W12 Immunological techniques Flashcards

1
Q

Antibodies have a Y-shaped structure

A

2 × heavy chains & 2× light chains
2 parts: Fc & Fab fragments
Fab region is specific for a protein (“antigen”)
5 different types of heavy chain in mammals generates 5 “isotypes” IgA, IgG, IgD, IgE & IgM

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2
Q

Antibodies or “immunoglobulins (Ig)” are

A

specialised proteins produced by B cells
Membrane-bound (B cell receptor)
Secreted

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3
Q

Antibodies are an important part of our immune response

A

Neutralisation

Opsonisation for phagocytosis

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4
Q

Raising antibodies

A

The process of producing an antibody specific for a target protein
These antibodies have a wide range of applications in immunological techniques

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5
Q

How do we raise antibodies?

A

Mice are immunised with the target protein
B cells are harvested & fused with tumour cells to form hybridoma
A hybridoma that produces antibody against the target protein is selected & cloned
The antibodies secreted by the cloned hybridoma are harvested & used in immunological techniques

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6
Q

Blood typing

A

Blood typing is an example of an “agglutination reaction” used to determine blood type

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7
Q

ABO system of blood typing

A
A & B glycoproteins on RBCs
Four types of blood A, B, AB & O
Rhesus factor (D protein or RhD) is also a protein on the surface of RBCs (positive or negative)
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8
Q

A, B & RhD are antigens that

A

that elicit immune responses in mismatched donor/recipient blood transfusions

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9
Q

How does the blood test work?

A

A blood sample is mixed with antibodies raised against A, B or RhD antigens
The sample is visually checked for agglutination (blood cells sticking together)
Agglutination indicates the presence of antigens in the blood sample

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10
Q

Flow cytometry

A

Flow cytometry is a technology used to analyse the proteins on cells that are in suspension

Often involves the use of commercially produced antibodies that are conjugated to fluorochromes

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11
Q

What can flow cytometry determine about a cell?

A

The cell size and density
Whether or not a cell expresses a target protein
The amount of expression of a target protein
The cells identity

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12
Q

Fluorochrome

A

A fluorescent molecule that absorbs light of a certain wavelength and in turn emits light of a certain wavelength

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13
Q

How a flow cytometer works

A

Fluorochrome-conjugated antibodies specific for the target protein are added to the cells

Cells are channelled past lasers that excite the fluorochrome (e.g. blue laser excites FITC which then emits green light and PE emits Red light)

The li`ght emitted from the excited fluorochromes is detected & plotted on a graph

Amount of light emitted = amount of antibody bound to protein = amount of protein expressed by the cell

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14
Q

Flow cytometer applications

A

Diagnostics - CD4 T cell counts in HIV
Diagnosis of haematological malignancies

Research - cell sorting
Identification & analysis of immune cells

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15
Q

B cell

lymphoma patient

A

(Reduced T cell & Granulocytes

Increased B cell clonality)

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16
Q

Confocal microscopy differences w/flow cytometer

A

The cells to be analysed are not in suspension

Used to analyse tissue sections or cells attached to a microscope slide

The light emitted by the fluorochrome-conjugated antibodies is observed under a microscope instead of plotted graphically

Confocal microscopy has the advantag`e of visualising where the protein is on the cell

17
Q

Confocal microscopy - applications

A

Mainly research
Identification & analyse cells within tissues
Co-localisation of different antigens

18
Q

IHC

A

IHC stands for ImmunoHistoChemistry

Used to show the distribution & localization of antigens in tissue sections using antibody-antigen interactions

19
Q

How does IHC work?

A

Thin sections of tissue are cut

Primary antibodies that recognise the target protein are added to the tissue

The antibody-antigen interaction is visualised using chromogenicdetection

A secondary antibody specific for the primary antibody conjugated to horseradish peroxidase (HRP) is added

HRP catalyses the conversion of the chromogen 3,3-diaminobenzidine (DAB) substrate to produce a brown precipitate at the location of the protein

The brown precipitate is then visualised using a light microscope

20
Q

Example of IHC

A

IHC is used to stain B-Raf protein in tissue sections from cancer patients and direct eligible for treatment with B-Raf inhibitors

21
Q

Cancer can be caused by mutations in tumour suppressor or tumour promoter genes

A

These genes encode proteins that are involved in suppressing or promoting cell division

Mutations in these genes can cause uncontrollable cell division

E.g. BRAF is a gene that encodes B-Raf protein that promotes cell division

Many cancers have BRAF mutations that result in over production of B-Raf and thus uncontrolled cell division

22
Q

ELISA

A

Enzyme-Linked ImmunoSorbant Assay

Quantifies the amount of a protein or antibody in liquid samples such as sera or tissue culture supernatants

23
Q

Four different types of ELISA

A

Direct
Indirect
Sandwich
Competitive

24
Q

ELISA applications

A

Antibody titres in patient serum e.g viral infections such as HIV & Hepatitis B

Detection of bacterial toxins in food such as Escherichia coli O157:H7

Home pregnancy testing detection of human chorionic gonadotropin hormone (HCG) in urine

Research quantification of cytokines/chemokines/growth factors in tissue culture supernatants

25
Q

Sandwich ELISA

A

Add capture antibody

Add sample (serum or TC spnt)

Add detection antibody (HRP - horse radish antibody)

Add substrate (TMB - a chromogenic substrate)

Determine optical density

Density of blue solution = amount of protein in sample

26
Q

Differences in the types of ELISA

A

Direct ELISA - subsrate + primary antibody conjugate

Indirect ELISA - substrate + secondary antibody conjugate

Sandwich ELISA - substrate + capture antibody

Competitive ELISA - substrate + inhibitor antigen

27
Q

Western blotting

A

a technique used to detect proteins
A Southern Blot (Edwin Southern) detects DNA
A Northern blot detects RNA

28
Q

Western blot involves four steps

A

Sample preparation
Electrophoresis
Transfer to membrane
Stain for protein of interest

29
Q

Sample preparation - western blot

A

Cells are lysed & proteins denatured

30
Q

Electrophoresis - western blot

A

Lysates are loaded onto a gel & proteins separated based on size

31
Q

Transfer to membrane - western blot

A

Fractionated proteins are transferred onto a membrane

32
Q

Stain for protein of interest - western blot

A

The membrane is incubated with a primary antibody specific for the target protein

The membrane is then incubated with a HRP conjugated secondary antibody specific for the primary antibody

A chemiluminescent HRP substrate is added to the membrane

The membrane is exposed to x ray film that “bleaches” when exposed to light

33
Q

Diagnostic use - western blot

A

The pathogen is lysed & its proteins are separated on a gel.

The proteins are transferred to a membrane.

The membrane is mixed with patient serum to capture antibodies.

Excess is washed off then secondary antibodies are added to visualise.
eg. Viral infections (HIV)
parasites (spirochete Borrelia burgdorferi that
causes Lyme disease)

34
Q

Research - western blot

A

Cell signalling proteins

Mechanism of action for cancer drugs