2. 110 The Immunoassay Handbook
where [Ab]=antibody concentration and K=the afﬁnity
constant governing the reaction.
Clearly F also depends on the values of K and [Ab]. Thus,
in conventional immunoassay designs it is imperative that
the total amount of antibody, the individual sample volume,
the total incubation volume, and K are identical in all the
incubation tubes. Assuming this is the case, the only vari-
able quantity in the system is the amount of analyte present
in the test sample. Following measurement of the ﬁnal ana-
lyte concentration in the incubation mixture (by reference
to the known concentrations present in a set of standards),
the amount of analyte in the sample is determined, and—
since the original sample volume is known—the unknown
analyte concentration in the sample may be derived.
In general, reduction of the amount of antibody used in
the system results in increased antibody occupancy (assum-
ing other factors remain constant). This conclusion is illus-
trated in Fig. 3, in which F is plotted against antibody
concentration (assuming the presence in incubation mix-
tures of the analyte concentrations shown).3 However, as
the amount of antibody (and hence the antibody concentra-
tion) employed approach 0, F tends to [An]/(1/K+[An]).
Thus, when the antibody concentration is low (less than
approximately 0.01/K–0.05/K) antibody fractional occu-
pancy is dependent only on K and the analyte concentration
in the medium to which the antibody is exposed, being
3 Note that antibody and analyte concentrations are expressed in units of 1/K, thereby generalizing the ﬁgure.
FIGURE 1 The “antibody binding site occupancy principle” of
immunoassay. All immunoassays implicitly rely on the measurement of
the fractional antibody binding site occupancy by analyte.
FIGURE 2 Immunoassays differ in the way fractional occupancy of antibody binding sites is determined. So-called “competitive” assays (left) rely on
indirect measurement of occupancy, by observation of unoccupied sites. Non-competitive or immunometric assays (right) rely on direct measurement of
binding site occupancy. Note that labeled antibody assays may be either of competitive or non-competitive design.
3. 111CHAPTER 2.5 Ambient Analyte Assay
independent both of the volume of the medium and of the
amount of antibody present.
In other words, when a small amount of antibody cou-
pled to a solid support is exposed to analyte-containing ﬂuid
of such volume that the resulting antibody concentration
is less than approximately 0.05/K,4 the fractional occu-
pancy of antibody binding sites solely reﬂects the preexist-
ing ambient analyte concentration per se (see Fig. 4), the
total amount of analyte in the test sample being irrelevant.
Analyte binding by antibody inevitably causes analyte
depletion in the medium, but because the amount so
bound is small, the reduction in the ambient analyte con-
centration caused by the introduction of antibody is insig-
niﬁcant. For example, if the sensor-antibody binding site
concentration is less than 0.01/K, the reduction in the
analyte concentration is less than 1% (irrespective of the
concentration’s magnitude) (see Fig. 3).
A close similarity exists between these concepts and the
operation of a simple mercury thermometer. A small
thermometer—when placed in a liquid in a container—
extracts heat from the liquid until it reaches thermal equilib-
rium, at which point the liquid’s temperature is indicated.
Provided the thermometer’s thermal capacity is insigniﬁcant
compared with that of the liquid, the indicated temperature
is independent both of the thermometer’s size and the vol-
ume of liquid in which it has been immersed. However, the
4 When antibody is coupled to a solid support, its concentration in the system is
given by the number of effective antibody binding sites divided by the volume of
ﬂuid to which the antibody is exposed. In other words, the location of antibody
within the medium is irrelevant under equilibrium conditions. Only the kinetics of
the binding reactions are affected when the antibody is restricted to a particular
zone or compartment, such as by its location on a solid surface (assuming the
absence of secondary effects, such as antibody denaturation).
introduction of a large thermometer of high thermal capac-
ity into the liquid will alter the latter’s temperature. The
ﬁnal recorded value will therefore depend both on the ther-
mometer’s size and the liquid volume (Fig. 5).
In these circumstances the only practical way of deter-
mining the initial temperature of liquid samples is by
ensuring that their volumes are standardized, that an iden-
tical thermometer (at the same initial temperature) is used,
and that the system is calibrated using water standards of
identical volume. By these means, a calibration curve may
be drawn relating the true (initial) temperature of water
samples to the ﬁnal, observed, temperature.
Such a procedure is closely analogous to the steps
adopted in conventional immunoassay protocols.
Applications of the Ambient
Analyte Assay Principle
Two of the most important practical applications of the
ambient analyte principle are “microspot” assays (leading to
the construction of multianalyte microarrays) and free hor-
mone immunoassays. (Note that the latter were conceived of
and developed before it was later recognized that they con-
stituted a particular example of a more general principle.)
These two areas of application each involve different addi-
tional concepts and are therefore best examined separately.
As indicated above, the ambient analyte assay conditions
prevail if an amount of “sensing” or “capture” antibody is
located on a solid support and exposed to an analyte-
containing medium of sufﬁcient volume that the antibody
concentration is less than approximately 0.05/K (or, prefer-
ably, less than about 0.01/K). Moreover, the antibody can be
conﬁned within a “microspot” such that the total number
of antibody sites is ideally less than v/K×10−5×N (where
v = sample volume (mL) and N = Avogadro’s number
(6 × 1023)). Thus if v = 1 and K = 1011 L/M, the number of
binding sites causing negligible disturbance (<1%) to the
ambient analyte concentration is 6×107 (see Fig. 6). Follow-
ing the microspot’s exposure to an analyte-containing sam-
ple, occupancy of antibody within the spot may be
determined by its exposure to a second (labeled) “develop-
ing” antibody reactive with either occupied or unoccupied
sites, these being equivalent to the non-competitive and
competitive measurement strategies adopted in conven-
tional immunoassays (Fig. 7).
Since ambient analyte assays rely on measurement of the
fractional occupancy of the sensor antibody (regardless of
the exact amount of the latter present), a useful stratagem
in this context is to label the sensor antibody itself with a
second label and to observe the ratio of signals emitted by
the two labeled antibodies5 (Fig. 8). Fluorescent labels are
5 Measurement of the ratio of the two signals obviates (inter alia) problems arising
from ﬂuctuations in the intensity of the exciting light source, variations in coating
density of the sensing antibody (or nucleotide) on the solid support, etc. Other
advantages of the use of two labels are that it enables full quality control of
manufactured microspot arrays and—if necessary—exact positioning of the signal
detector (e.g., confocal microscope) over each microspot in the array.
FIGURE 3 Fractional antibody binding site occupancy (F) plotted as a
function of antibody binding site concentration for different values of
analyte (antigen) concentration. (All concentrations are expressed in
multiples of 1/K, thereby generalizing the curves.) Note that for
antibody concentrations <0.01/K (approximately), percentage binding
of analyte is <1% for all analyte concentrations, and F is essentially
unaffected by differences in antibody concentration, being governed
solely by the analyte concentration (ambient analyte immunoassay
(AAI)). Note that radioimmunoassays and other “competitive”
immunoassays conventionally rely on antibody concentrations
approximating 0.5/K–1/K or above (implying binding of labeled analyte
alone (B0) of at least 33%). Non-competitive (immunometric) assays
are generally based on the use of considerably higher antibody
4. 112 The Immunoassay Handbook
FIGURE 4 If a probe bearing a small number of antibody molecules on its surface is placed in an analyte-containing medium of sufﬁcient volume
that ambient analyte conditions are fulﬁlled, then the fractional occupancy of antibody binding sites will be unaffected either by variations in the
volume of the medium or in the number of antibody molecules on the probe.
FIGURE 5 The thermometer analogy. A (cold) thermometer absorbs heat until reaching thermal equilibrium with its surroundings. The temperature
it records will not differ from the original ambient temperature if the amount of heat absorbed is an insigniﬁcant fraction of the total in the system.
5. 113CHAPTER 2.5 Ambient Analyte Assay
particularly advantageous in that they possess very high
speciﬁc activities and permit an array of “microspots” dis-
tributed on the surface of a “chip” or sample holder (Fig. 9)
to be optically scanned using a confocal microscope or
charge-coupled device (CCD) camera (Fig. 10). In princi-
ple, other labels, such as chemiluminescent labels, can also
be used, though their lower speciﬁc activities would, in
some circumstances, signiﬁcantly limit assay sensitivities.
Intuitively, one might expect that the sensitivity of an
immunoassay that uses a minute amount of antibody, con-
ﬁned to a spot so small as to be invisible and binding only
an insigniﬁcant fraction of the total analyte present in a
sample, would be extremely low. However, microspotFIGURE 6 When exposed to an analyte-containing medium greater
than shown, an antibody microspot assay conforms to ambient analyte
FIGURE 7 Following exposure to the analyte-containing medium of the “capture” (or “sensor”) antibody, a second labeled (developing) antibody is
used to determine sensor-antibody binding site occupancy using a non-competitive approach (left) or competitive approach (right).
FIGURE 8 Microspot immunoassay relying on ﬂuorescent-labeled antibodies. The ratio of α and β ﬂuorescent photons emitted reﬂects the value of
F. This ratio is solely dependent on the analyte concentration to which the probe has been exposed, being unaffected by the amount or distribution of
antibody coated (as a monomolecular layer) on the probe surface.
6. 114 The Immunoassay Handbook
assays can yield higher sensitivities than other formats.
This surprising ﬁnding requires explanation.
The reason may be readily understood without resort to
mathematical theory by consideration of the effects of
increasing the diameter of a microspot within which sen-
sor antibody molecules are located at maximum surface
density (see Fig. 11). If the amount of antibody in a small
microspot is such that the antibody concentration in the
system approximates 0.01/K, then—as indicated above—
less than 1% of analyte molecules in the solution to which
the spot is exposed will, at equilibrium, be bound to the
antibody. Enlarging the spot’s size increases the amount of
analyte bound (and hence the signal generated by labeled
antibodies attached to the analyte, assuming use of a non-
competitive “sandwich assay” approach) but decreases
antibody fractional occupancy. Thus, the increase in signal
emitted by analyte-bound labeled antibody molecules is
less than that of the area of the spot. On the other hand,
the background signal increases along with the size of the
spot. The signal/background ratio therefore decreases
with the increase in spot area (Fig. 12), this phenomenon
being likely to reduce sensitivity.6 Conversely, reduction
6 Assuming the random variation in the background signal (noise) is approximately
proportional to the background.
FIGURE 9 Antibody microarray. Each microspot is interrogated to determine its fractional occupancy by the analyte against which it is directed.
FIGURE 10 A laser-based confocal microscope provides a sensitive
method of determining ﬂuorescent signals emitted from the spots.
FIGURE 11 Increase in “capture” antibody-coated area results in
increased analyte capture, but analyte surface density (and the corre-
sponding signal generated by developing antibody) falls. Maximal
analyte surface density (and a maximal signal/background ratio) is
achieved when the antibody concentration falls below 0.01/K.
7. 115CHAPTER 2.5 Ambient Analyte Assay
in spot size below that yielding an antibody concentration
of 0.01/K will not signiﬁcantly increase the signal/back-
ground ratio and hence not increase sensitivity.7 Indeed,
progressive reduction in microspot area toward zero ulti-
mately reduces the number of captured analyte molecules
(and hence the signal emitted therefrom) to zero, in which
event the system will clearly be totally insensitive.
7 This conclusion applies only when the reaction is allowed to proceed to
equilibrium. It may be advantageous to reduce the antibody concentration below
0.01/K if the reaction is terminated before equilibrium is reached (see below).
Theoretical consideration of (non-competitive) micro-
spot immunoassay sensitivity (Ekins, 1989) permits predic-
tion of the sensitivities attainable using sensor antibodies
of varying afﬁnities as a function of the minimum detect-
able surface density of labeled antibody molecules (S*min)
(Fig. 13).8 Such analysis indicates that the achievement of
high microspot assay sensitivity requires a detector capable
of accurately measuring low surface densities of labeled
developing antibodies and close packing of sensor-antibody
molecules within the microspot area, maximizing the sig-
nal/background ratio. It also suggests that, assuming the
use of very high speciﬁc activity non-isotopic labels, sensi-
tivities yielded by microspot assays are unlikely to be infe-
rior and (depending on the measuring instrument used)
may be considerably superior, to the sensitivities achiev-
able in macroscopic assays of conventional design. For
example, my colleagues and I—using antibodies labeled
with ﬂuorescent microspheres and a commercially avail-
able confocal microscope—achieved, in early studies, sen-
sitivities in the order of 0.06 labeled antibody molecules/µm2.
Subsequently Boehringer Mannheim researchers, using
improved scanning equipment incorporated into proto-
type analyzers, achieved detection limits of approximately
0.01 molecules/µm2, suggesting that assay detection limits
in the order of 10−17 mol/L (i.e., 103–104 analyte mole-
cules/mL) are attainable using the ambient analyte
Predictions of high ambient analyte assay sensitivity can
readily be veriﬁed in practice (Ekins and Chu, 1993), see
Fig. 14. However, it must be emphasized that the label
used in microspot assays must be of such high speciﬁc
activity that photon-counting errors do not constitute the
principal source of signal variation limiting sensitivity.
8 The analysis does not take into consideration the statistical problems that arise
when analyte concentrations are so low that the probability of capturing a single
analyte molecule within the microspot area becomes the major determinant of
FIGURE 12 As microspot size increases (implying an increase in sensor antibody concentration) the signal deriving from analyte molecules bound to
the sensor antibody fades into the background.
FIGURE 13 Theoretically predicted non-competitive microspot
immunoassay sensitivities plotted as a function of the minimum
developing antibody surface density (S*min) detectable within the
microspot area. Values of capture antibody surface density (S) of 105
binding sites/µm2 and of developing antibody concentration of 1/K*
have been assumed. K, K* are equilibrium constants of capture (sensor)
and developing antibodies, respectively.
8. 116 The Immunoassay Handbook
These conclusions assume the establishment of thermo-
dynamic equilibrium in the system; however, the velocities
of binding reactions are reduced with a reduction of con-
centrations of one or both of the reactants and the time to
reach equilibrium increases. Moreover, it is well known
that diffusion constraints on binding reactions reduce
reaction velocities when capture antibody molecules are
linked to a solid support. Thus, the suggestion that
microspot assays are likely to be more rapid than conven-
tional assays may likewise be counter intuitive. However,
this prediction can again be readily understood by consid-
eration of a series of antibody microspots of increasing
diameter, each containing sensor antibody molecules at
the same surface density. As indicated above, antibody
fractional occupancy at equilibrium—and hence analyte
surface density—is highest when the microspot area is
small, the system therefore conforming to ambient analyte
assay conditions. Moreover, it is intuitively evident and
may be conﬁrmed theoretically (Crank, 1975) that the
smaller the antibody microspot, the lower the diffusion
constraints on analyte migration to it. Thus, the surface
density of analyte molecules within the microspot area
increases more rapidly as the microspot area is decreased.
Indeed, the thermometer analogy provides a good illustra-
tion of these concepts, it being evident that the smaller a
thermometer, the faster it reaches thermal equilibrium
with its surroundings.
Detailed theoretical analysis of the rate at which analyte
molecules migrate toward, and bind to, an antibody
microspot reveals that the (initial) antibody occupancy rate
(OR) (per unit area of microspot) is given by (Ekins, 1995):
where ka =association rate constant (cm3/s/molecule),
[An] = ambient analyte concentration (molecules/mL),
D=diffusion coefﬁcient (cm2/s), rm =microspot radius,
dAb =antibody surface density (binding sites/cm2).
Thus as rm tends to zero, the term πrm
small compared to 4Drm, implying that OR approximates
ka[An]dAb. In other words, the velocity at which antigen
molecules bind (per unit area) to antibodies attached to the
solid support increases with reduction in rm, ultimately
approximating that seen in a homogeneous solution.
Detailed computer models illustrate the events follow-
ing the introduction of a microspot into an analyte-
containing medium (Fig. 15), likewise demonstrating
that the surface density of analyte molecules within the
microspot area increases more rapidly (and equilibrium is
reached sooner) the smaller the spot diameter (Fig. 16).
In summary, microspot-based ambient analyte assays
are potentially more sensitive, and can be performed in
shorter times, than assays relying on conventional formats.
FIGURE 15 Computer simulation of analyte binding to microspots (assuming typical analyte diffusion and antibody binding constants) shows that
equilibrium is reached more rapidly, and that fractional occupancy of sensor antibody is at all times higher, with smaller spots.
FIGURE 14 Section of typical dose-response curve falling below
0.01µU/mL yielded in a two-step TSH dual-labeled microspot
“ratiometric” assay using Texas Red-labeled solid-phase sensor antibody
and a ﬂuorescent microsphere-labeled developing antibody. Sensitivity
(detection limit) derived from precision proﬁle.
9. 117CHAPTER 2.5 Ambient Analyte Assay
Practical realization of these potentially important advan-
tages nevertheless requires good instrumentation and
attention to key determinants of assay sensitivity, such as
non-speciﬁc binding of labeled reagents to solid supports,
minimization of background signals from the supports
themselves, etc. Likewise industrial implementation of
these ideas, and the development of reliable multianalyte
microspot assays, presents considerably greater problems
than that of conventional single analyte assay kits. Among
these are the development of methods for manufacturing
microarrays, for rapid and sensitive scanning of the arrays
and for fully automated analyzers incorporating the micro-
ﬂuidic and other sample processing systems required to
ensure reliability of assay results.
Current methods of constructing microarrays were
reviewed by Schena et al. (1998). The industrial method
evolved in the course of my own group’s collaboration
with Boehringer Mannheim relied on small disposable
polystyrene carriers (or “chips”) onto which microspots
were deposited using “inkjet” technology. Arrays compris-
ing 100–200 spots (each of diameter approximating 80µm
and spaced approximately 40µm apart) were deposited in
this manner on the ﬂat bottom (ca. 3mm diameter) of the
carrier wells (Fig. 17). Using prototype instrumentation,
microspot arrays (each individually quality controlled)
were produced at a speed of 10,000 arrays/h.
A fundamentally different approach applicable to oligo-
nucleotide (and polypeptide) microarray construction is
that originally developed (to generate large numbers of
candidate drugs) by Fodor et al. (1991) using combinatorial
synthetic techniques. These permit construction of numer-
ically large arrays, though (because the efﬁciency of syn-
thesis is only in the order of 85–95%) oligonucleotides
located within individual microspots are of lower purity
than those produced using pre-synthesized material. The
consequent need for “redundant” spots implies that the
potential information content of large arrays constructed in
this manner is considerably less than might appear at ﬁrst
sight. Nevertheless, the realization that such arrays could
potentially be used for diagnostic purposes led to the estab-
lishment of the US company Affymetrix in 1992, this hav-
ing since assumed a prominent position among the many
manufacturers now developing microarray-based technol-
ogies for DNA/RNA analysis. However, a number of other
companies subsequently developed equipment for the local
production of oligonucleotide arrays (e.g., Bowtell, 1999)
based on the use of inkjet dispensers, solid pens, or “quills.”
Although the principal use of oligonucleotide arrays is
DNA analysis, they may also be used as standard array
templates permitting individual researchers to construct
antibody arrays of their own design, using antibodies to
which complementary oligonucleotide sequences have
been linked (Ekins, 1998a).
Meanwhile, several manufacturers now market array-
scanning equipment (Bowtell, 1999), their published sen-
sitivities ranging from 0.5molecules ﬂuor/µm2 (ScanArray,
PerkinElmer Life and Analytical Sciences, Inc., Boston,
MA). [Editor’s note: there is a directory of companies pro-
viding products in the microarray ﬁeld at www.biochipnet.
The use of microarray technologies is nevertheless still in
its infancy, and the number of published applications is as
yet limited. Indeed, it has been said that the scientiﬁc litera-
ture contains more reviews about the technology than
papers reporting its use (Bassett et al., 1999). Nevertheless,
in the course of my own group’s collaborative studies with
Boehringer Mannheim, a variety of non-competitive and
competitive “immunoarray” systems were developed
(Finckh et al., 1998), comprising multiple sandwich assays,
labeled analyte back-titration assays for low-molecular-
weight analytes, and capture-antigen assays for the determi-
nation of serum antibodies. Assays have primarily related to
analytes within the ﬁelds of endocrinology, allergy, and
infectious disease, but similar techniques have also been
employed for the screening of therapeutic drugs. Routine
(15min) TSH assays have been of high sensitivity (detection
limits <0.01µU/mL). Close correlations with the results
FIGURE 16 Simulations of the kind portrayed in Fig. 15 reveal that
equilibrium is reached more rapidly, and that fractional occupancy of
sensor antibody is at all times higher, with smaller spots.
FIGURE 17 Typical antibody (and oligonucleotide) microarray
prepared by Boehringer Mannheim GmbH using inkjet deposition tech-
10. 118 The Immunoassay Handbook
obtained with the latest commercially available test kits have
been demonstrated for a variety of allergens (e.g., birch, cat
epithelia, house dust mite, α-amylase, bee venom, and total
IgE), assay precision, and sensitivity being superior. For
total IgE, a detection limit of <0.01IU/mL has been
achieved. Microarray-based assays relating to a number of
infectious diseases (e.g., HIV, HBsAg, anti-HBC, rubella)
have likewise been developed and have been shown to be
superior to the latest commercially available methods and
We also carried out a limited number of studies exem-
plifying the technology’s application to DNA analysis
(Finckh et al., 1998). For example Mycobacterium tubercu-
losis, which is resistant to Rifampicin (an efﬁcient ﬁrst-
line drug), was speciﬁcally selected for study because of
the technical challenges it poses (e.g., single-point muta-
tions, formation of strong intra-strand secondary struc-
tures, extremely GC-rich segments) as well as its clinical
relevance. Rifampicin inhibits the RNA polymerase by
binding to its β-subunit (rpo-β); however, various sin-
gle-base transitions clustered in a 27-codon segment of
the bacterium gene cause resistance. A study on 80
selected samples from two clinical centers specializing in
tuberculosis diagnosis showed a high degree of concor-
dance with a reference (culture) method.
In summary, miniaturized microarray-based assays con-
stitute a ubiquitous technology, applicable to a wide range
of analytes. Their need for small samples, their greater
sensitivity, speed and reliability,9 their reduced manufac-
turing costs, and the potential savings to clinical laborato-
ries arising from the simultaneous determination of many
different analytes in a single sample are among the impor-
tant advantages that would in any event be likely to lead to
their replacement of existing methodologies. But the most
compelling factor currently driving microarray develop-
ment is the perception of the potential diagnostic impor-
recently—proteomics. Major pharmaceutical manufactur-
ers are among the many that have realized the technology’s
implications, anticipating the future development of drugs
tailored to individual patients according to their genetic
makeup. There is therefore little doubt that the ligand
assay ﬁeld is presently on the brink of a revolution that is
likely to totally transform diagnostic medicine, drug devel-
opment, and other related areas within the next few years.
FREE (NON-PROTEIN BOUND) HORMONE
The direct measurement by immunoassay of the “free”
(non-protein bound) concentrations of thyroid and steroid
hormones has, in recent years, emerged as a standard diag-
nostic procedure in many clinical laboratories, it being
widely accepted that the free hormone concentration mea-
sured under equilibrium conditions in vitro constitutes the
determinant of the hormone’s physiological activity. This
concept, termed the “free hormone hypothesis,” derives
9 Note that microarray formats enable microspots to be included that enable detec-
tion in test samples of cross-reacting substances whose presence would be
unnoticed in conventional assay formats.
primarily from observations that, in subjects in whom
serum-binding protein concentrations are “abnormal,”
overall hormonal effects correlate closely with the free
hormone concentration. Nevertheless, doubts regarding
the hypothesis’ validity remain. These have stemmed in
part from the lack of explanation for the occurrence of spe-
ciﬁc binding proteins in mammalian blood and the charac-
teristic changes in their concentrations that accompany
pregnancy in certain species (see, e.g., Seal and Doe, 1966).
Such doubts have been reinforced by uncertainties regard-
ing the underlying physicochemical basis of the hypothesis
(Ekins et al., 1982; Ekins, 1985a), exempliﬁed by conﬂict-
ing views regarding the rate limitations on hormone efﬂux
from the microcirculation held by thyroidologists (follow-
ing Robbins and Rall, 1979) and by steroidologists (e.g.,
Tait and Burstein, 1964).10
Critics of the hypothesis have suggested that the bound
hormone concentration determines hormone delivery to
certain tissues, implying that serum-binding proteins ful-
ﬁll a speciﬁc tissue-targeting role. Changes in binding pro-
tein concentrations are thus postulated as redistributing
the hormone supply between target organs in the body,
albeit the suggested mechanisms underlying this putative
phenomenon differ. For example, Keller et al. (1969) visu-
alize that certain organs are permeable to bound hormone.
In contrast, Pardridge and his co-workers (see, e.g.,
Pardridge, 1987) suggest that “transient conformational
changes about the ligand-binding site within the microcir-
culation” cause changes in binding protein structure and
hence in hormone-binding afﬁnities, resulting in enhanced
hormone dissociation within certain tissues. Meanwhile—
relying on an analysis of the kinetics of bound hormone
dissociation, intracapillary hormone diffusion, and capil-
lary wall permeation—the present author has proposed
that bound hormone concentrations inﬂuence the mater-
nal hormone supply to the fetus in early pregnancy (Ekins,
1985b, 1990), the latter being postulated as of crucial
importance to fetal brain development.
Notwithstanding continuing debate attaching to the
physiological role (if any) of speciﬁc hormone-binding
proteins, the determination of serum-free hormone
(particularly free T4) concentrations is of considerable
diagnostic importance. Unfortunately, some of the immu-
noassay methods developed by kit manufacturers were
based on fallacious physicochemical concepts, such meth-
ods yielding misleading results in certain clinical situations
and creating major controversy regarding the basic princi-
ples of free hormone measurement. However, only a brief
summary of this topic (reviewed in greater detail elsewhere
(Ekins, 1990, 1998b)) can be appropriately presented here.
10 Robbins’ and Rall’s (1979) view is that, as blood ﬂows through target organ
capillaries, the intracapillary free hormone concentration is maintained at its in
vitro equilibrium value in the face of hormone loss into tissue by instantaneous
hormone dissociation from binding proteins. In contrast, Tait and Burstein (1964)
postulated that only hormone initially in the free state is available for tissue uptake,
implying a decline in the intracapillary free hormone level as blood transits the
target organ, and free hormone molecules are lost into the extravascular
compartment. This view is based on the supposition that release of hormone from
bound hormone complexes is negligible during capillary transit, and implies, inter
alia, that the rate of blood ﬂow through the target organ constitutes a major
determinant of its hormone supply.
11. 119CHAPTER 2.5 Ambient Analyte Assay
All current free hormone immunoassay methods rely on
the basic ambient analyte principle, i.e., that exposure of a
small amount11 of antihormone antibody to a test serum
sample results in occupancy of antibody binding sites to an
extent that reﬂects the ambient free hormone concentra-
tion in the sample (Fig. 18). Occupancy of binding sites
can be determined in three different ways, generally
1. the “labeled hormone back-titration” approach
(“two-step” free hormone immunoassay);
2. the “labeled hormone analog” approach (“single-
step” free hormone immunoassay);
3. the “labeled antibody” approach (likewise a “single-
The ﬁrst of these relies on determination of unoccupied
antibody binding sites (the antibody being generally
linked to a solid support) by their exposure to labeled
hormone following removal of the test serum (thereby
preventing reaction of the labeled hormone with serum
binding proteins which, if permitted, would distort the
The second obviates these sequential operations by the
use of a labeled hormone analog that must, in principle, be
totally unreactive with serum proteins (though retaining the
ability to bind to antibody). However, the ﬁrst commercial
kits of this genre were based on a different (and erroneous)
perception of these methods’ underlying principle, this
allowing a much higher degree of labeled analog binding to
serum proteins (i.e., ca. 99%) than is permissible in valid
11 That is an amount that binds no more than 5% of the total hormone present in
methods.12 Though—by the addition to kit reagents of
albumin and other such artiﬁces—free T4 values yielded by
these kits in normal and pregnant subjects were “engi-
neered” to be closely comparable, incorrect and misleading
results were frequently observed in other clinical situations.
Labeled analog methods therefore fell into considerable dis-
repute, though if a genuinely unbound analog was to be used
(i.e., one—in the case of T4—of an afﬁnity vis-à-vis serum
proteins some orders of magnitude less than analogs used in
the original kits), assay results would be generally accurate.13
Indeed, certain manufacturers continue to market labeled
analog kits, though the author has no recent information
regarding their analytical validity or diagnostic reliability.
The third (labeled antibody) approach also relies on the
use of a hormone analog, though kit manufacturers have
clearly been somewhat reluctant to disclose this fact, pre-
sumably wishing to avoid the suspicion that attaches to
analog-based methods. However, the analog used in
12 The developers of these kits considered that reduced analog binding to serum
binding proteins was required solely to avoid displacement of endogenous
hormone therefrom, thereby increasing the ambient-free hormone concentration.
They therefore postulated that, provided the afﬁnity of the analog for endogenous
binding proteins was sufﬁciently reduced as compared with that of the hormone
itself (i.e., to less than 10%), that little or no displacement of hormone from
binding proteins occurred. The analog could therefore be described as not
signiﬁcantly bound (Midgeley & Wilkins, 1985). Among other implications of this
entirely fallacious concept, major binding to serum albumin was permitted, on the
grounds that such binding would not displace hormone because of this protein’s
high binding capacity. Ironically, though the analogs employed were almost
entirely bound to the endogenous albumin present in test samples (causing
signiﬁcant errors when test sera contained unusual albumin levels or abnormal
albumins), such binding was essential for assays of this type to possess any
superﬁcial resemblance to a genuine free hormone assay. In other words, such
success as early labeled analog assays enjoyed was based on an artifact of the
13 Note however that the presence of endogenous hormone antibodies in test
samples can—as with most immunoassay methods—lead to incorrect results.
FIGURE 18 Basic principle of free hormone immunoassay. A variety of different strategies may be used to determine occupancy of
antibody binding sites.
12. 120 The Immunoassay Handbook
labeled antibody techniques is coupled to a solid support,
such attachment creating a “macro analog” and evidently
contributing to a further major reduction of analog bind-
ing to serum proteins. For this and other reasons, labeled
antibody-based kits appear to conform more closely to the
principles governing valid analog-based free hormone
immunoassays and generally yield correct and clinically
Though all three methods rely on the basic ambient
analyte assay principle, only the two-step method is fully
independent of incubation volume, because of the pres-
ence within single-step assay systems of another reagent
(i.e., analog) whose reactions with antibody are concentra-
tion and volume dependent. However, if the incubation vol-
ume remains essentially unchanged, variations in the
volume of sample added to the incubation mixture are
largely irrelevant (provided that ambient analyte assay
conditions are fulﬁlled), since differences in sample dilu-
tion have no affect on the ambient free hormone concen-
tration in these circumstances.
Free hormone assays differ from those in which the ana-
lyte is totally unbound in so far as the reservoir of analyte
maintaining near constancy of the ambient (free) analyte
concentration in the face of antibody uptake comprises the
rapidly-dissociating pool of protein-bound hormone pres-
ent in the sample. Thus, the total amount of hormone
ﬁnally bound to antibody may greatly exceed the amount
initially present in the free state. For example, in the case
of free thyroxine measurements, up to ca. 5% of the total
hormone in the sample may be bound to antibody at the
termination of the assay, albeit only ca. 0.2% of the hor-
mone in the incubation mixture is free (assuming a ﬁnal
10-fold dilution of the serum sample by buffer and other
assay reagents). The amount of hormone bound to anti-
body will nevertheless be proportional to the ambient free
hormone concentration in the original sample.
It should be perhaps be noted in this context that—
according to Robbins and Rall (1979)—it is precisely this
mechanism that operates during the delivery of thyroid
hormones to target tissues and cells in vivo. In other words,
such cells function as natural ambient analyte concentra-
OTHER APPLICATIONS OF THE PRINCIPLE
As indicated in the introduction of this article, the ambient
analyte assay principle is potentially applicable in many
situations in which the measurement of sample volume is
either impossible or inconvenient, such as the determina-
tion of analyte concentrations in vivo. A simple example of
such an application was the subject of a study by my col-
leagues and myself some years ago, albeit it was abandoned
before completion because of the competing demands on
our time and resources consequent on the commencement
of the collaboration with Boehringer Mannheim on micro-
As is well known, certain hormones, including steroid
hormones, are found in saliva, the salivary concentration
being claimed to reﬂect the free concentration present in
serum. Salivary steroid assays have therefore attracted con-
siderable attention in the past, particularly from partici-
pants in the WHO Human Reproduction Program, in the
context of which steroid hormone assays on subjects reluc-
tant to provide blood for religious and other reasons is fre-
quently a complicating factor.
Nevertheless, the collection of salivary samples also
poses logistic problems and is not without an attendant
health risk. In principle, these could be obviated by the use
of a small plastic probe bearing a small area of antibody at
its tip, the probe being sucked by the subject for a speciﬁed
time interval thereby permitting “sensing” of the ambient
salivary steroid concentration. It should be noted in this
context that ambient analyte assay conditions are fulﬁlled
if (assuming the presence of 105 molecules of antibody on
the microspot surface; the antibody having an afﬁnity con-
stant of 1011 L/M) the ﬂuid volume to which the antibody
is exposed exceeds ca. 1.7µL. Thus, the presence of an
extremely small amount of saliva sufﬁces to permit mea-
surements that are sample volume independent. However,
though initial studies using this approach yielded encour-
aging results, the study was halted (for the reasons indi-
cated earlier) before full validation and reliability tests
could be completed.
This example nevertheless illustrates one potential use
of what is, in effect, an ambient analyte sensor, albeit not
one embodying a transduction system permitting con-
tinuous monitoring (by electronic or other means) of
changing analyte concentrations.14 Other such uses in
medical practice and in other contexts can be readily
Summary and Conclusion
Ambient analyte assay represents a concept that is not
immediately apparent and often provokes initial disbelief.
(Indeed, the author was once challenged to prove its valid-
ity experimentally by a well-known Nobel Laureate, a
challenge which—given the concept’s solid theoretical
basis—was not difﬁcult to meet.) In this article, some of its
more important implications have been discussed, among
which the emergence and widespread use of miniaturized
multianalyte chip-based microarray methods (for DNA
analysis, for the determination of the products of gene
expression (proteomics) and for conventional immunodi-
agnostic applications) are likely to have the most signiﬁ-
cant and enduring consequences. Indeed, for the various
reasons indicated in this article, immunoanalyzers relying
on conventionally-formatted binding assays are ultimately
likely to be replaced by much smaller instruments based on
the use of microarrays, permitting the rapid determination
of multiple or single analytes as required. It should be
noted in this context that many hormones and other sub-
stances comprise heterogeneous mixtures, the only fully
satisfactory solution to their assay being the determination
of their principal components (Ekins, 1990).
Ambient analyte assay can, in short, be anticipated to
revolutionize the entire medical diagnostics ﬁeld in the
14 The slow kinetics of antibody–antigen reactions nevertheless preclude the
monitoring of rapidly-changing analyte concentrations notwithstanding the
emergence of satisfactory and sensitive transduction systems permitting
continuous measurement of antibody occupancy.
13. 121CHAPTER 2.5 Ambient Analyte Assay
EDITOR’S UPDATE FOR FOURTH
EDITION: RECENT DEVELOPMENTS
(BY DAVID WILD)
Parpia and Kelso (2010) independently tested the coun-
ter intuitive claims for ambient analyte assay that an
immunoassay’s limit of detection can be improved by
reducing the amount of capture antibody, and that the
results should be insensitive to the volume of sample as
well as the amount of capture antibody added. They used
ﬂow cytometric analysis to detect the binding between a
ﬂuorescent ligand and capture microparticles, since this
methodology can directly measure fractional occupancy,
the primary response variable in ambient analyte
After experimentally determining that the theoretical
requirements for ambient analyte conditions had been
achieved, comparisons were carried out between ambient
and non-ambient assays in terms of signal strength, limit
of detection, and sensitivity to variation in reaction volume
and number of particles.
The critical number of binding sites required for an assay
to be in the ambient analyte region was estimated to be 0.1
VKd, where V is the reaction volume in liters (L) and Kd is
the equilibrium dissociation constant in moles per liter.
The parameter b is used to deﬁne the point where VKd=1.
Parpia and Kelso created two assays, one in the ambient
analyte range with b=0.047 (surface area 0.000445cm2)
and the other at b=4.75 (surface area 0.045cm2).
As predicted by the theory, the ambient analyte assay
exhibited a superior signal/noise ratio and this reduced
the limit of detection, proving that ambient analyte assays
can be ultrasensitive, validating this part of the theory.
They demonstrated that when the ambient analyte criteria
for binding sites were achieved, the signal level was no dif-
ferent between sample volumes of 100 and 200µL,
whereas as the antibody binding site density was reduced,
beyond the ambient analyte requirements, the impact of
sample volume gradually became apparent. The signal
level in the ambient analyte version of the assay was also
unaffected by variations in the number of binding sites.
They concluded that ambient analyte theory is an excel-
lent guide to developing assays with superior performance
Hartmann et al. (2009) wrote a review on protein micro-
arrays for diagnostic assays that updates the subject since
Roger Ekins ﬁrst authored this chapter. Their excellent
summary includes many recent references and several use-
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