Transcript BIOT 184 Introduction to Biotechnology

BIOMAN 2010
IMMUNOASSAYS
Barbara Bielska
Northampton Community College
Tannersville, PA
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Antibody-mediated immunity
Antibody-mediated immunity is part of
acquired or adaptive immunity
• Antigen
• Antibody-mediated immunity involves B
cells, and T cells (lymphocytes).
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What is antigen
• Any substance that can elicit an immune response is said to be
immunogenic and is called an immunogen.
• There is a clear operational distinction between an immunogen and
an antigen.
• An antigen is defined as any substance that can bind to a specific
antibody.
• All antigens therefore have the potential to elicit specific antibodies,
but some need to be attached to an immunogen in order to do so.
• This means that although all immunogens are antigens, not all
antigens are immunogenic.
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Immunogen: adjuvant
In immunology, an adjuvant is an agent that may stimulate the immune
system and increase the response to a vaccine, without having any
specific antigenic effect in itself.
The word “adjuvant” comes from the Latin word adjuvare, meaning to
help or aid.
"An immunologic adjuvant is defined as any substance that acts to
accelerate, prolong, or enhance antigen-specific immune responses
when used in combination with specific vaccine antigens.“
http://en.wikipedia.org/wiki/Immunologic_adjuvant
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What Is Antibody
Antibody:
An immunoglobulin, a specialized immune protein, produced because of the
introduction of an antigen (immunogen) into the body, and which possesses
the remarkable ability to combine with the very antigen that triggered its
production.
The production of antibodies is a major function of the immune system and is
carried out by a type of white blood cell called a B cell (B lymphocyte).
Antibodies can be triggered by and directed at foreign proteins,
microorganisms, or toxins.
The term "antibody" dates to 1901.
Prior to that time, an "antibody" referred to any of a host of different
substances that served as "bodies" (foot soldiers) in the fight against
infection and its ill effects.
According to http://www.medicinenet.com/script/main/hp.asp
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What Is Antibody
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Immunoglobulins
• Immunoglobulins – Antibodies
IgD surface of many cells (mostly B)
IgM surface of B cells
IgG blood; the most abundant Ab
IgA digestive and respiratory systems
IgE skin, digestive and respiratory tract;
responsible for allergic reactions
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IgG: immunoglobulin G
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Antibodies
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Vaccine is made from an antigen
isolated or produced from the
disease-causing microorganism.
The vaccine is injected into the blood
stream.
The B cells in the blood stream
respond to the antigen by
producing antibodies.
The antibodies bind to the antigen to
"neutralize“ or inactivate it.
In addition, memory cells are
produced and remain ready to
mount a quick protective immune
response against subsequent
infection with the same disease
causing agent.
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Antibodies: Applications
• Diagnostic tests
• Research tool
• Drug delivery system
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Immunoassays
• Diagnostic and research tools
• Allow to detect very minute amounts of the analytes
• Highly sensitive and specific
• Multibillion dollar business
•
Examples: HIV test, hepatitis test, tests for numerous infectious diseases and other
disorders (thyroid dysfunction), test for H. pylori, numerous QC tests.
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Polyclonal and Monoclonal
Antibodies
• Polyclonal Ab recognize the same antigen but
different epitopes (antigenic determinants)
heterogeneous
made by the B cells
• Monoclonal Ab recognize the same antigen
and the same epitope, homogenous clones
made by mice hybridomas (tissue culture)
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Monoclonal Ab
The production of monoclonal anti-bodies was first invented by
Cesar Milstein,Georges J. F. Köhler and Niels Kaj Jerne in 1975.
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HAT medium is often used for preparation of monoclonal antibodies.
This process is called Hybridoma technology.
Laboratory animals (e.g., mice) are first exposed to an antigen against which we
are interested in isolating an antibody.
Once splenocytes are isolated from the mammal, the B cells are fused with
HGPRT negative, immortalized myeloma cells using polyethylene glycol or the
Sendai virus.
Fused cells are incubated in the HAT medium.
Aminopterin in the medium blocks the de novo pathway.
Hence, unfused myeloma cells die, as they cannot produce nucleotides by de
novo or salvage pathway.
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HAT medium is often used for preparation of monoclonal antibodies.
Cont’d
Unfused B cells die as they have a short lifespan. In this way, only the B cellmyeloma hybrids survive.
These cells produce antibodies (a property of B cells) and are immortal (a
property of myeloma cells).
The incubated medium is then diluted into multiwell plates to such an extent that
each well contains only 1 cell.
Then the supernatant in each well can be checked for desired antibody.
Since the antibodies in a well are produced by the same B cell, they will be
directed towards the same epitope, and are known as monoclonal antibodies.
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HAT Medium (Hypoxanthine Aminopterin Thymidine
medium)
is a selection medium for mammalian cell culture, which
relies on the combination of aminopterin, a drug that acts
as a folate metabolism inhibitor by inhibiting dihydrofolate
reductase, with hypoxanthine and thymidine, which are
intermediates in DNA synthesis—a purine derivative and a
deoxynucleoside, respectively.
The trick is that aminopterin blocks DNA de novo synthesis,
which is absolutely required for cell division to proceed, but
the other components provide cells with the raw material to
evade the blockage (the "salvage pathway"), provided that
they have the right enzymes, which means having
functioning copies of the genes that encode them.
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Therefore, the use of HAT medium for cell culture is a form of
artificial selection for cells containing working TK and HGPRT
(Hypoxanthine-guanine phosphoribosyltransferase).
The protein encoded by this gene is a transferase, which catalyzes
conversion of hypoxanthine to inosine monophosphate and guanine to
guanosine monophosphate via transfer of the 5-phosphoribosyl group from
5-phosphoribosyl1-pyrophosphate.
This enzyme plays a central role in the generation of purine nucleotides
through the purine salvage pathway.
Mutations in this gene result in Lesch-Nyhan syndrome or gout.
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Types of immunoassays
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ELISA: enzyme linked immuno-sorbent assay
Radio-immunoassays
Immunoassays with color or fluorescent tags
(colloidal gold, polymers, crystals)
Basic formats
Direct assay
Indirect assay
Sandwich assay
Competitive assay
Multiplex assay
Rapid assay: lateral flow chromatography
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Microtiter plate
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Direct ELISA
The direct ELISA uses the method of directly labeling the antibody itself.
Microwell plates are coated with a sample containing the target antigen, and the
binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or
fluorescent end-point.
Since the secondary antibody step is omitted, the direct ELISA is relatively quick,
and avoids potential problems of cross-reactivity of the secondary antibody with
components in the antigen sample.
However, the direct ELISA requires the labeling of every antibody to be used, which
can be a time-consuming and expensive proposition.
In addition, certain antibodies may be unsuitable for direct labeling.
Direct methods also lack the additional signal amplification that can be achieved with
the use of a secondary antibody.
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Indirect ELISA
The indirect, two-step method uses a labeled secondary antibody for
detection.
First, a primary antibody is incubated with the antigen.
This is followed by incubation with a labeled secondary antibody that
recognizes the primary antibody.
For ELISA it is important that the antibody enzyme conjugate is of high specific
activity.
This is achieved when the antibody is affinity purified and the enzyme
conjugation chemistry preserves antibody specificity as well as enzyme
activity.
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Sandwich ELISA
The sandwich ELISA measures the amount of antigen between two layers of antibodies.
The antigens to be measured must contain at least two antigenic sites (epitopes), capable
of binding to the antibody, since at least two antibodies act in the sandwich.
For this reason, sandwich assays are restricted to the quantitation of multivalent antigens
such as proteins or polysaccharides.
To utilize this assay, one antibody (the “capture” antibody) is purified and bound to a solid
phase typically attached to the bottom of a plate well.
Antigen is then added and allowed to complex with the bound antibody.
Unbound products are then removed with a wash, and a labeled second antibody (the
“detection” antibody) is allowed to bind to the antigen, thus completing the “sandwich”.
The assay is then quantitated by measuring the amount of labeled second antibody bound
to the matrix, through the use of a colorimetric substrate.
Major advantages of this technique are that the antigen does not need to be purified prior to
use, and that these assays are very specific.
However, one disadvantage is that not all antibodies can be used.
Monoclonal antibody combinations must be qualified as “matched pairs”, meaning that they
can recognize separate epitopes on the antigen so they do not hinder each other’s binding.
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ELISA: Enzyme Linked Immunoassay
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ELISA steps
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Competitive ELISA
When two “matched pair” antibodies are not available for a target, another option is the competitive
ELISA.
The enzyme may be linked to either the antigen or the primary antibody.
In this type of ELISA, there is an inverse relationship between the signal obtained and the
concentration of the analyte in the sample, due to the competition between the free analyte and the
ligand-enzyme conjugate for the antibody coating the microplate, i.e. the more analyte the lower the
signal.
Briefly, an unlabeled purified primary antibody is coated onto the wells of a 96 well microtiter plate.
This primary antibody is then incubated with unlabeled standards and unknowns.
After this reaction is allowed to go to equilibrium, conjugated antigen is added.
This conjugate will bind to the primary antibody wherever its binding sites are not already occupied by
unlabeled antigen.
Thus, the more unlabeled antigens in the sample or standard, the lower the amount of conjugated
antigen bound.
The plate is then developed with substrate and color change is measured.
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Competitive immunoassay
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Competitive immunoassay
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Multiplex ELISA
A logical progression of the widely used microtiter plate ELISA is toward a protein array
format that allows simultaneous detection of multiple analytes at multiple array addresses
within a single well.
Different types of multiplex ELISA have been developed.
Generally, multiplex ELISA can be achieved through antibody array, where different
primary antibodies are printed on glass plate to capture corresponding antigens in a
biological sample such as plasma, cell lysate, or tissue extract.
Detection method can be direct or indirect, sandwich or competitive, labeling or nonlabeling, depending upon antibody array technologies.
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Multiplex ELISA protein array
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Rapid test
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Test device component categories
Porous materials
Sample pads
Conjugate pads
Membranes Absorbent pads
Reagents
Capture antibodies and/or antigens (test line and control line)
Conjugate ligand (antibody or antigen)
Detector particle (e.g., colloidal gold)
Blocking agents, detergents, surfactants, stabilizers, buffers, etc.
Housing and lamination materials
Back laminate (for holding porous components together)
Top laminate (optional, to act as a "splash guard" or prevent evaporation and
back-migration of detector reagent)
Device housing (optional—if used, the housing typically comprises two,
snap-fit plastic pieces that envelop the reagent-loaded porous media
assembly and facilitate sample addition)
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Rapid immunoassay
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Rapid immunoassay
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Rapid lateral flow test
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Pregnancy
test
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Specifications for a pregnancy test
Following are the development guidelines given to the research and development manager responsible
for designing, developing, and scaling up to manufacturing a hypothetical lateral-flow diagnostic
test device for detecting human chorionic gonadotropin (hCG) in urine.
The hCG hormone is measured in milli-international units per milliliter (mIU/ml).
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More than 99% of specimens containing 25 mlU/ml will produce a visible signal at the test line.
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More than 99% of specimens containing 5 mlU/mL will not produce a visible signal at the test line.
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The test will have a control line that will always produce a visible signal if the test is performed
correctly and if all of the test components are functional.
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If the sample is positive (contains 25 mIU/ml hCG), the test must produce a visible signal at the
test line within 3 minutes of sample addition.
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The test result must be stable (not change) for at least 30 minutes after the sample is added.
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The test device will be contained in a plastic housing and designed to be compatible with "instream" sampling protocols.
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The packaged (unused) product must be stable for at least 18 months at ambient conditions (15°–
30°C).
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Total incremental manufacturing cost per test (excluding licensing and royalties) can not exceed
$0.25 US.
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Rapid test for drugs of abuse
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