Important terms
antigen - a high molecular weight substance that induce an immune response (a production of antibodies)
- is reversibly bound by its specific antibody (weak, noncovalent intermolecular forces)
antibody - protein that combines specifically with antigen
antigenic determinant - that portion of an antigen involved in a reaction with an antibody
hapten
- a low molecular weight substance that can induce an immune response only when coupled to high
molecular weight immunogenic molecules (proteins)
- can react with the antibodies separately, i.e. free hapten
antiserum - the serum of an animal that contains antibodies to an antigen
- usually contains a mixture of different populations of antibodies to the same antigen (to the multiple
antigenic sites)
affinity - the attraction that a binding protein (Ab) and its ligand (Ag) have for one another
valency - the effective number of antigenic determinants on an antigen molecule, i.e. number of antibody molecules
that can be bound to an antigen at the same time
cross-reactivity - binding of an antibody to an antigen other than the one initiating the immune response
precipitation - a reaction in which antigen and antibody are in the proper ratios so that a large insoluble lattice or
matrix is formed
flocculation - precipitation reaction producing large, loosely bound precipitate
agglutination - clumping or aggregating together by specific antibody of a particle, such as a red blood cell or latex bead to
which the specific antigenic determinant is attached
agglutinin - specific antibody that causes agglutination
Immunoglobulins (Ig)
- glycoproteins with antibody activity
- structure: 2 light chains (L) and 2 heavy chains (H)
- specific domains: structural or functional signification
- light chains: kappa () and lambda ()
- heavy chains: gamma (), alpha (), mu (), delta (), epsilon ()
classes:
subclasses:
IgG
4
IgA
2
IgM
2
IgD
IgE
-
-
* monomers: IgG, IgD, IgE, IgA (+ dimer)
* pentamer: IgM
How to use antigen - antibody reactions
in a laboratory?
* detection of antigens of different microorganisms (serology)
* blood groups determination
* isolation of varied compounds from biological materials (affinity chromatography)
* determination of antibody concentration in body fluids
* determination of concentration of varied proteins
* determination of concentration of hormones, drugs and the other low molecular weight compounds
analyte = antigen
reagent = antibody
advantages:
* high specifity
* low detection limit
* many applications
What is necessary for an immunoassay
performance ?
1) a specific antibody to the analyt to be determined
2) suitable method of complex Ag-Ab detection (indicator phase of immunoassay)  various techniques:
I. methods utilizing of low Ag-Ab complex solubility
- if their concentrations correspond to the zone of equivalence (different sample dilution within a test tubes
or dilution by diffusion within a gel)
* immunoprecipitation - from hours to days
* agglutination - from minutes to hours
II. indicator - labeled immunoassays
- indicator molecules attached to the reagent (radioactive compounds, enzymes, fluorescing molecules)
* high speed and sensitivity
* automatic analyzers
What are we interesting in utilizing
immunoassays?
1) is the analyt present within a sample?
i.e. QUALITY
* gel immunodiffusion
* immunoelectrophoresis
2) what is the amount of an analyt within a sample?
i.e. QUANTITY
* immunoprecipitation in a solution
* radial immunodiffusion
* radio immuno assay (RIA)
* enzyme immuno assay (EIA)
* fluoro immuno assay (FIA)
* lumino immuno assay (LIA)
Qualitative techniques
1) immunodiffusion (Ouchterlony)
- determination of antigen determinans differences
- purity of antigens or antibodies
2) immunoelectrophoresis
- blood serum protein elfo + immunoprecipitation
of the separated proteins and anti-whole human serum
Quantitative techniques
1) immunoprecipitation in a solution
- detection of small aggregates of Ag-Ab complexes by:
a) light-scattering (nephelometry), e.g. determination of IgG, IgA, IgM blood serum concentrations
b) turbidity meassuring (turbidimetry), e.g. determination of circulating immunocomplexes
2) radial immunodiffusion (Mancini)
- diffusion of an antigen to be determined into the gel which contains a specific antibody
- the diameter of precipitate is proportional to concentration
3) indicator - labeled immunoanalysis
- high sensitivity and velocity
- varied methods
generally:
competitive
= binding assays (Ag or Ab is labeled)
noncompetitive = sandwich assays (Ab is labeled)
types of labels:
 high specific activity
* radioisotopes (125I)
* enzymes (peroxidase)
* fluorophores (FITC)
separation techniques:
a) heterogeneous assays - labeled ligand bound by antibody must be physically separated from the free labeled ligand
b) homogeneous assays - do not require physical separation of bound and free lebeled ligand
RIA
(Radio Immuno Assay)
* heterogeneous methods only (there is no changing of radiation intensity between the free and labeled form of ligand)
* competition between the two type of identical compounds:
the antigen to be determined and the labeled antigen of standard concentration added as a reagent
* both Ag and Ag* can react with the specific antibody:
Ag (sample) + Ab + Ag*  Ag-Ab + Ag*-Ab
* determination of low or high molecular weight compounds (e.g. estradiol, thyroxine)
disadvantage: radioactive material
modification: IRMA (Immuno Radio Metric Assay) - „sandwich“
(e.g. determination of thyrotropin, cortisol, C-peptide)
RRA (Radio Receptor Assay)
- determination of biological effective compound or its receptor
- no antibody but a specific receptor is used which reacts with a ligand
- analyte can be added into the cell suspension
advantage:
disadvantage:
directly a biological efficiency is meassured
low stability of isolated receptors
application:
e.g. *determination of estrogen or progesterone receptors within a breast carcinoma
* determination of LH, FST, ACTH
- especially for scientific purposes
EIA
(Enzyme Immuno Assay)
* heterogeneous (both: competitive, noncompetitive) or homogeneous techniques
* low limit of detection because of an amplification of signal (enzyme produces a lot of products)
* disadvantage: possibility of an enzyme inhibition by some compouds occuring in biological samples
(e.g. salicylates)
* detection:
according to the substrate is used (spectrophotometry, nephelometry, fluorimetry)
1) sandwich techniques (i.e. noncompetitive assays)
* antibody is labeled
* microtiter plates or plastic test tubes
* direct proportionality between the enzyme activity and the analyte concentration
a) antigen-measuring system (large Ag, e.g. proteins)
Ab1 (immobilized) - Ag (sample) - Ab2*
b) antibody measuring system (e.g. IgE determination)
Ag (immobilized) - Ab1 (sample) - Ab2*
2) competitive-binding assays
ELISA (Enzyme Linked Immunosorbent Assay)
* heterogeneous system, Ag or Ab labeled
* substrate: chromogen or fluorogen (100 - 1000x lower detection limit, incubation time is shortened)
* inverse proportionality between the enzyme activity and the analyte concentration
* determination of drugs or hormones
EMIT (Enzyme Multiplied Immunoassay Technique)
* homogeneous system, antigen labeled
* binding of Ab to the enzyme-labeled ligand changes the enzymatic activity of the label
* direct proportionality between the enzyme activity and the analyte concentration
* determination of low molecular weight compounds
LIA
(Lumino Immuno Assay)
luminescence = radiation generated by excitated electrones transitions to the ground state
a) fluorescence - a short-time luminescence (it starts during an excitation by excitatory irradiation)
b) phosphorescence - a long-time luminiscence (excitation by excitatory irradiation)
c) chemiluminiscence - excitation by chemical reaction (oxidation)
ILMA (Immuno Lumino Metric Assay)
* noncompetitive technique
* a pair of a specific antibodies is used
* label: isoluminol
FIA
(Fluoro Immuno Assay)
* heterogeneous or homogeneous systems
* competitive or noncompetitive techniques (analogy of EIA)
* the measuring of fluorescence can be influenced by the composition of biological material
* higher sensitivity than a radioactivity measuring
* determination of both low and high molecular weight compounds
label: conjugated or covalently bond fluorochrome
* fluorescein-isothiocyanate (FITC)
* umbelliferone
* chelates of rare-earth metals (Eu, Tb, Sm)
detection: fluorimeter
* exciting radiation
200 - 400 nm (1)
* fluorescence
400 - 700 nm (2)
requirements to labels:
* high extinction coefficient
* large difference between an absorption and emission wavelenghts
FPIA (Fluorescence Polarization Immuno Assay)
* homogeneous, competitive-binding assay, antigen labeled
* based on the amount of polarized fluorescent light detected when the fluorophore label is excited with plane-polarized
light: - small molecules rotate freely  fluorescent light emitted by the molecule is relatively depolarised
- when an antibody binds a low molecular weight labeled ligand the fluorescence polarization is increased
(rotation is much more slower)
* inverse proportionality between the enzyme activity and the analyte concentration
* determination of low molecular weight compounds (e.g. drugs, vitamines, some hormones)
DELFIA
* competitive or noncompetitive techniques
* microtiter plates
* measuring of time-modulating fluorescence of a stable chelates of the lanthanoids (Eu, Tb, Sm)
 stable chelate is change to the fluorescence compound when the antigen binds to antibody (340 nm / 620 nm)
SLFIA (Substrate Labeled Fluorescent Immuno Assay)
* homogeneous competitive technique
* label: fluorogenic enzyme substrate (-galactosyl-umbelliferone)
* when the antibody binds to the labeled antigen, reaction of enzyme with its substrate is stericly prohibited
 no fluorescence
* direct proportionality between the enzyme activity and the analyte concentration