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Identification of Breast Cancer Peptide Epitopes Presented by HLA-A* 0201

By Sreedharan Lakshminarayanan

outline
Breast cancer

Statistics
Risk factors Types of Breast cancer

Peptide purification and Isolation


ELISA Mass spectrometery

Western Blotting

Breast cancer
Cancerous growth in breast tissue

Multiply uncontrollably
The cause is unclear Several risk factors

Some risk factors


Gender

Age
Genetic Family history

Menstrual periods
Breast radiation early in life Alcohol

Smoking
Lack of exercise

Statistics
1.5 million worldwide.

The United States is more prone to breast

cancer. 1 in 8 women and 1 in 1000 men.

http://www.komennyc.org/site/PageServer?pagename=breasthealt h_statistics

Types of Breast cancer


Ductal carcinoma Breast cancer

Lobular Carcinoma Breast Cancer


Rarest form: Inflammatory Breast cancer

How cancer occurs?


Malignant cells escapes from immune

recognition and destruction. HLA can act as biomarker but it cannot convey information. Class I MHC responsible for conveying the intracellular to immune system. Then peptides are recognized by cytotoxic T lymphocytes.

Peptide isolation and purification


Breast cell lines (MDA-MB-231, BT-20, MCF-

7) and non cancer cell line (MCF 10 A) are cultured in a suitable medium. sHLA obtained by deletion of transmembrane and cytoplasmic domain. sHLA and breast cell line and non cancer cell line are mutated for a certain period.

ELISA
As the name describes enzyme-linked

immunosorbent assay, where the reaction of antigen and antibody takes place in vitro and those reactions are monitored by enzyme measurements. Ninety-six microwell plates is used. Antibody is immobilized on each well of micro plate

Peptide separation
Purified peptide

Separated through Reverse phase HPLC


Jupiter proteo C12 column Gradient elution

Detected by UV absorption

Mass Spectroscopy
Peptides are Ionized by elecrospray

ionization. Mass to charge ratio. Peptides are confirmed by identical peaks.

Western Blotting
Also known as immunoblotting or protein blotting. Used to confirm the protein expression. Steps involved in western blotting:- Sample preparation - Electrophoresis - Transfer of proteins and staining

Sample preparation
Tissue are lysed to release protein

Maintained in a buffer
Centrifuged Proteins were used for further separation.

Gel electrophoresis
- Polyacrylamide gel electrophoresis are being

used in this experiment - Proteins are separated according to the charge in first dimension and according to the mass in second dimension on gel electrophoresis by applying electrical current. - Separated proteins are transferred or blotted.

By applying electric field proteins are transferred

from gel to a sheet of blotting paper called nitrocellulose. The proteins will move out from the gel onto the surface of the nitrocellulose membrane. Copy of protein pattern will be on the membrane. Dyes are added to the gel to confirm the transfer of protein. Membrane is incubated with an specific antibody to the particular protein.

Continuous.
Then antibody coupled with enzyme and

incubated with the substrate. After reaction with enzyme, luminescent will be produced which is used for the identification.

http://www.virology.ws/2010/07/07/virology-toolbox-the-western-blot/

Comparison
ELISA gives a +ve or ve test result based on

the antibody. Whereas western blotting is more specific and it allows to visualize the antibodies against the viral protein.

References
O. E. Hawkins, R. S. VanGundy., Identification of Breast Cancer

Peptide Epitopes Presented by HLA-A*0201 Journal of Proteome Research 2008, 7, 14451457. J.P. Carralot, C. Lemmel, Mass spectrometric identification of an HLA-A*0201epitope from Plasmodium falciparum MSP-1., International Immunology, 2008 Vol. 20, No. 11, pp. 14511456. ONeil, K. A.; Miller, F. R.; Barder, T. J.; Lubman, D. M. Profiling the progression of cancer: separation of microsomal proteins in MCF10 breast epithelial cell lines using nonporous chromatophoresis. Proteomics 2003, 3 (7), 125669.