Transcript Immunochemical methods in biochemistry

Immunochemical methods in
biochemistry
Jana Novotna
Characteristics of antigens
• Antigen - macromolecular substance of natural or synthetic
origin, that the immune system recognizes as foreign. The
presence in the organism - stimulation of antibody production
(to induce an immune response).
• Immunogenicity – property of substance (immunogens or
antigens) to induce a detectable immune response.
• Antigenicity – given by a surface structure of immunogen antigenic determinants. The organism responds only to those
that are foreign to it.
Characteristics of antigens
• Antigenic specificity – property of antigen molecule (or its part) to
react with the specific antibody.
• The number of antigenic determinants of the antigen depends on
its size and chemical complexity of macromolecule
– egg ovalbumin, MW 42 000 - 5 antigenic determinants
– thyroglobulin, MW 700 000 - many than 40 antigenic determinants
• Hapten – small molecule that can elicit immune response only when
attached to a large carrier such as a protein.
• In general, only large molecules, infectious agents, or insoluble
foreign matter can elicit an immune response in the body.
Characteristics of antigens
• Chemical nature of antigens:
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proteins
polysacchrides
lipopolysaccharides
nucleoproteins
glycoproteins
steroid hormones
bacterial cells, viruses
synthetic polypeptides
synthetic polymers
Characteristics of antigens
• The antibody recognizes an epitope - defined segment of the
antigen on the macromolecular carrier (about 5-8 amino acids)
• Immunoreactivity of epitope depends on
– primary, secondary and tertiary stricture
– variability of epitope
Antibodies (immunoglobulins) and their
composition
• Immunoglobulins produce
plasma cells, which
differentiate from B cells.
• Proteins with specific binding
activity to the antigen
(antigenic determinant),
formed after it´s stimulation.
• Immunoglobulins account for ~
20% of the total plasma
proteins.
• The chemical nature glycoproteins
• The antibody binding site
occupies 5% of the antibody
molecule, distinguishes very
fine differences in the antigenic
determinants.
• The structure of the binding
site is exactly complementary
to the antigenic determinant
(lock and key).
• The variety of specific
antibodies in the body is more
than 108.
Characteristics of antibodies
(immunoglobulins)
• Variability of antibodies is subject to 5-classes of Ig: G,
A, M, D, E
• Heavy chains – g, a, m, d, e
• Light chains – k, l
• Subclasses of immunoglobulins:
– IgG – g1, g2, g3, g4
– IgA – a1, a2
– IgM - m1, m2
Characteristics of antibodies
(immunoglobulins)
Flexibility and motion of imunoglibulins
The forces binding antigen to
antibody
• Electrostatic : between attraction
oppositely charged ionic group – (NH3-) of lysine and (-COO-) of aspartate.
• Hydrogen bonding – relatively
weak and reversible hydrogen
bridges between hydrophilic group (OH, -NH2, COOH).
• Hydrophobic– non-polar,
hydrophobic side chains of Val, Leu,
Ile (hydrophobic groups come close
together and exclude water
molecules between them. The force
of attraction increases.
• Van der Waals – forces which
depend upon interaction between
the external „electron clouds“. Nonspecific attractive forces.
Polyclonal antibodies
• Host animals ca be used to raise
antibodies against a given antigen
• Each epitope in the antigen
molecule stimulates the production
of antibodies to a single clone of B
lymphocytes (complete antigens
having multiple epitopes, some
clones of activated B-cells).
• Polyclonal antibody consists of
mixture of monoclonal antibodies
having specificity for complex
antigens.
• They are conventional produced by
immunization of animals with
antigen.
Monoclonal antibody
• Products of single clone of
plasma cells by B
lymphocytes;
• Mostly prepared in laboratory.
They are directed against
single epitope – identical
copies with same structure and
antigen specificity.
• They have excellent specificity
but poor ability to precipitate
antigen.
Antibody affinity
antibody + antigen
k2
 immunokomplex [AbAg]
k1
[AbAg]
Kekv =
[Ab] [Ag]
=
k1
k2
equilibrium constant
Affinity of antibody the equilibrium constant that describes the antigenantibody reaction.
Affinity is the strength of the reaction between a single antigenic
determinant and a single combining site on the antibody (the sum of the
attractive and repulsive forces operating between the antigenic
determinant and the combining site of the antibody).
Antibody avidity
• Avidity is a measure of the
overall strength of binding of
an antigen with many antigenic
determinants and multivalent
antibodies.
• Avidity refers to the overall
strength of binding between
multivalent antigens and
antibodies.
Specificity and cross reactivity of
antibody
• Specificity of antibody - the ability of an individual antibody
combining site to react with only one antigenic determinant or the
ability of a population of antibody molecules to react with only one
antigen.
• Antibodies can distinguish differences in:
– the primary structure of an antigen
– isomeric forms of an antigen
– secondary and tertiary structure of an antigen
• Cross reactivity - the ability of an individual antibody combining site
to react with more than one antigenic determinant or the ability of a
population of antibody molecules to react with more than one
antigen.
Imunoprecipitation reaction
• Used for qualitative and
quantitative detection of antigens
and antibodies:
– phase 1– formation of primary
complexes with low MW
– phase 2 – interconnection of
Ag and Ab to the three
dimensional network
(formation of insoluble
aggregates )
Quantitative (Heidelbergova)
immunoprecipitation curve
Immunochemical tests for detection of
antigen-antibody reaction
• Use of reaction antibody - antigen in vitro is the basis of
immunochemical methods.
• Factors affecting reaction:
– the affinity of antibody,
– the ratio of Ab/Ag
– the physical form of the antigen (particulates or antigen in
solubilized form)
Agglutination tests
• Agglutination (from Latin, agglutino – to glue/ attach) - specific
antibody reacts with the corpuscular antigen.
• Agglutination reaction is based on the formation of bridges between
bivalent (IgG) or multivalent (IgM) antibodies and antigenic particles
with multiple epitopes.
• Among all other antibodies, IgM is a good agglutinin, since it has
high affinity to different antigens.
• Hemagglutination is a variant of agglutination technique in which
red blood cells are used as the antigen bearing particles.
Agglutination tests
• Agglutination reactions are performed on slides, in test tubes or
microtiter plates.
• They are more sensitive in comparison with immunoprecipitation
methods.
• The agglutination methods produce qualitative or semiquantitative
results.
• Agglutination may be either:
– Qualitative test – to identify the presence of an antigen or an antibody.
– Quantitative test – to measure the level of antibodies to particulate antigens;
• Serial dilutions of sample is used.
• Agglutination assays may be classified as direct or indirect tests.
Microtitration plates - variety of materials
(polystyrene, polypropylene, polycarbonate)
Direct agglutination
• The antigen is an integral part of the cell surface (red blood cells,
bacteria).
• A suspension of particles is directly agglutinated by specific
antibodies present in the examined sample.
• This assay is frequently used in the hematology for the
– determination of blood group
– in the immunological diagnostics for detection of specific antibodies directed
against naturally occurring antigens on the surface of some microbes (for
example against Salmonella typhi – the Widal test).
Indirect agglutination
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Indirect agglutination assay utilizes particles with the antigens that have
been passively attached to their surface.
Particles already coated with antigens (antibodies) to determine the
antibody (antigen) in given sample. Eg:
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Pregnancy test – using hormone secretion (human chorionic gonadotropin),
diagnosis of rheumatoid arthritis (rheumatoid factors are anti-immunoglobulin antibodies
directed against Fc-fragment of IgG usually class IgM)
Immunoassays
Highly sensitive techniques
High sensitivity is achieve by labeling of one reacting component
(antibody or antigen) with a substance which detection is more
sensitive
1) radioisotope – radio immunoassay (RIA)
2) enzyme – enzyme immunoassay (EIA)
3) fluorescent dye (e.g. fluorescein)
• Detection limits of can be as low as 10−15 – 10−20 mol/l
• Enzyme immunoassays utilize enzymes, usually peroxidase or
alkaline phosphatase, to detect and quantify immunochemical
reactions.
Heterogeneous enzyme immunoassay
• Enzyme-linked immunosorbent assay (ELISA)
• One of the immunochemical reactants (antigen or antibody) is first
non-specifically adsorbed to the surface of a solid phase (tubes,
wells of microtiter plates or magnetic particles)
Competitive enzyme immunoassay
• Always performed under condition of antigen excess
• A limited quantity of specific antibodies bound to the solid phase.
• The enzyme-labeled antigen (conjugate) is mixed with serum
sample containing the unknown amount of antigen.
• The serum antigen and enzyme-labeled antigen compete for
binding sites of antibody.
• Labeled and non-labeled antigen bind to the antibody in the same
proportion  the more non-labeled antigen is contained in the
mixture the less labeled antigen is bound
Competitive enzyme immunoassay
The probability of the antibody binding the labeled antigen is inversely proportional to the concentration of
unlabelled antigen.
Non-competitive enzyme immunoassay
(sandwich methods)
• Heterogeneous immunoassay - Used for the determination of
antigens or antibodies. Antibody (or antigen) is bound to the surface
of solid phase (plastic microwell surface) which must always be in
excess over the analyte being measured.
• Suitable for the determination of large antigens having multiple
binding sites for the antibody (e.g. human chorionic gonadotropin,
α1-fetoprotein, total IgE).
1. Non-competitive enzyme immunoassay for determination of antigen.
2. Non-competitive enzyme immunoassay for determination of
antibody
Antigen determination
Two different molecules of antibodies
directed against various epitopes are
necessary.
The first antibody is in excess adsorbed
to a solid phase.
The serum sample or calibrators
containing the desired antigen are added to
the well with immobilized antibody 
incubation  all the non-reacting material is
washed away  a second enzyme-labeled
antibody (different from
the first antibody) is added in excess
(another antigen epitope binds to
the second labeled antibody).
“Sandwich complex“ consisting of solidphase antibody – antigen – enzyme labeled
antibody complex is formed. After washing
of all the unreacted enzyme-labeled
antibody, the substrate is added.
The intensity of the finally measured colored
product of the enzyme reaction is directly
proportional to the amount of antigen.
Antibody determination
Especially useful for detection of
specific serum antibodies against
viruses and parasites, antibodies and
specific IgE antibodies to individual
allergens.
The antigen must be first immobilized
on the solid phase.
After adding test samples or
calibrators the antibodies in the
sample react with the immobilized
antigen and forms immunocomplexes.
The next steps of the assay procedure
are similar to that described for the
measurement of antigens.
Immunochemical methods in fast
diagnostics
• Fast and tentative “point-of-care” or “bed-side” tests based on “dry
chemistry”
• Reagents are anchored in a porous substrate that is attached on a
plastic pad or inserted in a frame with windows for sample
application and result reading.
• Examples of application:
– estimation of human chorionic gonadotropin (hCG) for diagnostics of pregnancy,
– troponine T for acute heart infarction,
– tests for narcotics
Detection of narcotics in urine or saliva
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Test is based on competition of a drug in
the sample with the same drug
immobilized in the detection area of the
test for a limited amount of specific
antibody.
The sample is mixed with antibodies in
the reaction zone.
The antibodies are labeled with stained
micro-particles.
Both the sample and antibodies move by
capillary action through the reaction zone
to the detection area (contains the
immobilized drug).
If the sample contains molecules of the
drug it fully saturates the binding sites of
color-labeled antibodies.
The antibody molecules cannot then react
with the immobilized drug and color of the
detection area stays unchanged.
Detection of human chorionic
gonadotropin, hCG (pregnancy test)
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Three different antibodies are used.
The first sample area contains specific
anti-hCG antibody labeled with micro
particles of colloid gold or blue latex
(Ab1).
Urine sample is applied, molecules of Ab1
flow to the second, detection area where
another specific antibody against hCG
(Ab2) is anchored.
If hCG is contained in the sample a
combined immunocomplex is formed with
both labeled and anchored antibody (Ab2hCG-Ab1), a colored band displays in the
detection area.
Excess of the labeled antibody is caught
in the third (control), with immobilized
antibodies against labeled anti-hCG two
bands are interpreted as a positive result.
Western blotting
• is a widely used analytical technique used to detect
specific proteins in a sample of tissue homogenate or
extract.
• It uses gel electrophoresis to separate native or
denatured proteins.
• The proteins are then transferred to a membrane
(typically nitrocellulose), where they are stained with
antibodies specific to the target protein.
Electrophoretic profile of separated collagen fraction isolated from
peripheral pulmonary arteries of rats exposed to hypoxia and its
Western blot with antibodies to collagen type I
References
• http://www.nios.ac.in/media/documents/dmlt/Biochemistry/Lesson24.pdf
• https://www.google.cz/search?q=imunoglobulin+struktura&biw=1280
&bih=862&source=lnms&sa=X&ei=igfzVOD4K4HsUM7Hgng&ved=0
CAYQ_AUoAA&dpr=1#q=immunochemical+techniques
• http://elte.prompt.hu/sites/default/files/tananyagok/practical_biochem
istry/ch07s05.html
• http://www.microbiologybook.org/mayer/ab-ag-rx.htm
• http://ulbld.lf1.cuni.cz/file/1610/immunochemical-methods.pdf