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ANALYTICA

CHIMICA
ACTA
Analytica Chimica Acta 347 (1997) 177-I 86 ELSEVIER
Antibodies for immunosensors
A review
Bet-told Hock*
Technical University of Miinchen at Weihenstephan, Faculty qf Agriculture und Horticulture, Department qf Botany,
D-85350 Freising, Germany
Received 12 September 1996: received in revised form 4 February 1997: accepted 14 February 1997
Abstract
Immunosensors (1%) are miniaturized measuring devices, which selectively detect their targets by means of antibodies
(Abs) and provide concentration-dependent signals. Ab binding leads to a variation in optical properties, electric charge, mass,
or heat, which can be detected directly or indirectly by a variety of transducers. The quality of ISs primarily depends upon the
selectivity and affinity of the Abs used in the receptor unit. Since sensitivity is directly related to the affinity of the ligand
binding, reversibility excludes high sensitivity. Consequently, detection units for single-use and quasi-continuous measuring
devices are presently preferred. An important future development is seen in the field of multianalyte sensors. The most
attractive application is based on Abs with sufficiently different cross-reactivities. The response pattern provides information
on the presence and concentration of structurally similar analytes, as they are found for instance in pesticide groups such as the
s-triazines. It is obvious that this development will be significantly accelerated by applying recombinant techniques for Ab
production. The next step is directed toward the generation of recombinant Ab libraries, which will provide a huge repertoire,
generated at the DNA level, for new Ab types.
Keywords: Immunosensors; Immunoassays; Multianalyte system; Recombinant antibodies; Pesticides
1. Introduction
Immunosensors (1%) are considered a major devel-
opment in the immunochemical field and connected
with high expectations. This technology has been
extensively reviewed, e.g. [l-5]. However, there
appears a considerable gap between the final goal
and reality. This situation is mirrored by a lack of
commercial applications although the instrumentation
is well advanced.
*Corresponding author. Fax: +49 8161 71 4403
This review is intended to shed some light on the
present situation of 1% by analyzing the possibilities
as well as the limitations of IS technology. It is shown
that the main limitation is related to the antibodies
(Abs) and their properties. Whereas the shortage of
Abs is likely to be abolished by recombinant techni-
ques, other limitations, especially the lack of rever-
sible operations of Ab-guided sensors, is of theoretical
nature and can only be circumvented. The same holds
true for alternative ligands such as RNA or DNA
probes.
0003-2670/97/$17.00 $J 1997 Elsevier Science B.V. All rights reserved
PII SOOO3-2670(97)00167-O
178 B. Hock/Analytics Chimica Acta 347 (1997) 177-186
A B C D
l *
Sample , n
4*
n
Fig. 1. Composition of an immunosensor: (A) A receptor unit
(bioreceptor) with antibodies which bind the ligand and provide a
signal, (B) a transducer, which transforms the response into an
electrical signal, and (C) an electronic part, which is involved in
data processing, including the display (D).
2. Components of immunosensors
Immunosensors (1%) belong to the biosensors
because an essential component, the Ab, is derived
from a biological process. ISs with their tripartite
composition and their individual parts in close contact
(Fig. 1) resemble the other biosensors: (1) The bio-
logical component, here the Ab, conveys selectivity
and sensitivity to the sensor. The Ab binds the analyte
with high affinity and is therefore able to detect the
analyte in the presence of other substances. This
biological component is normally immobilized at or
near the second component, (2) the transducer. The
transducer converts the particular biochemical or bio-
physical event, here the binding of the analyte to the
Ab, into an electrical signal. This is amplified and
digitalized by (3) an electronic part.
3. Types of immunosensors
The merits of ISs are clearly related to the selec-
tivity and affinity of the Ab-analyte binding reaction.
There are several possibilities to derive a measurable
signal from Ab binding. They can be related to two
basic approaches: (1) The use of a tracer, which
provides the signal. This approach has been derived
from immunoassay (IA) technology. It significantly
facilitates the problem of signal generation but it
generates new problems related to the synthesis, sta-
bility and costs of appropriate tracers. The tracer either
labels the occupied binding sites of the Abs or the free
ones. The first alternative corresponds to non-compe-
titive IAs, the second one to competitive IAs. In spite
of distinct advantages of the non-competitive
approach, e.g., lower detection limits and robuster
assays, it cannot be applied for haptens because it
requires as analytes multivalent antigens with more
than one epitope. (2) Tracer-free procedures pose
much higher demands to the Abs and to the transducer
part as the tracer variant.
The alternatives (1) and (2) are known in the
literature as (1) indirect ISs and (2) direct ISs. This
distinction has an entirely different meaning as the
terms direct and indirect in the IA field, which both are
tracer-related. They are distinguished by whether Ab
binding is directly detected, e.g., after an enzyme-
labelled Ab is bound to an immobilized coating con-
jugate or an enzyme tracer to an immobilized Ab, or
whether the detection only takes place after a second
binding reaction, e.g., if an enzyme-labelled second
Ab is used to label a first Ab bound to an immobilized
coating conjugate.
Independent from the distinction between direct
and indirect ISs, there are several ways how the
analyte binding by the Ab can be exploited to change
a physical parameter. Table 1 lists the most important
types. The presently available transducers can be
broadly divided into optical, electrochemical, mass-
sensitive, and thermal devices. Consequently there are
optical, electrochemical, mass-sensitive and thermal
Table 1
Categories of immunosensors according to different physical
principles
1. Optical immunosensors
Surface plasmon resonance (SPR)
Grating couplers
Differential interferometry
Reflectometric interference spectroscopy (RIR)
Double beam waveguide interferometers
Resonant mirror (mode coupling)
2. Electrochemical immunosensors
Potentiometric
Immunofield effect transistors (immunoFET)
Amperometric
Voltametric
3. Mass sensitive immunosensors
Piezoelectric
SAW technique (acoustic surface waves)
4. Thermoelectric immunosensors (thermistors)
B. Hock/Analytics Chimica Acta 347 (1997) 177-186 179
ISs. At present, there seems to be a preference for
optical ISs. Although there are a few commercial
sensors available using optical and mass-sensitive
transducers, no commercial applications with ISs have
been reported so far.
4. Tasks of immunosensors
In contrast to the basic structure of an IS, there is
less agreement about which requirements should be
fulfilled by an IS. This topic leads to the central
question: Are the same Ab properties required as
for immunoassays (IAs) or should additional proper-
ties be considered?
Independent from the respective answer, there
should be some progress if one proceeds from the
simple level of IAs to highly sophisticated ISs. This
requires a closer look into the tasks a biosensor (and
consequently an IS) should fulfill. Table 2 shows that
there are different views on this subject. However, the
main question is: Should an IS operate reversibly and
therefore be able to provide continuous measurements
as it is required for a chemical sensor or do these
requirements not apply to an IS? It is of little use to
argue which definition is correct. It is more important
Table 2
Criteria for sensors (biosensors)
Karst et al. [6]
Miniaturised measuring devices, which selectively detect their
targets (e.g. molecules or ions) and provide reversible and
concentration-dependent signals
A measuring device with a preferentially miniaturised construc-
tion, which converts chemical informations in correspondingly
proportional electrical signals
Scholtissek et al. [8]
The characterization of a sensor as a continuous and reversible
measuring device is used in physics and does not necessarily apply
to biosensors.
Canh, T.M. [9]
(1) A biosensor must provide a signal that has a direct
relationship with the quantity under investigation.
(2) The following requirements must be met: repeatability,
reproducibility, selectivity, sensitivity, a linear region of response,
and a good reponse time.
to pay attention to the tasks a biosensor such as an IS
can accomplish! Does it include continuous and rever-
sible measurements? What can be expected from
sensitivity and reproducibility?
5. Immunosensors as advanced immunoassays
5. I. Interlaboratory tests with immunoassays
Considering the expectations placed on ISs, their
high level of sophistication and their considerable
developmental costs, it appears to be reasonable to
set at least the same standard for ISs in terms of
sensitivity, selectivity and robustness as for IAs. IAs
are well established in clinical analysis, and they are of
growing importance in the environmental field, espe-
cially in pesticide analysis. An example is given in
Fig. 2 illustrating the potential of enzyme immunoas-
says. An interlaboratory trial was carried out for
atrazine in 1995 by the Immunoassay Study Group
[lo] with 16 participants who volunteered after a
public announcement of the action. Each participant
analyzed three atrazine samples with a test kit supplied
by Riedel de Haen AG. The data demonstrate the
excellent correspondence between the nominal atra-
zine concentrations, obtained in sample b and c by
spiking, the gas chromatography/mass spectrometry
(GUMS) and IA data. This test was used as basis for a
DIN (Deutsches lnstitut fiir Normung e.V., German
Standards Institute) guideline for selective immunoas-
says [Ill. The data clearly show that the requirements
of the guideline, the quantitative determination of a
single pesticide, can be met.
5.2. Pegormance of immunosensors
It is clear from this discussion that ISs should also
reach meaningful detection limits if they are applic-
able for practical work. Many examples are now
available which prove that this goal can be achieved.
Only one example is given below for an SPR sensor,
presently the most widely used type of ISs. The
detection principle relies on the optical phenomenon
of surface plasmon resonance (SPR), which detects
changes in the refractive index of the solution close to
the surface of the sensor chip. This is in turn directly
related to the concentration of solute in the surface
180 B. Hock/Analytics Chimica Acta 347 (1997) 177-186
0.3
s‘
s
3
k! 0.2
.-
2
c CA 0.1
0.0
-I
tap
water well, Kreuz-
spiked
stollen,
spiked
n A frazirte (nominal)
A trazine (GC/MS)
q Atrazine (ELISA)
aa a bb b cc c
Sample No.
Fig. 2. Interlaboratory test for atrazine, organized in 1995 by the Immunoassay Study Group in DIN NAW I,4 [5]. Natural water samples were
supplied by the Stadtwerke Maim AG (Dipl. Min. A. Meitzler) and assayed for atrazine by GUMS. Samples b and c were spiked with
atrazine. The error bars indicate standard deviations.
layer. The operation principle requires that one ligand
(usually the Ab) is immobilized (in a dextran matrix)
on the sensor chip, which forms one wall of a micro-
flow cell. The sample contains the other reaction
partner(s) and is injected over the surface in a con-
trolled flow. Any change in surface concentration
resulting from interaction is detected as an SPR signal,
expressed in resonance units (RU).
Minunni and Mascini [ 121 used the BIAcoreTM
technology for sensitive atrazine analysis. Due to
the low molecular weight of the herbicide the signal
change was relatively small when immobilized Abs
were loaded with low amounts of atrazine. On the
other hand, an enzyme tracer-dependent procedure
was to be avoided. The compromise was the approach
of competitive immunoassays with immobilized coat-
ing conjugate, however, using unlabeled Abs. Fig. 3
shows the details. A coating conjugate with an
exposed atrazine residue was covalently bound to
the sensor chip surface (through carboxymethylated
dextran mediated by carbodiimide). Then a stream
carrying atrazine monoclonal Abs together with the
herbicide was directed over the surface. Here a com-
petitive reaction takes place. In the presence of an
excess of atrazine all Ab binding sites would be
10
t t,t
I I I
t t.
0 2 4 6 8
time ,m,n,
Fig. 3. Indirect multi-step procedure for the detection of atrazine
with a SPR sensor. An atrazine derivative is immobilized on the
sensor chip surface and a solution containing monoclonal
antibodies mixed with the herbicide (sample) flows over the
surface (1). To enhance the primary response a second antibody
anti-mouse Fc flows over the surface and binds mAbs’ Fc fragment
(2). The cycle ends with the regeneration of the sensor chip surface
(3). From [12].
occupied with atrazine and consequently no Abs bind
to the coating conjugate. With decreasing analyte
concentrations, however, increasing amounts of Abs
are bound. Although the procedure does not need an
enzyme-labeled tracer, it proved to be necessary to
B. Hock/Analytics Chimica Actu 347 (1997) 377-186 181
1000
E
I I I I I
I I I I
0.0 0. 2 0. 4 0. 6 0. 8 1. 0 1. 2
atrazine concentration [ ppbj
Fig. 4. Standard curve for atrazine obtained with a SPR sensor.
0.25 pg ml- ’ anti-atrazine mAb are mixed with the sample. The
secondary antibody (anti-mouse Fc 125 pg ml-’ in acetate buffer
pH 5) is injected over the surface. The surface is regenerated with
100 mM NaOH +20% acetonitrile. 5 measurements for each
concentration are compared. From [8].
enhance the primary response by a second Ab (anti-
mouse Fc). The cycle ends with the regeneration of the
sensor chip surface (20% acetonitrile in 100 mM
NaOH solution). Fig. 4 shows the standard curve of
the optimized assay in tap water. Each point represents
the mean is of 5 replicates. The variation coefficient
is below 5%, the detection limit 0.05 ug 1-l. The
analysis time for each cycle amounts to 15 min. This
example together with many other cited in the litera-
ture shows that IS are capable of sensitive and fast
measurements. Selectivity depends on the applied Abs
and will be discussed in Section 7.
6. Trade-off between sensitivity and reversibility
Reversibility is sometimes considered an important
issue for future ISs. However, fast reversibility and
high sensitivity (in the sense of low detection limits)
exclude each other. This becomes obvious if the
relation between the affinity constant K and the middle
of a competitive assay is considered. The middle of an
assay indicates the range where the assay operates and
consequently the sensitivity. It also corresponds to the
region where the most accurate measurements are
possible.
In the equilibrium reaction between the free analyte
H (here a hapten) and the (monovalent) antibody Ab
towards the analyte-antibody complex HAb (=bound
analyte)
H+Ab=HAb
(1)
the affinity constant K determines the concentration
ratio between the antibody-bound hapten and the free
reaction partners
[H Abl
K = kdkff = ,Hl x ,Abl (lm01~‘).
At the middle of the test, half of the Ab binding sites
are occupied. Therefore
[HAb] = [Ab].
(3)
In this case (Eq. (2)) becomes
K= ,Hk,
(4)
with [HO.s]=free hapten concentration at 50% occu-
pation of the Ab binding sites (half saturation). It is
inversely proportional to the affinity constant. This
means that at high K values only low hapten concen-
trations are required for half saturation.
Therefore it is of practical interest to relate the
analyte concentration required for half saturation to K
and the Ab concentration. For this purpose, the total
hapten concentration [H,,,], which is equal to the
analyte concentration in the sample, is related to K
and the total Ab concentration [Ab,,,]. Since
[H,,t] = [HI + [HAb] (5)
it can be derived for half saturation (Eq. (3)) from
Eqs. (4) and (5) that
[Hos.tot] = l/K + [Ab], (6)
=
1 /K + [Abtot/21 (7)
with [H0.5,tot]=total hapten concentration (=analyte
concentration in the sample) resulting in half satura-
tion.
This means that the analyte concentration at half
saturation is inversely related to the affinity constant K
and directly related to the Ab concentration. Optimiz-
ing assay sensitivity therefore must consider high
affinity Abs and low Ab concentrations.
Eqs. (4)-(7) were derived for tracer-free systems.
Some modifications are required if tracers are
182 B. Hock/Analytics Chimica Acta 347 (1997) 177-186
included (e.g. enzyme-hapten tracers of systems with
immobilized Abs). But again there is an inverse rela-
tion between K and [Ha.51 as shown by Schwalbe-Fehl
[ I 31
[Ho.51 = $ + [Ttot],
8
K=
3 . ([Ho.51 - [Ttotl)
with [T,,,]=total tracer concentration.
Since a sensitive assay has its half saturation at low
[Ho,5] and is therefore characterized by a high K, the
analyte binding by the antibody is virtually irrever-
sible.
It is most interesting to take a look at receptors,
binding molecules which are usually designed for
reversible binding. Otherwise, new receptors would
have to be synthesised after each ligand binding. There
is a surprising solution: Receptors are restored after
ligand binding. This always requires an additional
biochemical apparatus. It includes for instance in
the case of the acetylcholine receptor the enzyme
acetylcholinesterase, which splits acetylcholine into
acetate and choline and therefore restores the receptor
to its original state.
The immune system did not adopt this fancy
machinery simply because it was not necessary.
Abs are used by the immune system to tag foreign
material in order to get it eliminated from the body by
specialized immune cells. It is us who want to obtain
Abs for sensitive assays, which at the same time
should operate on a reversible basis. This task among
others (signal amplification) is fulfilled in nature by
receptors.
6.1. Quasi-reversible approaches
There are several strategies to approach reversible
operation in the immunochemical field. The most
simple and effective way is a quasi-reversible strategy.
It was employed for the first time in the field of
immunosensing by the group of Rolf D. Schmid with
a sensing device, the flow injection immunoanalysis
(FIIA [ 141). This technology is a further development
of FIA (flow injection analysis), introduced in 1975 by
Ruzicka and Hansen [ 151. FIA is now widely used as a
method for the continuous, automated performance of
chemical and biochemical detection methods. It is
Fig. 5. Flow injection immunoanalysis. (FIIA). From [14].
P=pump; M=mixing chamber; L=Lee valve (3/2-way valve);
D=detector (fluorimeter combined with an integrator and/or
computer).
based on the injection of a liquid sample into a
continuous flow of a liquid carrier. The sample is
introduced through an injection valve with a loop with
an accurately known volume and is transported to the
biosensor.
In the following example, the FIA principle was
combined with a competitive IA for the detection of
atrazine [ 141. An atrazine-peroxidase conjugate was
used as a tracer. The experimental setup is shown in
Fig. 5. The central part is a membrane reactor with an
immobilized atrazine Ab. The following components
are consecutively pumped through the membrane
reactor: sample (or standard), enzyme tracer peroxide
(H202), and the second substrate hydroxyphenylpro-
pionic acid. The enzyme activity of the Ab-bound
tracer is determined fluorimetrically. Fig. 6 shows
typical calibration curves for atrazine with two dif-
ferent Abs. It is obvious that sensitivities are obtained,
which are comparable to IAs. But even more impor-
tant, the membrane reactor, a special development for
the FIIA, enables the automated change of the Ab
membrane after the completion of the assay. Therefore
this sensing device could in principle be used for the
continuous measurement of real samples.
ISs could also be operated in a quasi-continuous
mode. However, the same result is usually achieved by
other means, namely the regeneration of free Ab
binding sites, which is due to a change of the Ab
affinity in the presence of the regeneration medium.
Experiences with optical ISs (e.g. BIAcore) have
shown that up to 100 regeneration cycles can be
carried out with a single preparation of immobilized
Abs. But it seems to be advantageous, at least in
B. Hock/Analytics Chimica Acta 347 (1997) 177-186 183
0 0.001 0.01 0.1 1 10
atrwine concentration l/.fg/L]
Fig. 6. Calibration CU~Y~S for atrazine, obtained with FIIA. From
[ 141. l =polyclonal antibody C193; V=monoclonal antibody
K4E7.
environmental analysis, to apply this procedure to
immobilized coating conjugates and to use fresh
Abs after each regeneration cycle. The exposed ana-
lyte residues are usually less sensitive to the regenera-
tion conditions than Ab binding sites.
Finally it is tempting to consider a theoretical
possibility for continuous measurement, which oper-
ates beyond the equilibrium, but requires of course
low affinity Abs. Investigations in the group of Wilson
[ 161 on Fab fragments derived from mouse IgG
showed distinct conformation changes at the contact
of the l&and with the free Ab binding site. This
demonstrates that a rigid key-keyhole model is not
sufficient for the recognition mechanism. Crystallo-
graphic data were used to calculate the Fab surface of
the Ab binding site in the free and the occupied stage.
The free binding site forms a bowl-like pocket,
whereas the occupied binding site forms a distinct
furrow, connected with a deep pocket, which perfectly
fits the antigen. If such conformational changes were
used for signal generation, true reversible measure-
ments could be carried out with ISs. However, it is not
clear, yet, whether such conformation changes gen-
erally occur in Abs and whether they are large enough
to be applied for analytics. Moreover, if such a
mechanism were feasible, it most probably would
have evolved in the receptor field. But continuous
sensing (and signal amplification) is always achieved
in this case by a receptor restoration and a sophisti-
cated signal transduction chain.
In conclusion Ab properties required for ISs are
comparable to those applied in IAs. However, this
does not mean that ISs are merely advanced IAs
because of their faster speed. In reality there is a great
potential for constructing multianalyte systems by
combining several ISs.
7. Multianalyte systems
Although there have been several approaches to
extend single analyte IAs to multianalyte systems (e.g.
[17]), this is the true domain of ISs. The theory of
multianalyte systems has already been laid down in
the 1980s by Ekins [ 181. He suggested to coat silica
chips with up to 10000 Ab microspots per cm’. If
fluorescent sensor Abs are used, the signal could be
generated in the case of non-competitive assays (with
antigens) after the immunoreaction by labelling the
bound antigen with a second tracer Ab, equipped with
a different fluorescent dye (sandwich approach). Com-
petitive assays (with haptens) could use a fluorescent
anti-idiotypic Ab, labelling the free binding sites of
the primary Ab. Since sensor and tracer Abs are
differently labelled, the fractional occupancy of the
sensor Ab binding sites and therefore the analyte
concentrations in the sample can be derived from
the signal ratio (‘ratiometric assay’). Since the Ab
concentrations are kept to a minimum at the micro-
spots, the signal ratio does not depend anymore from
the Ab concentration. Laser scanning confocal fluor-
escence microscopy can be used for optical scanning
of the silica chip.
The current status of immunochemically-based
simultaneous multi-analysis for environmental appli-
cations has been competently reviewed by Brecht and
Abuknesha [ 191. The required devices for signal gen-
eration and read-out systems are principally available.
But it is obvious that larger multianalyte systems are
severely restricted at present time by the availability of
Abs. If this bottleneck can be eased, significant pro-
gress is expected. especially with respect to quasi-
184 B. Hock/Analytics Chimica Acta 347 (1997) 177-186
continuous modes of operation and label-free
approaches.
mRNA isolation from B-lymphocp s
The most attractive perspective is seen in the field of
IS arrays using cross-reacting antibodies, which are
targeted against a group of closely related analytes. If
the cross-reactivities are sufficiently different, conclu-
sions on the composition and analyte concentrations
of the sample can be drawn from the response pattern
of the sensor array. If many different patterns are
available, generated from standards of different com-
position and concentrations, spectrum libraries can be
obtained and used for the identification of unknown
samples. This approach is commonly used in chemo-
metrics and pattern recognition [20]. It is obvious that
neuronal networks provide an attractive support for
trainable systems.
CH "L CL
1
cDNA synthesis
- +m
+-Is-
“H Cti "L CL
1
separate amplification of
VL and VH
“H
“L
1
ligation and cloning
into plasmid vector
This means that the power of ISs can be fully
exploited with sensor arrays that are able to interpret
response patterns similar to biological systems.
H chain library
I
L chain library
8. Antibodies for sensors
t
transformation of E. coli
Considering the enormous potential of future ISs, it
is clear that the traditional approaches for Ab produc-
tion are not satisfactory. New Abs, whether polyclonal
or monoclonal, always depend on new immunisations,
i.e. lengthy and tedious procedures, which do not
always guarantee success. Significant changes are
expected from the recombinant approach to Ab gen-
eration and diversification. In this case, generation of
new Ab variants by new immunization is replaced by
selection from suitable Ab libraries, which can be
constructed within shorter time and reach virtually
unlimited size [e.g. [21]]. The main bottleneck is the
handling and screening of large libraries for Ab genes.
1
random combination
1
DNA library with
paired H and L chain fragments
1
expression in E. co/i
1
The basic technology for the production of recom-
binant Abs (rAbs) has been developed in the medical
field for the production of humanized Abs to circum-
vent allergic reactions during therapy. Presently, med-
ical applications still play a major role, especially in
HIV and cancer research. However, significant steps
were also undertaken in environmental analytics (for
review cf. [22]). Fig. 7 presents a general scheme for
the production of rAbs and their diversification, leav-
ing ample space for modifications. It starts with the
isolation of mRNA from B-lymphocytes from the
spleen of immunized animals, which is still considered
screening
1
recombinant antibody fragments
or
scFv
Fig. 7. General scheme for the production of recombinant
antibodies.
Fab
B. Hock/Analytics Chimica Acta 347 (1997) 177-186 1x5
the most effective method to generate high affinity millions of years of evolution, seems to have a clear
Abs. After cDNA synthesis the desired immunoglo- lead. However, it is within the scope of recombinant
bulin sequences, e.g. the variable regions, are ampli- technologies to apply in-vitro evolutionary strategies
fied by PCR and cloned into a suitable vector, followed for an optimal design of binding peptides. It is clear
by transformation of the host, usually Escherichia that this concept also applies to totally different
coli. Each transformed bacterium contains an Ab ligands such as RNA or even DNA probes. Even
coding DNA sequence, which can be multiplied and artificial guest-host systems have entered into compe-
transferred to the progeny, then representing a recom- tition putting biology-derived ligands and therefore an
binant bacterial clone. essential part of biotechnology on the test.
As the B-lymphocytes are a heterogeneous cell
population, this procedure distributes the entire reper-
toire of Ab coding DNA sequences to different clones,
which already represent a simple Ab library. The
individual heavy and light chain fragments can be
combined subsequently by random combination,
expressed and screened for functionality. Further
diversification and fine tuning is achieved by varying
the CDR regions within the variable regions, which are
responsible for antigen or hapten binding, for instance
by error-prone PCR. This approach generates syn-
thetic libraries of huge sizes for the selection of new
Ab properties. It is obvious that evolutionary strategies
will speed up and simplify in the future the screening
problem.
Acknowledgements
I would like to thank the Deutsche Forschungsge-
meinschaft for the grant Ho 383/3 1- 1. Dr. M. Minunni
and Prof. M. Mascini (University of Firenze) and Prof.
R. Schmid (University of Stuttgart) kindly provided
some of the figures. I am grateful to Stefanie Rau-
challes for typing the manuscript.
References
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production and diversification, the possibility of pro-
ducing fusion proteins. With respect to ISs, it is
desirable to equip the rAb fragments with suitable
tags, e.g. his-tags or strep-tags, which not only facil-
itate the purification but also the orientation of rAbs at
the sensor surface.
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9. Outlook
Future progress in the IS field is expected mainly
from a combination of two different developmental
lines, the construction of IS arrays and the acceleration
and expansion of Ab production by recombinant
techniques. It would not be surprising to experience
a gradual transition from Abs to synthetic peptides as
ligands for analyte binding. The main criterium for the
final choice not only depends on the affinity and
selectivity ranges provided of one system or the other
but also on the costs and ease of generation a new
spectrum of ligands.
III
121
131
141
[51
IsI
171
181
I91
1101
The immune system with its possibilities for affinity
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