Antibodies for Immunosensors

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 techniques, 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 biological 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-competitive 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 reversible 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, especially in pesticide analysis. An example is given in

Fig. 2 illustrating the potential of enzyme immunoassays. 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 atrazine 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 immunoassays [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


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 coating 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 competitive 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 literature 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 concentrations 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 saturation.

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


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

[I31

[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 important, 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 analyte 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 measurements 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 sophisticated 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 microspots, 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 generation 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 progress is expected. especially with respect to quasi-


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 composition and concentrations, spectrum libraries can be obtained and used for the identification of unknown

samples. This approach is commonly used in chemometrics 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 production 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 generation 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, medical 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 repertoire 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 synthetic 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.

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