immunological applications - pedersen science · multiplex assays that use beads and fluorescent...

Immunological

ApplicationsChapter 8: Background

Biotechnology: A Laboratory Skills Course | explorer.bio-rad.com2

The Immune System

Types of Immunity

– Innate

• The natural immunity present at birth

– Acquired

• A specific response to foreign substances. Some

cells “remember” invaders and can respond quickly to

future infections

• Immunizations trigger this response so that recipients

are prepared for future infections


The Immune System

Antigens

– Something that elicits an immune response

• Can be viruses, bacteria, toxins, drugs, DNA, RNA, and other

chemicals

Antibodies

– Proteins produced by white blood cells that bind to antigens

Epitope

– A single shape on the antigen that an antibody will bind to


The Immune Response

Pathogen has antigen on surface, is contacted by

macrophage and engulfed through phagocytosis

The macrophage displays antigen on surface with

major histocompatibility complex (MHC)

T cells relay the message to B cells

B cells start producing antibodies and become

plasma cells

Some become memory B cells


The Immune System


Antibodies

Antibodies are also called

immunoglobulins (Ig)

– Types

• IgA: first line of defense with

invaders found in tears,

saliva, milk

• IgD: initiates B cells and

activates other immune cells

• IgE: allergic reactions

• IgG: most abundant,

produced when infected

• IgM: found in serum,

responsible for primary

immune response


Immune Response

Primary response

– Initial production of antibodies

when infected

– Response is slow

Secondary response

– Response after an initial

exposure

– Much more rapid and robust


Antibodies as Tools

Two main types of antibodies produced to be

used as tools in biotech

– Polyclonal antibodies are produced by injecting an

animal with an antigen and harvesting the serum that

contains the antibodies


Antibodies as Tools

Monoclonal

antibody production

– Extract B cells from

an inoculated animal

– Fuse with a tumor

cell to make

immortal

– Clone the immortal

cells and collect

antibodies


Antibodies as Tools

Antibodies to

deliver treatment

to specific cells

– Humanized

recombinant

antibodies

combine part of a

human and part of

a mouse antibody

sequence


Immunoassays

Tests that use

antibodies as a

component

– Most common is

pregnancy test

• Urine containing hCG is

added to strip

• Mobile colored

antibodies bind and

move

• They bind to a stationary

set against hCG

• Or continue to bind to a

control set


Other Types of Assays

Enzyme-linked immunosorbant assays (ELISA)

Ouchterlony or double diffusion assays

Western blots

Immunohistochemical and immunocytological assays

performed on slides or in tissues for microscopic

analysis

Multiplex assays that use beads and fluorescent

sorting also involve antibodies


ELISA

Uses antibodies to detect an antigen or antibody of interest by

exploiting the tendency of proteins to adsorb (stick) to a plastic

microplate

Can detect either antigens or antibodies

Can be either direct (using only a primary antibody) or indirect

(using a secondary antibody to detect the first antibody)


ELISA Types

Direct ELISA

– Antigens from sample coat the well

– Enzyme-linked antibodies bind to the antigen of interest if

present

– Colorimetric substrate is added

– Enzyme linked to the antibody cleaves the substrate, turning

the solution blue, which indicates a positive test


ELISA Types

Indirect ELISA

– Antigens from sample coat the well

– Primary antibodies bind to the antigen of interest if present

– Enzyme-linked secondary antibody is added which binds to

the primary antibody

– Substrate color change indicates a positive test


ELISA Types

Direct sandwich ELISA

– Primary antibodies are bound to wells

– Sample is added that may contain the antigen


the antigen if present



ELISA Types

Capture serum antibody

– Purified antigens are bound to wells

– Serum sample is added that may contain antibodies to the

purified antigen


serum antibodies if present



Instruments for ELISA

Microplate readers

– Use spectrophotometric methods to measure the

amount of absorbance by the developed substrate


Ouchterlony or Double Diffusion Assay

Antibodies are placed into

the center well and diffuse

outward

Antigens placed in the

surrounding wells diffuse

inwards

Interactions form a

precipitation event


Western Blotting

SDS-PAGE results can be

used to estimate the mass of

proteins, but cannot identify

them directly

Antibodies can be used in the

same way as an ELISA test

when the proteins from an

SDS- PAGE experiment are

transferred to a membrane

– Electroblotting is the general

method used


Western Blotting

Once proteins have been transferred to the

membrane, the technique is similar to the indirect

ELISA


Western Blot Detection

Colorimetric

– Visible color left after

substrate interaction

Chemiluminescent

– Substrate can emit light

when enzyme reacts

– Sometimes different

molecules linked to the

secondary antibody emit

different colors. This

allows for multiple

detections


Other Applications for Western Blots

Viral protein detection

– Diagnosis of HIV infection


Other Applications for Antibodies

Immunohistochemistry

and

immunocytochemistry

– When antibodies are

used to study tissues

and cells

• Immunofluorescence is

when an antibody is

labeled with a

fluorescent molecule

and will emit a different

wavelength of light than

the antibody being

stimulated



Multiplex beads

– Beads that have a color themselves can be used

– If different antibodies are linked to various colors then

as they move past a detector, the color of the bead

showing an interaction can be used to determine what

was bound



Fluorescent-activated

cell sorting (FACS)

– Different antibodies are

labeled with different

fluorescent molecules

enabling sorting due to a

charge that is applied to

the cell


Chapter 8 Summary

Background

• The Immune System

• Types of Immunity

• Immune Response

Antibodies

• Structure

• Types

• Responses

Antibodies as Tools

• Treatment

• Immunoassays

• Other Applications that Utilize Antibodies

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