Principles of Immunochemical
Techniques
Introduction
• Immunochemical reactions form the basis of a
diverse range of sensitive and specific clinical
assays.
• identification and quantitation of specific
substances
• an antibody is used as a reagent to detect an
antigen of interest.
• The exquisite specificity and the high affinity of
antibodies for specific antigens, coupled with the
unique ability of antibodies to cross link antigens,
BASIC CONCEPTS
• ANTIBODIES
– Immunoglobulins consist of five general classes
designated as IgG, IgA, IgM, IgD, and IgE.
Schematic diagram of IgG antibody molecule showing carbohydrate (Cbh). Disulfide bonds (-S-S-). and major
fragments produced by proteolytic enzyme treatment (F[ab′]2. Fc,Fab,Fd).
ANTIBODIES
• Polyclonal
– a heterogeneous mixture of antibodies produced
by many cell clones in response to immunization.
• Monoclonal
– the product of a single clone or plasma cell line
Production of antibodies
• Polyclonal antiserum
– raised in a normal animal host in response to
immunogen administration.
• The usual method of production of
monoclonal antibodies
– Fusing sensitized lymphocytes from the spleens or
lymph nodes of mice that have been immunized,
with a murine myeloma cell line
– Phage display
Antigens
• Antigens
– Natural and synthetic, including
– Proteins, carbohydrates, nucleic acids, lipids,and
other molecules.
• epitope (antigenic determinant).
– Each unique region of the antigen molecule that
will bind a complementary antibody
• Natural immunogens are usually complex and
have several epitopes.
• Hapten
– a chemically defined determinant that when
conjugated to an immunogenic carrier stimulates
the synthesis of antibody specific for the hapten
Immunogenicity
• Some general properties required for immunogenicity include:
•
•
•
•
(I) areas of structural stability within the molecule,
(2) randomness of structure,
(3) a minimal molecular weight of 4000 to 5000,
(4) ability to be metabolized (a necessary but not sufficient
criterion for some classes of antigens),
• (5) accessibility of a particular immunogenic configuration to the
antibody-forming mechanism,
• (6) the foreignness of structure to the host.
• The overall shape and charge of the molecule in most cases have
very little influence on the ability to induce antibody formation.
• The strength or energy of interaction between
the antibody and antigen is described by two
terms
• Affinity
– the thermodynamic quantity defining the energy of
interaction of a single antibody-combining site and its
corresponding epitope on the antigen.
• Avidity
– the overall strength of binding of an antibody and an
antigen
ANTIGEN ANTIBODY BINDING
• BINDING FORCES
– van der Waals-London dipole-dipole interaction,
hydrophobic interaction, and ionic coulombic bonding
• Distance,Temperature (van der Waals)
• aqueous or other polar solutions (hydrophobic)
• dielectric constant (aqueous solutions / added salt)
– layer of charged particles tend to shield
• pH, temperature, and ionic strength of the
reaction medium should influence the binding of
antigen and antibody.
REACTION MECHANISM
• n is the number of epitopes per molecule
• a&b
– numbers of antigen and antibody molecules per
complex.
FACTORS INFLUENCING BINDING
• ion species, ionic strength,and polymers.
– The order of inhibition
• Cs+ > Rb+ > NH+4 > K+> Na+ > Li+
• CNS- > NO-3 > I- > Br- > Cl- > F-
• Polymer
– Increases the rate of immune complex growth,
especially, early phase of the reaction.
FACTORS INFLUENCING BINDING
TYPES OF REACTIONS
• The precipitin reaction
• at a solid-liquid interface.
Schematic diagram for precipitin reaction.
Schematic diagram of precipitin curve illustrating zones of antibody excess (A), equivalence (8), and antigen
excess (C). The parameter measured may be quantity of protein precipitated, light scattering, or another
measurable parameter. Antibody concentration is held constant in this example.
Reactions at a Solid-Liquid Interface
• the antigen or antibody of interest is bound to
a solid phase,
– such as a cell membrane, or to a synthetic particle
(polystyrene or cellulose)
• provide lower detection limits
– additional low-avidity antibody binding occurs
QUALITATIVE METHODS
• Passive gel diffusion,
• Immunoelectrophoresis (IEP),
• Western and dot blotting.
• Antigen antibody ratio, salt concentration,and
polymer enhancement have the same
influence on the antigen antibody reaction in
gels as they have on reactions in solution.
• Passive diffusion
– simple diffusion
– double diffusion.
• a concentration gradient is established for both
reactants (antigen and antibody).
• Ouchterlony method.
Double immunodiffusion in two dimensions by the Ouchterlony technique. A,
Reaction of identity: B, reaction of non identity; C, reaction of partial identity; D,
scheme for spur formation. Ag, Antigen; Ab, antibody.
• the position of the precipitin band is in part a
function molecular masses of both antigen
and antibody.
• a negative reaction does not necessarily imply
absence of antibody or antigen.
– too small for the detection limit
– antibody may be nonprecipitating.
Immunoelectrophoresis (IEP)
• for the study of antigen mixtures,
• the evaluation of the specificity of antiserum,
• the evaluation of human gammopathies.
– applied to the evaluation of human myeloma proteins.
• Electrophoretic separation
• Formation of precipitin arcs
– shape and position are characteristic of the individual
separated proteins in the specimen
– By comparison with a known control separated on the
same plate,
Configuration for immunoelectrophoresis. Sample wells are punched in the agar and/or agarose, sample is
applied, and electrophoresis is carried out to separate the proteins in the sample. Antiserum is loaded into the
troughs and the gel incubated in a moist chamber at 4 °C for 24 to 72 hours. Track x represents the shape of the
protein zones after electrophoresis; tracks y and z show the reaction of proteins 5 and 1 with their specific
antisera in troughs C and D. Antiserum against protein 1-6 is present in trough B.
Crossed Immunoelectrophoresis (CRIE) also known as Two-dimensional electrophoresis.Diagram (A) shows the
configuration for the first dimension of CRIE. The segment of the gel denoted by the dashed lines in diagram
(A) is cut out and placed on a second plate. An upper gel containing antibody is added as shown in diagram
(B). Electrophoresis is now carried out at 90° relative to the first dimension run.
Counterimmunoelectrophoresis (CIE)
CIE showing positive reaction between anti-Haemaphilus influenzae B (upper well)
and a cerebrospinal fluid (CSF) sample containing H. influenzae B (Iawer well).
WESTERN BLOTTING
• electrophoresis step followed by transfer of
the separated proteins onto an overlying strip
of nitrocellulose or a nylon
• Using antibodies labeled with probes,
• limits of detection can be 10 to 100 times
lower than by using direct
immunoprecipitation and staining of proteins.
DOT BLOTTING
•
•
•
•
•
•
bypasses the electrophoretic separation step
a small "dot" protein mixture.
Dried
labeled antibody
Washing
detected with a photometric or
chemiluminescent
QUANTITATIVE METHODS
• Gel-based methods
– Radial immunodiffusion(RID)
– Electroimmunoassays
• turbidimetric & nephelometric assays
• labeled immunochemical assays.
RID
• a concentration gradient is established for a
single reactant, usually the antigen. The antibody
is uniformly dispersed in the gel matrix.
• Antigen passively diffuse into the gel until
antibody excess exists and immune precipitation
occurs
• A ring of precipitation around the antigen well
form.
• a calibration curve of ring area or diameter
versus concentration is plotted.
• the ultimate area of the precipitin ring at
equilibrium is dependent on the antigen
antibody concentration ratio.
• RID can be made more sensitive using PEG to
enhance precipitin line formation or by using
125I or enzyme-labeled reagents.
Electroimmunoassay
• as in RID,
• An applied voltage is used to drive the antigen
from the application well into a homogeneous
suspension of antibody in the gel
• a lower limit of detection
• The height of the resulting rocket-shaped
precipitin line is proportional to the antigen
concentration.
Electroimmunoassay
Rocket immunoelectrophoresis of human serum albumin. Patient samples were
applied in duplicate. Standards were placed at opposite ends of the plate.
Electroimmunoassay
• Electroimmunoassay methods produce the
best results with antigens having a strong
anodic mobility and intermediate to low
molecular weight.
• pH 8.6 (the most common pH used for the
method)
TURBIDIMETRIC & NEPHELOMETRIC
ASSAYS
• In many clinical laboratories, gel-based
methods are restricted to qualitative studies.
• Quantitative data are more commonly
obtained by turbidimetric and nephelometric
methods, radioimmunoassays, enzyme
immunoassays (EIAs), and fluorometric
immunoassays.
TURBIDIMETRIC & NEPHELOMETRIC
ASSAYS
• the rate of formation of immune complexes
are measured
• the reaction between antigen and antibody
begins within milliseconds and continues for
hours.
• Rate assays (Kinetic)
– Change (dls/dt) in intensity of scattered light (Is)
within the first few minutes of the reaction
TURBIDIMETRIC & NEPHELOMETRIC
ASSAYS
• Equilibrium(pseudo equilibrium) assays
– wait 30 to 60 minutes
• Improvement
(Turbidimetric & Nephelometric assays)
– addition of water-soluble linear polymers
• increasing the reaction rate
– This allows the use of much lower reactant concentrations
– results in a more stable immune complex suspension.
– Using a latex-enhanced procedure
• for low molecular weight proteins (e.g., myoglobin,MW
17,800), assay detection limits can be lowered, based on
antibody-coated latex beads.
LABELED IMMUNOCHEMICAL ASSAYS
• The previously discussed methods rely on
evaluating the immune complex formation as
an index of antigen antibody reaction.
• the initial binding of the antibody and antigen
has been used with labeled antigens and
antibodies
k1 = the rate constant for the forward reaction
k-1 = the rate constant for the reverse reaction
K = the equilibrium constant for the overall reaction
LABELED IMMUNOCHEMICAL ASSAYS
• the concentration of Ab, Ag, and AbAg will be
dependent on the magnitude of k1 and k-1
• For polyclonal antiserum, the average avidity
of the antibody populations will determine K
(typically 108 to 1010L/mol)
• the magnitude of k1 in comparison with k-1
will determine the ultimate limit of detection
attainable with a given antibody population.
LABELED IMMUNOCHEMICAL ASSAYS
• Radioactive labels
– safe handling and disposal
• Nonisotopic labels
• Methodological Principle
– Competitive & noncompetitive reaction formats
Immunoassay designs. L, Label.
A schematic diagram of the dose-response curve for a typical immunoassay. The
analytically useful portion of the curve is bracketed by points a and b.
• sequential immunoassay has lower detection
limit compared with that of a simultaneous
assay, provided k1 >> k-1
• In noncompetitive assays, the capture and
labeled antibody can be either polyclonal or
monoclonal.
• If monoclonal antibodies having specificity for
distinct epitopes are used, it is possible to
incubate the sample and conjugate
simultaneously with the capture antibody,
thus simplifying the assay protocol.
• Noncompetitive immunoassays can be
performed in either a simultaneous or
sequential mode.
• Hook effect (simultaneous mode)
– a situation can occur in which a high
concentration of analyte can saturate both the
capture and labeled antibodies.
• the assay response drops
– reduces the number of complexes formed and produces a
falsely low result.
• Prone to Hook effect
– Assays for analytes for which the normal
pathological concentration range is very wide
(e.g., hCG, AFP)
• Dilutions
• Sequential assay format
Heterogeneous Vs Homogeneous
lmmunochemical Assays
• Immunochemical assays that require a
separation of the free from the bound label
are termed heterogeneous
• The most widely used of these techniques are
precipitation and liquid phase and solid phase
adsorption.
Homogeneous Assays
• In this type of assay, the activity of the label
attached to the antigen is directly modulated
by antibody binding, with the magnitude of
the modulation being proportional to the
concentration of the antigen or antibody
being measured.
• technically easier and faster
Analytical Detection Limits
•
125I
has low specific activity compared with
enzyme labels and chemiluminescent and
fluorescent labels
• Enzyme labels provide an amplification
– can be improved by
• a chemiluminescent or bioluminescent reaction.
• Fluorescent labels
– background signal from the detector, assay reagents,
and nonspecific binding of the labeled reagent.
• strategies to lower the analytical detection
limits of immunoassays
– biotin-avidin system
• Ag: Ab-biotin: avidin label
– use of streptavidin-thyroglobulin conjugates and
macromolecular complexes of multiplelabeled
thyroglobulin and streptavidin-thyroglobulin
Examples of Labeled Immunoassays
• Radioimmunoassays (RIAs)
– Labels
•
125
I, 131 I & tritium(3H)
– Radiolabeling of antigen
• cause changes in reactivity
• requiring a quantity of purified antigen
– Labeled antibody assays (immunoradiometric
assay)
– Combinations of labels (e.g., 57Co & 125l)
• simultaneous assays
Enzyme Immunoassay
• enzyme-labeled antibodies or antigens (i.e.,
conjugates) are first allowed to react with
ligands, and enzyme substrates are
subsequently added.
• enzyme labels
– Alkaline phosphatase, horseradish peroxidase,
glucose-6-phosphate dehydrogenase, and βgalactosidase
– Enzyme cascade reactions
• two enzymes
Enzyme Immunoassay
• Detection systems
– Photometry
• versatile, reliable, simple to operate, and relatively
inexpensive.
– fluorescent & chemiluminescent measurement
• High sensitivity
Types of enzyme-linked immunoassay
• Enzymel-inked immunosorbent assay (ELISA)
• Enzyme multiplied immunoassay technique
(EMIT)
• CEDIA
ELISA
• Antigens or Antibodies in a sample can be
quantitated
• a heterogeneous ElA technique
• Sandwich complex" of solid phase Ab-Ag-Abenzyme.
• solid phase Ag-Ab-Ab-enzyme.
• substrates that produce visible products
– in screening, point-of-care, and home testing
applications.
Enzyme Multiplied Immunoassay
Technique.
• A homogeneous EIA (does not require a separation step)
• Simple to perform, easily automated
– used to develop a wide variety of drug, hormone,
and metabolite assays
Cloned Enzyme Donor Immunoassay
• Homogeneous EIA
• Competition between antigen and the enzyme
donor antigen conjugate for a fixed amount of
antibody in the presence of the enzyme
acceptor
EA,Enzyme acceptor; ED, enzyme donor.
Enzyme; β-galactosidase
Fluoroimmunoassay
• Limitations of this technique
– background fluorescence
– From drugs, drug metabolites, and protein-bound
substances, such as bilirubin
– Resolution
• use of rare earth (lanthanide) chelates and background
rejection (time-resolved)
• A europium chelate label is excited by a pulse of excitation
light (0.5 µs), and the long-lived fluorescence emission from
the label is measured after a delay (400 to 800µs). By this
time, any short-lived background signal has decayed.
Chemiluminescence Immunoassay
• Light emission produced during a chemical
reaction
• Chemiluminescent labels
– Isoluminol and acridinium esters
• Oxidation of isoluminol by hydrogen peroxide
produces a relatively long-lived light emission at
425 nm
• Oxidation of an acridinium ester by alkaline
hydrogen peroxide produces a rapid flash of light
at 429 nm.
Simplified Immunoassays
• for use in physicians' offices or the home
• Early efforts
– based on agglutination and inhibition of
agglutination using labeled red blood cells or
latex particles in a slide format.
• Sandwich immunoassays
ICON immunoassay device (a, immobilized antibody membrane; b, separating membrane; c, container; d,
adsorbent pad). hCG, Human chorionic gonadotropin; AS, monoclonal antibody to hCG.
Simplified Immunoassays
• Simultaneous multianalyte immunoassays
– more analytes are detected in a single assay
– based on either
• Discrete reaction zones or
• combinations of different labels.
• Discrete reaction zones
– Each test zone consists of antibodies for a specific
(drug) immobilized on the membrane surface.
Simplified Immunoassays
• Combinations of distinguishable labels
– Europium (613 nm, emission lifetime 730 μs) and
samarium (643 nm,emission lifetime 50 μs)
chelates
• Protein Microarrays
– Arrays of hundreds or thousands of micrometersized dots of antigens or antibodies immobilized
on the surface of a glass or plastic chip
• Facilitates simultaneous multianalyte
immunoassays
INTERFERENCES IN IMMUNOASSAYS
• Falsely low results
– Hook effect, at high antigen concentrations
• False negative or false positive results
– sample contains antianimal antibodies.
– To minimize this problem
• Nonimmune serum or IgG from the species used to
raise the antibodies has been a popular choice for this
purpose.
OTHER IMMUNOCHEMICAL
TECHNIQUES
• Cytochemical
• agglutination assays
Immunocytochemistry
• labeled antibody reagents as specific probes
for protein and peptide antigens
– evaluate single cells for synthetic capability
– for identification of various cell lines.
AGGLUTINATION ASSAYS
• qualitative & quantitative measurement of
antigens and antibodies.
• the visible clumping of particulates, such as cells
and latex particles, is used as an indicator
• IgM antibodies are more likely to produce
complete agglutination
– Size & valence
• When IgG
– Chemical enhancement or an antiglobulin
agglutination method.
AGGLUTINATION ASSAYS
• the ratio of antigen and antibody is critical.
– Extremes result in inhibition of aggregation.
• incomplete agglutination
– a weak antigen antibody reaction
– only IgG is involved
• enhancement
– Lowering the ionic strength
– Introducing polymeric molecules, such as
• polymerized albumin (5% to 30%), dextran, polybrene,
polyvinylpyrrolidone, or PEG.
AGGLUTINATION ASSAYS
• Hemagglutination
– the antigen is located on an erythrocyte.
• Erythrocytes
– good passive carriers of antigen
– easily coated with foreign proteins
– can be easily obtained and stored.
• Direct testing
– for blood group
AGGLUTINATION ASSAYS
• indirect or passive hemagglutination
– the erythrocytes are used as a particulate carrier
of foreign antigen (and in some tests of
antibodies)
• Other materials (antigen carriers)
– Latex
• difficult to coat, standardize, and store
AGGLUTINATION ASSAYS
• Hemagglutination inhibition
– the ability of antigens, haptens, or other
substances to specifically inhibit hemagglutination
of sensitized (coated) cells by antibody is
determined.
• In general,
– the agglutination methods are quite sensitive but
are not as quantitative as other immunochemical
methods
• Nonisotopic immunoassays,
– as convenient as agglutination, replacing
agglutination methods