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8 Principles ofImmunochemistry
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high concentration of Ab* can be critical for the effectiveness of the removal system of the labeled not involved in the
Ab-Ag-Ab* complex and, therefore, increase the background signal of the reaction.
The main advantages of the non-competitive architecture
are:
• The high specicity (the Ag is recognized in at least two
epitopes), which allows for distinguishing also macromolecules with a high homology, such as pituitary
hormones
• The high analytical sensitivity due to the direct proportionality between Ag concentration and emitted analytical
signal.
The limitations of non-competitive methods are:
• The fact that it cannot be used for the determination of
small molecules such as haptens. Indeed, the Ag must
have two different epitopes
• Possible underestimation of extremely high analyte concentrations due to the hook effect.
Heterogeneous Phase Methods
Heterogeneous-phase methods are dened as methods in
which the assessment of the amount of Ag-Ab complexes
formed, specically Ag*-Ab complexes, should be performed only after physical separation because the tracer
emitted signal is independent of whether Ag* is bound to
free Ab or not. The process of free-ligand separation
should be as complete as possible. It should not alter the
balance between the forms to not decrease the sensitivity
and precision of the determination. The simplicity and
reproducibility of the free-ligand separation step are fundamental parameters for the quality of an immunometric
method.
Free-Bound Separation Methods
The methods of separation of the free and bound phases have
also undergone considerable evolution. Initially, in competitive methods, carbon was used, which, by adsorbing small
molecules, allowed the separation of free Ag, both labeled
and unlabeled, from those engaged in the complex with the
Ab. Binding selectivity toward Ag of low molecular weight
can be obtained by coating the carbon particles with dextran
of appropriate molecular mass. The coated carbon then acts
as a molecular sieve that adsorbs only molecules whose size
is less than or equal to that of the dextran used for coating; all
molecules with larger sizes are excluded. After centrifugation and removal of the supernatant, the carbon (or carbondextran) is recovered and the analytical signal emitted from
the free form of the marker is measured.
Prior to the introduction of solid-phase separation methods, which are currently the most widely used, the following
techniques were used for non-automated assays.
Non-specic Precipitation
Based on the difference between the molecular size of the
free Ag fraction and the Ag-Ab complex, the latter can be
separated by chemical precipitation. The addition of a critical concentration of the precipitating agent obtains the precipitation. After centrifugation, the Ag-Ab complexes
become insoluble, then the free fraction of the labeled,
remains in the supernatant. The most used precipitating
agents are ammonium sulfate, ethanol, and PEG (polyethylene glycol) of molecular mass 6000 or 8000kDa (PEG 6000
or PEG 8000).
According toAb
They consist of precipitation of the Ag-Ab and Ag*-Ab complexes by an anti-Ig Ab in case of Ag determination. For
example, if the rst Ab is obtained by rabbit immunization, it
is possible to use a second Ab from mutton or goat, directed
against rabbit Ig. Binding with this additional Ab increases
the difference in molecular size of the bound complex with
respect to the free Ag fraction, facilitating its separation by
centrifugation.
Protein A
It is a constituent of the wall of Staphylococcus aureus, and
it possesses a particular afnity for the Fc fragment of human
IgG, except IgG3, and forms insoluble complexes that allow
free fractions to be separated after centrifugation.
The solid-phase separation technique is used, almost
exclusively, for all immunochemical determinations, automated or manual, as it allows to distinguish more effectively
the bound fractions from the free one, eliminating almost
completely the non-specic bonds of the Ag with other molecules and minimizing the analytical background signal. In
addition, solid-phase separation allows:
• To make the separation of free Ag and Ag-Ab complexes
as simple as possible and fully automatable
• Facilitate and make homogeneous the chance of meeting
between the molecules involved in the determination (Ag,
Ag*, and Ab)
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• To realize solid phase that allows the simultaneous determination of several parameters on the same support (biochips) or on different supports (microspheres with a size
of a few microns)
The different types of solid phases used are very numer-
ous and differ in size and in the technology to carry out the
separation between free and bound fraction but especially in
the kinetics of Ag-Ab complex formation. The use of solid
phase consisting of smaller particles, which remain in suspension in the reaction environment, makes more homogeneous the possibility of encountering the molecules that
must react. In contrast, in the other types of solid phases, the
binding agent is immobilized on the wall of the container in
which the reaction takes place. favoring the binding of the
molecules near the edges of the container with respect to
those further away. Chromium dioxide particles, latex microparticles, microparticles or paramagnetic particles, magnetic microparticles, cellulose foam, cones, and test tubes,
microwells, marbles, etc can constitute solid supports. In
technologies involving the determination of more than one
analyte in the same sample reaction environment, the solid
phases used are polystyrene microspheres and ceramic
biochips.
Some methods employ “universal” solid phases that bind
the captured Ab indirectly using the avidin-biotin system. In
this case, the capture reagent is conjugated to biotin, and the
solid support’s surface is coated with avidin. The binding of
the capture reagent to the solid phase is ensured by the
extraordinary afnity of avidin for biotin (very high-afnity
constant). In this case, the same solid phase can be used for
all determinations in which Ab is conjugated with biotin.
Table 8.3 Characteristics of heterogeneous and homogeneous immunochemical methods
Heterogeneous methods Homogeneous methods
More complex execution (need for
separation of free and bound fractions)
Can be automated only by dedicated
systems
High analytical sensitivity
Wide range of measurable
concentrations
Reduced sensitivity to sample matrix
interference
Quick and easy execution
Ease of complete
automation
Higher detectable
concentrations (low
sensitivity)
Narrow range of measurable
concentrations
High sensitivity to sample
matrix interference
procedures, and by the peculiar characteristics of the
tracers:
• In Ab defect determinations, the bond established between
Ab and Ag must lead to the highest degree of inhibition of
signal emission by the tracer. In the case of enzymatic
tracers, e.g., EMIT® (Enzyme Multiplied Immunoassay
Technique) and CEDIA® (Cloned Enzyme Donor
Immunoassay) technologies, it is necessary to carry out
the marking in such a way that the steric conguration
assumed by the Ag-Ab complex can hinder the binding
between the tracer enzyme and its specic substrate
(Fig.8.3);
• In Ab excess determinations, the binding between the two
Abs and the Ag must occur in such a way that the interaction of the Ab-bound tracers is sufciently close for them
to interact effectively (Fig.8.4).
Ag-Ab Complex Formation andEquilibrium
Reaction
Homogeneous-Phase Methods
The homogeneous-phase methods differ from the
heterogeneous- phase methods (Table8.3) in the detection of
Ag-Ab complexes formed in the immunochemical reaction,
which does not require the separation of free and bound fractions: all the molecular forms involved remain in the reaction
environment until the analytical signal is measured. For the
realization of these methods particular tracers, or more correctly tracer systems, which modify their ability to emit a
detectable signal as a function of the involvement in the formation of the Ag-Ab complex must be used.
This technology was initially introduced in the 1980s for
the determination of drugs with automated instruments and,
subsequently, applied for the determination of other analytes, both haptens and proteins. The efciency of this
method is limited by the permanence in the reaction environment of all potentially interfering species and by the heterogeneous-phase methods that can be removed by separation
The immunochemical methods, based on the unique characteristics of selectivity and avidity of the bond between Ab
and Ag, allow the realization of highly specic methods for
quantifying of the analyte, even in complex matrices. The
avidity of the Ag-Ab bond is measured by the afnity constant (K
), which is, to all intents and purposes, a thermody-
aff
namic constant of equilibrium (Keq). An Ab, to be suitable for
the development of immunochemical methods, must be
characterized by a Keq value greater than 109–1012M−1.
The binding reaction between Ag and Ab can be described
by the following equilibrium:
K
+
K
a
Ag Ab
[]
d
d
a
AgAb
eq eq
eq
Ab Ag AgAb
K
K
==
eq
K
the absorbance of
n
Green laser
ab
Bound ligand
of receptors)
[]
[]
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DE
++ ++
Ab Ag analyte
AE
S
Measurement of
the product
λ = 532 nm
Fluorescence
λ = 532 nm
positivity of
Ag-Ab reaction
Red laser
λ = 633 nm
The λ of the
fluorescence allows the
identification of the bead and
hence the relevant analyte
Ag
P
Phycoerythrin
Fig. 8.4 Measurement of Ag or Ab by immunometric methods based
on Luminex® technology. (Copyright EDISES 2021. Reproduced with
permission)
oerythri
Ab capture
Fluorescent bead
Ab analyte
Ag bound
to donor
enzyme
fragment (DE)
Substrate (S) Product (P)
Fig. 8.3 Schematic representation of the principle underlying the
CEDIA® technology: the binding of Ag * to Ab prevents the reassociation of enzymatic fragments (donor enzyme and acceptor
enzyme) and, therefore, the restoration of catalytic activity. The amount
of reactivated enzyme, directly proportional to the measured signal,
increases with increasing concentration of the Ag analyte. (Copyright
EDISES 2021. Reproduced with permission)
Acceptor
Enzyme
Fragment
(AE)
Bound ligand
Free ligand
Bound ligand
Free ligand
where Ka and Kd are the constants of association and dissociation rates of the complex, and Keq is the equilibrium constant of the process. The afnity of the antibody for the
analyte can be determined graphically with the Scatchard
diagram, which is obtained experimentally by incubating
increasing amounts of the analyte with a constant amount of
Ab and measuring, under conditions of equilibrium of the
reaction of formation of the Ag-Ab complex, the concentra-
Fig. 8.5 The Scatchard diagram corresponding to a monoclonal antibody is a straight line where slope=−K
The Scatchard diagram corresponding to a polyclonal antibody, that is
a heterogeneous class of antibodies with different K
it derives from the superposition of different antigen-antibody association processes, each characterized by its own K
calculated for this system
tion of free Ag and Ag bound to the Ab:
AgAb
eq
=−
KK
Ab AgAb
Ag
[]
eq
eq teq
eq
abscissa the concentration of the Ag-Ab complex (bound or
unbound) and in ordinate the ratio between the concentrations of free and bound Ag (bound/free) (Fig.8.5).
The knowledge of the characteristics of the Ab used in an
where Abt=[Ab]eq+[AgAb]eq is the total concentration of
Ab. The Scatchard diagram is obtained by representing in
immunochemical determination is essential to assess the
sensitivity of the method and, consequently, the range of
Bound ligand
High
affinity
Slope = –K
Low
Affinity
eq
eq
Max bound
ligand (total
no.
Ab
; intercept on the x axis=Ab.
, is curvilinear, as
eq
. An apparent K can be
eq
72
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1.0
0.8
0.6
0.4
bound antigen
0.2
–11
10
K = 1011 L mol
Abt = 10
K = 1010 L mol
Abt = 10
Fig. 8.6 Theoretical bond curves for a competitive immunoassay
method. A reduction in Ab shifts the curve to lower analyte concentrations, but at the expense of sensitivity (comparison between green and
blue curves), while an increase in K increases sensitivity (comparison
between blue and red curves)
–10
10
Concentration of antigen
in the sample. [An] (M)
–10
M
–9
M
10
–1
–1
–9
K = 1010 L mol
Abt = 10
–10
10
–8
M
–7
10
–1
analyte concentrations that can be measured. For example, in
a competitive method, the same Ab used at lower concentrations may allow lower concentrations of analyte to be
detected (increased sensitivity), or an Ab with a greater afnity constant than another at the same concentration gives a
higher analytical signal at the same Ag concentration
(Fig.8.6). The theoretical denition of the characteristics of
an Ab is fundamental for predicting, in principle, the analytical performance of an immunochemical method. However,
the real performance obtained is the result of all the factors
involved in carrying out the assay, both in terms of the
reagents used (Ab, tracer, labeled, etc.) and the physical conditions of the reaction (time, temperature, matrix), as well as
instrumental (level of automation, separation of free and
bound phases, etc.).
Table 8.4
a tracer
Analytical principle Method
Agglutination Hemagglutination
Precipitation Ouchterlony double immunodiffusion
Immunosensors Resonant mirror biosensor (IAsysTM)
Classication of immunochemical methods that do not use
Latex agglutination
Rocket immunoelectrophoresis
Immunoturbidimetry/nephelometry
Surface plasmon resonance (BIAcoreTM)
forms are present. Therefore, by using a constant concentration of Ab as a reagent, it is possible to detect a specic Ag
and, vice versa, by using an Ag, it is possible to detect the
presence of a specic Ab. If cells or very small particles are
coated with the reagent (Ab or Ag), their binding with Ag or
Ab in the sample can cause precipitation or agglutination,
even at very low concentrations. Many methods still in use
are based on the simple principle of precipitation. However,
the most relevant for their application are turbidimetry and
nephelometry. The main limitation of these methods is the
reduced sensitivity and, in some cases. Additionally, an
extremely high concentration of analyte can inhibit the formation of the complex.
Other label-free immunochemical methods are based on
biosensors. Currently, and there are several commercial systems available that allow direct detection of Ab-Ag
interaction.
Many of these are based on the generation of an evanescent wave from the surface of a sensor on which the ligand is
immobilized, with the possibility of continuously monitoring the kinetics of Ag-Ab binding. These technologies, with
the availability of specic low-cost instrumentation, will
allow in the future a widespread application of immunochemical methods.
Turbidimetry andNephelometry
Immunochemical Methods Without Tracer
In label-free immunochemical determinations, the formation
of the Ag-Ab complex can be detected directly, without
resorting either to the separation of unbound Ag or Ab forms
or to the measurement of the analytical signal emitted by a
special tracer (Table8.4).
In these methods, if the concentration is sufciently high,
the complexes formed as a result of the Ag-Ab binding are
detectable by the naked eye, or lower concentrations can be
quantied by exploiting their ability to deect a light beam.
The formation of the complex indicates that both molecular
Turbidimetry and nephelometry are the currently most
widely used immunochemical techniques that do not require
the use of a tracer.
These techniques are based on the evaluation of scattered
light by a heterogeneous system, such as a colloidal solution
or a suspension. Two limiting cases can be presented:
• The light is absorbed to a considerable extent so that the
intensity of the emerging beam is particularly attenuated.
• The light is considerably diffused by the suspended par-
ticles by a series of phenomena of reection and refrac-
tion of the suspended particles (Tyndall effect), originating
the so-called opalescent light.
()−()
51
LIN
LOG
Abscissa: DOSE
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The two phenomena always coexist, but their intensity is
always quite different. When absorption prevails over diffusion, it is preferred to evaluate the entity of the absorption by
the dispersed phase: in this sense, a turbidimetric measurement is carried out. In turbidimetry, the intensity of light transmitted by a suspension is measured: normal colorimeters or
UV-VIS spectrophotometers can be used to measure the transmittance or absorbance percentage.
When, on the other hand, the scattering phenomenon is
much more intense than the previous one, in the case of
excellent ne dispersed phase, then the evaluation of the
light scattered by the suspension at 90°, with respect to that
of the incident radiation, is carried out by performing a nephelometric measurement. Therefore, the detection system
must be positioned at 90° to the beam entering the sample
cell.
Turbidimetry is applied when the size of the particles
causing turbidity is of the order of a micrometer or more, a
condition in which absorption prevails over diffusion. When
dealing with smaller particles, tens or hundreds of nanometers, the diffusive effect prevails and, therefore, nephelometry is used. Nephelometric measurements are very sensitive,
and their accuracy is conditioned by many factors, such as
the pH of the medium, and the presence of interfering substances or that can adsorb on colloidal particles of the
suspension.
LOGIT
1 2
LIN LOG
Ordinate: cpm BOUND
Fig. 8.7 Graphic description of the “concentration and analytical signal” relationship and modication of the graph obtained by transforming the scale: (1) linear-linear; (2) linear-logarithm; (3) logit-logarithm
LIN
3
According to the way of expression of the marker, the
shape and the course of the curves describing the relation
between concentration and analytical signal are modied.
Given the curvilinear course of the function, which is
obtained in any case, in order to “stretch” the curve more and
make the function as linear as possible, at least in the central
part, even the representation of the analyte concentration can
be transformed from a linear scale to a logarithmic one
(Fig.8.7). This expedient proved to be indispensable until
the early 1980s when, as electronic processors were not yet
available, the only method available for calculating the
results of an assay was manual graphic representation.
Relationship Between Analytical Signal
andConcentration
For immunochemical determinations, the description of
the relationship between analyte concentration and measured signal (calibration curve), achieved by measuring
samples of known concentration (calibrators), is quite
complex as it is not described by a linear function and,
even if it does not involve analytical steps, it has a decisive influence on the precision and accuracy of the
measurement.
The data processing process aims to dene a mathematical function that can interpolate, all the points relating to the
calibrators’ measurements in best possible way. The expression of the analytical signal is rst referred to as the measured labeled fraction: free (F) or bound (B). Or through
transformations in direct ratios or referred to as the total signal (T) of the marker used in the assay: B/F, B/T, F/B, 1/B,
1/F, T/B, T/F. In other cases, indirect transformations are
used, which refer to the measured signal for the fraction B to
the signal obtained in the reaction system in the absence of
unlabeled analyte (B0): B/B0, B0/B and logit(B/B0), where
the following expression denes the logit function:
logit YYY
ln /
Interpolation andRegression Methods
The techniques used to search for mathematical functions
that correctly describe the relationship between concentration and analytical signal, can be grouped into two
categories:
• Interpolation
• Regression
With interpolation, the experimental points obtained by
the measurement of the calibrators are joined with a broken
line, being heavily affected by possible random errors. The
regression technique tends to identify a mathematical model
able to describe in a continuous way the relationship between
concentration and signal, approximating the experimental
points and mediating eventual measurement errors.
In the case of both interpolation and regression methods,
several approaches have been developed, ranging from simple empirical observation of experimental data to models,
which take into account the physicochemical aspects of the
interactions between the species involved in the immunological reaction. According to these criteria, methods for
describing the concentration–signal relationship can be
grouped into the following categories (Table8.5):
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Table 8.5
between concentration and analytical signal
Empirical methods
Manual graph
Linear interpolation
Polynomial expression
Approximate rational methods
Logit-log
4-parameter logistics
5 or more parameters logistics
Exact rational methods
Scatchard
Sips
Ekins
Feldman
Fernandez-Loeb
Rodbard
Wilkins
Classication of methods for processing the relationship
• Empirical methods
• Approximate rational methods
• Exact rational methods
The empirical methods include all the interpolation and
regression techniques, more or less simple, which can
describe the trend of the concentration–signal relation for
several immunochemical assays. The choice among the various possibilities must be based on the relationship between
the curve t the experimental points and the “logical” trend
of the relation, avoiding excessive degrees of curvature.
Rational methods tend to describe the experimental reality
based essentially on the law of mass action that regulates the
reaction between Ag and Ab. However, in order to make the
data processing and the implementation of the calculation
algorithms easier, some simplications have been introduced
that consistently move away from the experimental reality. In
particular, in many of the applied models, it is considered that:
• The kinetics of the reaction are of rst order.
• There is only one type of binding between Ag and Ab, i.e.,
there is no heterogeneity of epitopes and binding sites.
• The equilibrium constants for binding of the marker and
analyte to the capture system are identical, i.e., have the
same afnity.
• The signal measurement is carried out when thermody-
namic equilibrium is reached.
• The separation of the signal from labeled bound is abso-
lute in comparison to the free signal.
• In competitive assays, the labeled molecular weight shall
be identical to that of the analyte.
• The mass of the tracer is known.
• The signal due to non-specic binding of the labeled is
correctly measured.
• The signal due to non-labeled-specic binding is identical
in each sample measured.
Hardly all these conditions are completely reached in an
immunochemical determination. The exact rational methods,
in addition to the achievement of a good denition of the
concentration-signal curve, allow the determination of the
main parameters of the immunological reaction, such as the
equilibrium constant, the concentration of the binding sites,
the concentration of the labeled, the magnitude of the signal
the Ab, etc.
The use of electronic processors, facilitating the elaboration of complex algorithms, has allowed for intervention on
fundamental aspects of the experimental by the introduction
of statistic criteria, such as the attribution of different weight
factors in function of the concentration because the properties of the analytical system confer variable precision characteristics in relation to the quantity of analyte measured and
the possibility of exclusion from the procedure of extrapolation of the curve of points that suffer from random experimental errors (aberrant).
Since there is no absolute criterion to identify a data processing method with universal validity, the choice between
empirical and rational models must be made according to the
characteristics of the analyte and the properties of the binding system (Ab). This choice signicantly inuences the
quality of the results obtained by immunochemical determination, as different algorithms may associate different analyte concentrations with the same signal value. The
introduction of fully automated systems for these determinations has greatly limited or eliminated, the possibility for
operators to choose the interpolation algorithm based on the
characteristics of the reagents.
In almost all the completely automated systems for carrying out immunoassays, in addition to limiting the choice of
the interpolation algorithm of the relationship between analytical signal and concentration, the direct measurement of
all calibrators is not carried out, but the so-called recalibration is applied: only two calibrators are measured in
the laboratory (usually with low and high analyte concentration) to assess any variation of the signal obtained by the
instrument in use compared to that of a full calibration curve,
obtained from the manufacturer by averaging the signal of
repeated measurements of an appropriate set of calibrators
performed on several analyzers.
Interferences inImmunochemical Methods
For all the phases of an immunochemical determination, the
analytical conditions must be carefully studied and monitored, starting from the collection of the sample up to the
measurement of the analytical signal, to determine the concentration of the analyte to be measured.
8 Principles ofImmunochemistry
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75
Immunochemical methods are subject to interference
both in forming the Ag-Ab complex and in the detection
phase of the analytical signal. The leading causes are related
to the presence of molecules in the sample that can bind nonspecically the Ab (cross reaction), or interfere in the formation of the Ag-Ab complex (matrix effect), or that compete
with the Ab for Ag binding (e.g., auto-Ab and heterophilic
Ab).
Particular attention must be paid to the chemical–physical
conditions under which the immunochemical reaction takes
place. The architecture of these methods, particularly competitive ones, is based on the identical probability of Ag and
Ag* being able to be bound by the Ab: any alteration of the
conditions leading to a departure from the equilibrium leads
to an alteration of the results.
Recommended Readings
Adison GM, Hales CN (1971) The immunoradiometric assay. In:
Kirkham KE, Hunter WM (eds) Radioimmunoassay methods.
Churchill Livingstone, Edinburgh, pp447–461
Bazin H, Preaudat M, Trinquet E et al (2001) Homogeneous time
resolved uorescence resonance energy transfer using rare earth
cryptates as a tool for probing molecular interactions in biology.
Spectrochim Acta A 57:2197–2211
Berson SA, Yalow RS (2006) General principles of immunoassays. Clin
Chim Acta 369:125–143
Boscato LM, Stuart MC (1986) Incidence and specicity of interfer-
ence in two-site immunoassays. Clin Chem 32:1491–1495
Capolaghi B, Truchaud A (2004) L’Immunoanalyse automatisée au
début du XXI siècle. Spectra Biol 141:55–65
Cohen R. Cours immunodosages, DES biologie medicale, module de
biochimie, faculté de pharmacie. Université Claude Bernard, Lyon
Dudley RA, Edwards P, Eklns RP etal (1985) Guidelines for immuno-
assay data processing. Clin Chem 31:1264–1271
Ekins RP (1998) Ligand assays: from electrophoresis to miniaturized
microarrays. Clin Chem 44:2015–2030
Ekins RP, Newman GN, O’riordan JLH (1970) Saturation assays. In:
Mc Arthur JS, Colton T (eds) Statistics in endocrinology. MIT
Press, pp345–392
Gao Y, Yuan Z, Yu Y etal (2007) Mutual interference between serum
thyroglobulin and antithyroglobulin antibody in an automated che-
miluminescent assay. Clin Biochem 40:735–738
Gosling JP (2000) Immunoassays. Oxford University Press, NewYork
Henderson DR, Friedman SB, Harris JD etal (1986) CE-DIA®. A new
homogeneous immunoassay system. Clin Chem 32:1637–1641
Kohler G, Milstein C (1975) Continuous cultures of fused cells secret-
ing antibodies of predened specicity. Nature 256:495–497
Le analisi immunometriche: basi teoriche e applicazioni cliniche.
Padova: Piccin, 2014
Lequin RM (2005) Enzyme immunoassay (EIA)/enzyme linked immu-
nosorbent assay (ELISA). Clin Chem 51:2415–2418
Long DA (2002) The Raman effect: a unied treatment of the theory of
Raman scattering by molecules. Wiley, NewYork
Malvano R, Giraudi G (1998) L’immunochimica analitica. Ed. DiaSorin
Mathis G, Socquet F, Viguier M etal (1997) Homogeneous immuno-
assays using rare earth cryptates and lime resolved uorescence:
principles and specic advantages for tumor markers. Anticancer
Res 17:3011–3014
McCreery RL (2000) Raman spectroscopy for chemical analysis. Ed.
Wiley-Interscience, NewYork
Miles LEM (1977) Immunoradiometric assay (IRMA) and two-site
IRMA systems. In: Abraham G (ed) Handbook of radioimmunoas-
say. Macel Dekker, NewYork, pp131–177
Miles LEM, Hales CN (1968) Labelled antibodies and immunological
assay systems. Nature 219:186–189
Miles LEM, Hales CN (1970) Immunoradiometric assay procedures:
new developments. In: In vitro procedures in medicine. IAEA,
Vienna, pp483–489
Moalic V, Mercier B, Ferec C (2004) Technologie Luminex: principe,
applications et perspectives. Immunoanal Biol Spec 19:181–187
Price CP, Newman DJ (1997) Principles and practice of immunoassay.
Macmillan Press, London, pp443–480
Purvis DR, Pollard-Knight D, Lowe CR (1997) Principles and practice
of immunoassay. Macmillan Press, London, p511
Robbins J, Rall JE (1960) Proteins associated with the thyroid hor-
mones. Physiol Rev 40:415–489
Romano L, Marseglia S (1986) Aspetti termodinamici del dosaggio
RIA.Ed. Radim
Romano L, Zofra S, Giglio G (1995) Guida pratica agli immunodos-
aggi. Ed. Poli industria chimica
Rubenstein KE, Schneider RS, Ullmann EF (1972) Homogeneous
enzyme immunoassay. A new immunochemical technique. Biochem
Biophys Res Commun 47:846–851
Ullman EF, Kirakossian H, Switchenko AC etal (1996) Luminescent
oxygen channelling assay (LOCITM): sensitive, broadly applicable
homogeneous immunoassay method. Clin Chem 42:1518–1526
Walker WHC, Keane PM (1977) TheoriÃÄtical aspects of radioim-
munoassay. In: Abraham G (ed) Handbook of radioimmunoassay.
Macel Dekker, NewYork, pp87–130
Wide L (1983) Non competitive versus competitive binding assay. In:
Odell WD, Franchimont P (eds) Principles of competitive protein-
binding assay. Wiley, NewYork, pp243–254
Wu JT (2000) Quantitative immunoassay: a practical guide for assay
establishment, troubleshooting, and clinical application. AACC
Press
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Tests andBiomarkers
GiuseppeLippi, ElisaDanese, andMartinaMontagnana
9
Introduction toLaboratory Tests
andBiomarkers
The term “laboratory test” conventionally refers to a medical
procedure consisting of the analysis of a biological uid,
such as blood, urine, or other biological matrices, as
described in Table9.1. Thus, the laboratory examination is
not necessarily aimed at the measurement of a substance but
rather at the quantication of a biological signal. Laboratory
tests have become essential in the screening, diagnosis, prediction, and monitoring (including therapeutic) of most
diseases.
According to the Biomarker Denition Working Group of
the National Institute of Health (NIH) of the United States of
America, a biomarker (or “biological marker”) is instead a
characteristic that can be directly determined (measured) and
used as an indicator of (i) normal biological processes; (ii)
pathological processes; (iii) response to any therapeutic
intervention.
In general, although it is not obvious how the two denitions differ, it is preferred to use the term “laboratory test” to
identify any analysis aimed at determining a biological signal in a matrix (regardless of the type of measurement). For
example, the prothrombin time (a coagulation test) measures
the overall clotting time of a plasma sample but does not
provide direct information on the concentration of individual
clotting factors that contribute to the coagulation process.
The term “biomarker” is conventionally reserved to identify
a directly quantiable molecule in the biological sample
(e.g., a biochemical parameter, a protein, and an enzyme). In
a literal sense, prothrombin time is, therefore, a laboratory
test, but it cannot be categorized as a biomarker. On the contrary, cardiac troponins are determined directly in serum or
plasma (by monoclonal antibodies) to diagnose cardiac dam-
G. Lippi (*) · E. Danese · M. Montagnana
Section of Clinical Biochemistry, University Hospital of Verona,
Verona, Italy
e-mail: giuseppe.lippi@univr.it
Table 9.1
medicine
Blood (whole, plasma, or serum)
Urine
Feces
Saliva
Sweat
Tears
Nasal secretion
Sputum
Pus, exudates, transudate
Cavity uids (physiologically or pathologically present)
Cerebrospinal uid
Pleural uid
Pericardial uid
Synovial uid
Bile
Stones
Liquids extracted from cysts and other neoformations
Skin and skin appendages
age and are, therefore, to all intents and purposes, both laboratory tests and biomarkers.
nite, as evidenced by the evolution of their discovery, characterized by an almost exponential growth over the years.
Nevertheless, of the several thousand biomarkers that are
discovered each year, only a minority (an estimated ~1 out of
5000) enter clinical practice. This is due to a particularly
long (it can last 5–10 years, or even longer) and complex
process (“biomarker pipeline”), ranging from discovery to
commercialization and requiring a series of intermediate
steps, including verication, preclinical validation, clinical
validation, development of commercial methods, clinicalanalytical validation of commercial methods, introduction
into guidelines or recommendations, and clinical use
(Fig.9.1).
human, and organizational) in preparation for the introduction of new biomarkers in clinical practice and the unfortunately very frequent nding of the disappearance of many
Biological matrices commonly analyzed in laboratory
The number of potential biomarkers is now almost in-
This largely justies the signicant efforts (economic,
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. Ciaccio (ed.), Clinical and Laboratory Medicine Textbook, https://doi.org/10.1007/978-3-031-24958-7_9
77
78
5000
1
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G. Lippi et al.
Discovery
Verification
Preclinical
validation
Clinical
validation
Marketing
Clinical-analytical
validation
Guidelines
Clinical
practice
Fig. 9.1 Evolution of biomarkers, from discovery to clinical use.
(Copyright EDISES 2021. Reproduced with permission)
biomarkers during the process of translation from the laboratory bench to the patient’s bedside (“loss in translation
between bench and bedside”).
The Ideal Characteristics ofaBiomarker
According to the NIH denition, the determination of a biomarker is nowadays essential for screening, diagnosis, prognosis, and monitoring of most (if not all) human diseases. Thus, it
is evident that the biological characteristics and the analytical
techniques used for its determination substantially inuence
the diagnostic performance in each of the potential elds.
In general, the ideal characteristics of a biomarker are
summarized in Table9.2.
The rst essential feature is the presence of high concentrations in the target organ/tissue. This is related to the fact
that if the biomarker is present in a low concentration in the
target organ/tissue, its increase in circulation (or in biological uids) following a pathology/organ damage will not be
detectable by simple, rapid, and inexpensive techniques. The
lower the concentration of the biomarker in the circulation
(or biological uids), the greater the analytical complications
for its determination, resulting in the concept of low diagnostic “sensitivity” (many false negatives). In other words, if the
amount of biomarker contained in the damaged tissue is
small, its concentration in the biological uid under
Table 9.2
Present in high concentrations in the organ/tissue target
Absent in non-target organs/tissues
Absent in the biological uids of healthy subjects
Rapidly released into the biological uid as a result of pathology
Persist in the biological liquid for a period sufcient to ensure an
adequate diagnostic window
Show increases in biological uid related to the severity of the
damage and prognosis
Measurable in the biological liquid simply, quickly, and
economically
Ideal characteristics of a biomarker
investigation will be equally modest, thus making its measurement quite complex.
The second ideal feature, a logical consequence of the
rst, is that the biomarker should be absent in non-target
organs/tissues. The higher the concentration of the biomarker
in other organs/tissues, the lower the possibility of correlating its increase to the damage to the target organ/tissue,
which results in the concept of low diagnostic “specicity”
(many false positives). Typical examples are the virtually
ubiquitous enzymes, such as lactate dehydrogenase (LDH)
and gamma-glutamyltransferase (GGT), whose blood concentrations increase following cellular damage in a myriad
of organs and tissues. Their increase is certainly a sign of
cellular damage but understanding their origin and cause
(especially in the absence of specic clinical signs or symptoms) is virtually impossible.
The third ideal characteristic is also substantially consequential to the rst and refers to the opportunity for the biomarker to be absent in the blood or other biological uids of
presumably healthy subjects. This aspect appears to be of
substantial importance in optimizing the diagnostic sensitivity of the biomarker. An example is presented in Fig.9.2 that
shows the comparison between two hypothetical biomarkers
of liver damage: biomarker “A,” which is already physiologically present in the circulation under normal conditions, and
biomarker “B,” which is not normally present in the circulation under physiological conditions. In the rst case, a reference range is available for the biomarker concentration
(since biomarker “A” is present in the circulation also in
healthy subjects), while in the second case, a diagnostic
threshold is used corresponding to the analytical sensitivity
limit of the method (since biomarker “B” is absent in the
circulation and therefore usually not measurable in healthy
subjects). Following organ damage, both biomarkers are
released from the target tissue and enter the circulation. The
concentration of biomarker “A” will progressively add to
that already present in the circulation, and an unavoidable
period must elapse before the concentration exceeds the
upper limit of the reference range. In contrast, the concentration of biomarker “B” will almost immediately exceed the
diagnostic threshold since its concentration in circulation is
virtually undetectable in healthy subjects.
Hours
Biomarker concentration
10
Hours
12
Hours
Biomarker concentration
9 General Information onLaboratory Tests andBiomarkers
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Fig. 9.2 Comparison of the
diagnostic performance of
biomarkers physiologically
present in the circulation
(“A”), or whose concentration
is not detectable in the
circulation in the absence of
organ damage (“B”).
(Copyright EDISES 2021.
Reproduced with permission)
16
14
12
10
8
6
4
2
0
012345678910
16
14
«A» «B»
12
10
8
6
4
2
Biomarker concentration
0
0123456789
79
Fig. 9.3 Kinetics of lactic
acid (lactate), cardiac
troponin, and lactate
dehydrogenase (LDH)
following acute myocardial
infarction in relation to the
relative diagnostic window.
(Copyright EDISES 2021.
Reproduced with permission)
A fourth essential characteristic of the biomarker is its
rapid release into the circulation (or biological uids) following organ/tissue pathology. Obviously, this characteristic
is always related to the timeliness of the request to reach a
diagnosis and establish a therapy. As an example, the diagnostic timeliness translates into minutes (maximum hours) in
pathologies in which an urgent intervention is required, as in
the case of acute myocardial infarction or cerebral ischemia,
in which the precocity of the therapeutic intervention cannot
be delayed beyond 3–4hours. In the case of the diagnosis of
malignant neoplastic diseases, instead, the concept of precocity expands over days (or weeks) because the progression
of the disease is much slower, and interventions are less radical. It seems, therefore, evident that the kinetics of biomarker
release becomes the foundational element of diagnostic earliness, as long as the kinetics is not too rapid. This is illustrated in Fig.9.3, which shows the kinetics of three potential
Lactate Troponin
LDH
Measurement
012345678910 11
Cut-off
Diagnostic window
biomarkers of cardiac damage in relation to an acute event
(e.g., acute myocardial infarction) manifested at point 0. The
effective recognition of this pathology (in terms of timely
and efcient therapeutic management) requires a diagnostic
window between 30 minutes and 3 hours from the event,
depending on the fact that patients arrive at the emergency
room after an average time of about 1hour from the event,
and the need to intervene to manage the most severe forms of
infarction no later than 3hours from the onset of symptoms.
The concentration of the rst biomarker (lactic acid)
increases in the circulation immediately after the acute
infarction, but it is often so early that when the patient arrives
at the emergency room to perform diagnostic tests, its concentration may have already fallen within the reference interval (the half-life of lactic acid is about 10–15minutes). The
third test, LDH, demonstrates the opposite kinetics, with
release into the circulation very late with respect to the acute
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