibody

Prepared by:

AMER SAAD AL-ALIABDUALLAH SAUD AL-SHETELY

TAREQ NAFEA AL-HARBY

Discovery

In the 1970s, the B-cell cancer myeloma was known, and it was understood

that these cancerous B-cells all produce a single type of antibody. This was

used to study the structure of antibodies, but it was not possible to produce

identical antibodies specific to a given antigen.

The process of producing monoclonal antibodies invented by Georges

Köhler and César Milstein in 1975; they shared the Nobel Prize in

Physiology or Medicine in 1984 for the discovery.

The key idea was to use a line of myeloma cells that had lost their ability to

secrete antibodies, and come up with a technique to fuse these cells with

healthy antibody producing B-cells.

Definition MCA are antibodies that are identical because they were produced

by one type of immune cell (B cell), all clones of a single parent

cell. Given any substance, it is possible to create monoclonal

antibodies that specifically bind to that substance; they can then

serve to detect or purify that substance. This has become an

important tool in biochemistry, molecular biology and medicine.

PRODUCTION OF MONOCLONAL ANTIBODYHYBRIDOMA TECHNOLOGY

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)

Step 2: - Screening Of Mice For Antibody Production

HYBRIDOMA TECHNOLOGY

After several weeks of

immunization

Serum Antibody Titre Determined

(Technique: - ELISA / Flow cytometery)

Titre too low

BOOST(Pure antigen)

Titre High

BOOST(Pure antigen)

2 weeks

PRODUCTION OF MONOCLONAL ANTIBODY

Step 3: - Preparation of Myeloma Cells

HYBRIDOMA TECHNOLOGY

Immortal Tumor Of Lymphocytes

+ 8 - Azaguanine

Myeloma Cells

High Viability & Rapid Growth

HGPRT-

Myeloma Cells

PRODUCTION OF MONOCLONAL ANTIBODY

Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells &

Selection of Hybridoma Cells

HYBRIDOMA TECHNOLOGY

FUSION

PEG

MYELOMA CELLSSPLEEN CELLS

HYBRIDOMA CELLSELISA PLATE

Feeder CellsGrowth Medium

HAT Medium

1. Plating of Cells in HAT selective Medium

2. Scanning of Viable Hybridomas

PRODUCTION OF MONOCLONAL ANTIBODY

Step 5: - Cloning of Hybridoma Cell Lines by “ Limiting Dilution” or Expansion

HYBRIDOMA TECHNOLOGY

A. Clone Each +ve Culture

B. Test Each Supernatant for Antibodies

C. Expand +ve Clones

Mouse Ascites Method

Tissue Culture Method

PRODUCTION OF MONOCLONAL ANTIBODY

HYBRIDOMA TECHNOLOGY

PRODUCTION OF MONOCLONAL ANTIBODY

Mca.swf

Applications of Monoclonal Antibodies Diagnostic Applications

Biosensors & Microarrays Therapeutic Applications

Transplant rejectionCardiovascular disease CancerInfectious DiseasesInflammatory disease

Clinical ApplicationsPurification of drugs, Imaging the target

Future Applications Fight against Bioterrorism

Monoclonal antibodies for cancer treatment

Three mechanisms that could be responsible for the cancer

treatment.

A. mAbs act directly when binding to a cancer specific

antigens and induce immunological response to cancer

cells. Such as inducing cancer cell apoptosis, inhibiting

growth, or interfering with a key function.

B. mAbs was modified for delivery of a toxin, radioisotope,

cytokine or other active conjugates.

C. it is also possible to design bispecific antibodies that can

bind with their Fab regions both to target antigen and to a

conjugate or effector cell

mAbs treatment for cancer cells

ELISA

Enzyme Linked Immunosorbent Assay

What is ELISA?

ELISA is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample.

The ELISA has been used as a diagnostic

tool in medicine and plant pathology.

The principle of (ELISA)

It is a solid phase assay that requires the

separation of reagents.

The principle depend on the type of

ELISA(direct or indirect)

Applications

Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as with the HIV test) and also for detecting the presence of antigen.

It has also found applications in the food industry in detecting potential food allergens such as milk, peanuts and eggs.

The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV.

It has a high sensitivity. In an ELISA test, a person's serum is applied to a plate to which HIV antigens have been attached. The plate is then washed to remove other components of the serum. Then an antibody applied to the plate, followed by another wash. This antibody is chemically linked in advance to an enzyme.

A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence.

ELISA results are reported as a number either positive or negative result.

Above is ELISA data from three patients. Numbers are expressed as optical density at 450 nm:

Positive result ≥0.500. 0.300 to 0.499 are indeterminate and need to be retested. Negative ≤0.300.

Positive Control

Negative Control

Patient APatient BPatient CAssay

Control

1.6890.153O.0550.4121.9990.123

Anim0100.swf

ReferenceMedLinePlus. "HIV ELISA/western blot." U.S. National Library of Medicine. Last

accessed April 16, 2007.

http://www.nlm.nih.gov/medlineplus/ency/article/003538.htm

Lequin R (2005). "Enzyme immunoassay (EIA)/enzyme-linked immunosorbent

assay (ELISA).". Clin. Chem. 51 (12): 2415-8. PMID 16179424.

Köhler, G., and Milstein, C. Continuous cultures of fused cells Secreting

antibodies of predefined specificity. Nature, 256: 495, (1975).

Koprowski, H., Steplewski, Z., Herlyn, D., and Herlyn, M. Production of

monoclonal antibody against human melanoma by somatic cell hybrids. Proc.

Nat. Acad. Sci. USA. 75: 3405, (1978).