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

7/29/2019 Identification of Breast Cancer Peptide Epitopes Presented by HLA-A* 0201

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By

Sreedharan Lakshminarayanan

Identification of Breast Cancer PeptideEpitopes Presented by HLA-A* 0201


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outline

Breast cancer

Statistics

Risk factors

Types of Breast cancer Peptide purification and Isolation

ELISA

Mass spectrometery Western Blotting


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Breast cancer

Cancerous growth in breast tissue

Multiply uncontrollably

The cause is unclear

Several risk factors


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Some risk factors

Gender

Age

Genetic

Family history Menstrual periods

Breast radiation early in life

Alcohol Smoking

Lack of exercise


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Statistics

1.5 million worldwide.

The United States is more prone to breast

cancer.

1 in 8 women and 1 in 1000 men.


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

h_statistics
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Types of Breast cancer

Ductal carcinoma Breast cancer

Lobular Carcinoma Breast Cancer

Rarest form:

Inflammatory Breast cancer


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How cancer occurs?

Malignant cells escapes from immunerecognition 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.


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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 transmembraneand cytoplasmic domain.

sHLA and breast cell line and non cancer cell

line are mutated for a certain period.


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ELISA

As the name describes enzyme-linked

immunosorbent assay, where the reaction of

antigen and antibody takes place in vitro andthose reactions are monitored by enzyme

measurements.

Ninety-six microwell plates is used.

Antibody is immobilized on each well of micro

plate


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Peptide separation

Purified peptide

Separated through Reverse phase HPLC

Jupiter proteo C12 column

Gradient elution Detected by UV absorption


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Mass Spectroscopy

Peptides are Ionized by elecrosprayionization.

Mass to charge ratio.

Peptides are confirmed by identical peaks.


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Western Blotting

Also known as immunoblotting or proteinblotting.

Used to confirm the protein expression.

Steps involved in western blotting:-- Sample preparation

- Electrophoresis

- Transfer of proteins and staining


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Sample preparation

Tissue are lysed to release protein

Maintained in a buffer

Centrifuged

Proteins were used for further separation.


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Gel electrophoresis

- Polyacrylamide gel electrophoresis are beingused in this experiment

- Proteins are separated according to the

charge in first dimension and according to themass in second dimension on gel

electrophoresis by applying electrical current.

- Separated proteins are transferred or blotted.


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By applying electric field proteins are transferred

from gel to a sheet of blotting paper callednitrocellulose.

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.


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Continuous.

Then antibody coupled with enzyme andincubated with the substrate.

After reaction with enzyme, luminescent will be

produced which is used for the identification.


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Comparison

ELISA gives a +ve orve test result based onthe antibody.

Whereas western blotting is more specific and

it allows to visualize the antibodies against theviral protein.


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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. Proteomics2003, 3(7), 125669.


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Documents

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