Journal oflmmunological Methods, 150 (1992) 5--21

5

© 1992 ElsevierSciencePublishers B.V. All rights reserved 0022-1759/92/$05.00

JIM06325

Enzyme immunoassay techniques An overview T. P o r s t m a n n and S.T. Kiessig Department of Medical Immunology, Medical School (CharitY), Humboldt Unit'ersity Berlin, Berlin, Germany

(Accepted 12 February 1992) In spite of the great variety of enzyme immunoassays (EIA) they can be classified into two groups 'analyte-observed' and 'reagent-observed' assays, depending on their reaction principle. The latter are favored by use of monoclonal antibodies and are characterized by a greater sensitivity, a larger measuring range, a lower susceptibility to disturbing influences. They can be used only for detection of macromolecules. For heterogeneous EIAs to be used on laboratory scale, simple adsorption of antigens and antibodies is still recommendable though affinity constants decrease by at least one order of magnitude and antibody density at the solid phase and analyte binding capacity are not parallel due to increasing steric hindrance. For this reason, the antibody with the higher affinity constant should therefore always be used as solid-phase antibody. Microparticles used as solid phase for heterogeneous assays, due to their very high binding capacity for the analyte and extremely short diffusion distances, guarantee 'one step' assays of only a few minutes. Of the limited number of enzymes suitable as markers in immunoassays, horseradish peroxidase is the enzyme of choice followed by alkaline phosphatase. Although enzyme and enzyme-labelled reagents are detectable by fluorogenic product measuring with a sensitivity, which is 10-1000 times higher than using chromogenic substrates, the sensitivity of the assays can be increased only by factor 2-10. Labelling enzymes cannot only be covalently bound to the antibody, but also via anti-enzyme antibodies. Pros and cons of the different methods of coupling the enzyme/anti-enzyme complex to analyte-containing immune complexes are discussed. Different EIA variants to detect specific antibodies are reviewed. Among them only capture EIAs permit precise isotype analysis of antibodies of a distinct idiotype. Homogeneous EIAs are widely spread for hapten determination but even variants based on proximal linkage are no alternatives to heterogeneous EIAs for determination of macromolecules. Different parameters are defined which permit to assess the quality of an immunoassay and which should be used in routine assays as internal controls in the laboratory. Key words: Assayprinciple; Bound-free separation; Marker enzyme;Conjugates-substrate reaction; Assay parameter

Introduction Correspondence to: T. Porstmann, Department of Medical

Immunology, Medical School (Charit6), Humboldt University Berlin, Schumannstr. 20/21, P.O. Box 150, O-1040 Berlin, Germany (Tel.: 37-2-286.6484; Fax: 37-2-286.6475).

There is hardly any other development of methods that has reached a variety comparable to

that of enzyme immunoassays (EIA) based on the reaction between antigen and antibody. Nevertheless, new variants are being steadily developed with the aim of (1) increasing sensitivity; (2) increasing specificity; (3) reducing the duration of assays; (4) facilitating the performance of assays. This overview shall provide an insight into the state of the art, outline tendencies of fundamental techniques and be helpful for the development of EIAs for laboratory use.

Basic principle of immunoassays Immunoassays can be generally divided into unlabelled and labelled ones. Most unlabelled techniques are based on secondary immune reactions, as, e.g., precipitation and agglutination. They are measured by light scattering or particle counting methods. Labelled immunoassays are based on the primary immune reaction. There are two types, depending on the reaction principle: (1) type I 'reagent-observed'; (2) type II 'analyte-observed' (Ekins, 1981a). Whereas in the type I assay there is excess of antibody as binder, it limits the reaction in the type II assay with excess of analyte. Type II assays have got a maximum theoretical sensitivity of about 10 -14 m o l / I (Ekins, 1981b) and are therefore one to two orders of magnitude less sensitive than type I assays. The maximum sensitivity of type I assays, which are also called 'twosite assay' or 'sandwich assay', ranges between 10 -15 and 10 -t6 m o l / l (Jackson et al., 1983). Unlike type II assays, which are used to determine both haptens and substances of high molecular weight, type I assays require substances to be used as analyte with at least two different epitopes clearly distinct from each other in order to permit epitope-different labelled and unlabelled antibodies to bind without steric hindrance. Type I assays are thus unsuitable for hapten quantification. The different characteristics of type I and type II assays are listed in Table I. The decision to use one or the other assay principle is mainly influenced by the following aspects:

TABLE I CHARACTERISTICS OF TYPE I 'REAGENT-OBSERVED' AND TYPE I! 'ANALYTE-OBSERVED' ASSAYS (From Nakamura. R.M., Voller, A. and Bidwell, D.E., 1986) Type I 'reagent-observed' (excess antibody) (1) Maximal sensitivity is attained as amount of antibody approaches infinity. (2) Theoretical sensitivityof assay is one molecule of analyte. (3) Cross-reaction antigens will be equipotent with excess antibody system. (4) Antigen-antibodyreaction is less influenced by substances such as salt and urea. (5) Assaytime is relativelyrapid with labelled antibody procedures. Type 11 'analyte observed' (excess analyte) (1) Maximal sensitivityis attained when antibody concentration approaches zero. (2) This saturation assay is regulated by the equilibrium constant of the reaction between analyte and antibody. (3) Sensitivity of the assay is dependent upon the affinity constant of the antibody. (4) Cross-reactiveantigen will demonstrate a relative potency dependent upon the rate of the equilibrium constants of the analyte and cross-reactiveantigen. (5) Assay reaction is slow since equilibrium must be reached.

size, availability and labelling capacity of analytes, sensitivity and speed of analyte determination. If the analyte is small, easy to isolate, synthesize and label, there are no special requirements on assay sensitivity, its determination in a type II assay using labelled antigen will be obvious. If, on the other hand, the analyte is of high molecular weight, difficult to purify for labelling in sufficient quantity and requires a high sensitivity, the type I assay using labelled antibodies (Miles and Hales, 1968) will be the method of choice. To determine the extent of the immune reaction, the unbound labelled reagent has to be either separated from the bound one (heterogeneous assay) or the activity of the label has to be changed to the extent of the immune reaction in a way to make separation of bound and free reactants (bf separation) superfluous (homogeneous assay). -

-

Separation of bound and free reactants (bf separation)

The signal produced by the label is generally measured in the immune complex. Signals of free labelled reagent misclassified as bound are subtracted from the signals of the immune complex. Since bf separation has got an essential influence on precision, sensitivity and the handling of a test, the following requirements have to be met: (1) removal of free antigen and free antibodies without disturbing the equilibrium of the reaction; (2) reproducible quantitative separation of unbound molecules from the immune complexes; (3) no impairment of marker enzyme activity; (4) good and cost-efficient adaptation to automation. The different principles of bf separation are listed in Table II.

Solid phase techniques introduced by Wide and Porath (1966) Itave won through against the other methods due to the following advantages: (1) universal applicability for each system independently of the analyte's nature; (2) nearly complete separation of the immune complexes formed ( > 95% of bound labelled reagent); (3) easy and quick feasability, full automation depending on the system, and lower cost. For developing assays to be used in laboratories, protein adsorption on plastic surfaces (Catt and Tregear, 1967) is the easiest way. Binding is achieved by rate-limiting diffusion to the solids followed by a rapid and irreversible adsorption and is completed within 12-15 h. Dimethyl solfoxide (DMSO) and sodium dodecyl sulfate (SDS) inhibit solid-phase adsorption. However, unsoluble proteins (e.g., recombinant proteins) solubilized by SDS can be adsorbed by addition of 0.1-0.5 mol/! KzHPO 4 to the wells, tubes or

~' 250-

V//////////////////////////A

~ 200-

I:t:r 7~d

~ 150-

~

iate loG

~//////////A 100. High IgG

V//////////////////////////M density

~

50-

0

~

I

I

I

I

I

2.5

5

10

20

40

Antibody concentration used for adsorpt/on [mg//]

........ .

@ @~ .~ .,. '

....

"

Overloaded IgG

removed by washing

~'/////////.4,//.-'/////////////A

Fig. 1. Binding capacity of solid-phase antibodies in relation to antibody concentration used for adsorption determined with antibodies to cq-fetoproteinand t251-1abelledAFP (left) and artist compositionof solid-phaseantibodies(right).

8

TABLE II PRINCIPLES AND METHODS OF BF SEPARATION

balls, which precipitates SDS as potassium dodecyl sulfate. T h e precipitate is removed by washing the solid phase after protein adsorption. A d s o r p tion is relatively i n d e p e n d e n t o f pH. In phosphate buffer, p H 7 - 8 , the protein d~nsity r e a c h e d is the same as in carbonate buffer at p H 9.5. T h e rate and extent o f adsorption is only slightly influenced by t e m p e r a t u r e (McGinlay and Bardslcy, 1989). For solid-phase adsorption, the diagnostically relevant antigen or the specific antibody should be as p u r e as possible since in protein mixtures proteins start to c o m p e t e for free binding sites at a coating density o f 150 n g / c m 2 protein (Pesce et al., 1977). In contrast to antibody adsorption, the antigen binding capacity and thus the measuring range o f the assay can be enlarged by oriented solid-phase binding o f anti-

(1) Fractionated precipitation - Polyethylene glycol 6000 (final conc. 15%) - Ammonium sulphate (final cone. 35-50%) - Cold ethanol (final cor~c.60%) (2) Immunological precipitation (double antibody technique) -Anti-species antibodies together with inert immunoglobulin (3) Adsorption - Ion-exchange membranes - Dextran coated charcoal - Staphylococcus aureus bacteria strain Cowan 1 or purified insolubilized protein A (4) Solid-phase systems - Surface solid-phase systems (tubes, balls) - Particulate solid-phase media (cellulose, agarose beads m a g n e t i c particles)

Absorbance 492 nm 2.0

,,.0"

,e

1.5

1.0 /

i tl

,,.,..O

Mort9

/

AFP

Mon38

0.5 ~.e

,'~

.

,

,~...,~...&....a.

..... ....~...A..&..k'~"" ,

2

-

,

20

~_ ,

200

AFP[IIg/I]

Mort38

AFP

Mon9

Fig. 2. Dose-response curves in an enzyme immunometric assay for AFP and dependency on the affinity constant of the monoclonal antibody used f3r solid-phase adsorption and for enzyme labelling: KA (I.mol -t) M o n 9 unlabelled M o n 9 - H R P labelled

1.5x I0 I0 1,8× 109

Mon 9 solid-phase coated Mon 38 unlabened Mon 38-HRP labelled Mon 38 solid-phase coated

1 × 109 7.6x 10e 1 x 107 1.2 × 10s

The other factors like agitation or enhancement of reaction temperature rather accelerate the rate of diffusion, which limits their effect at a given geometry of the reaction vessel (coated tube, coated well, coated bail) (King et al., 1990). If the undirected molecular movement is transformed into a directed one by filtration, as in solid-phase immunofiltration assays, the incubation time can be reduced to a few minutes only (IJsselmuiden et al., 1989).

& .......... ..... A" ¸¸"¸¸at..¸.¸¸¸¸.

o

o.s

1

1.s

2

2.s

a

3.s

4

4s

I n c u ~ t ~ r i ~ /h/

Fig. 3. Time courses of analyte bindingto solid-phaseinsolubilized ligandsin relation to surface area. • ~ *, ligand coated polystyrenebead with a surface area of ~ 1.25 cruZ; • e, ligand coated latex microparticleswith a surface area of ~ 10 cm2 usinga final concentrationof 0.125% solids. Reprinted from King et al. (1990 in: lmmunodiagnosisof Cancer, p. 86, by courtesyof Marcel Dekker.)

bodies via the Fc portion. There are two procedures of choice: (1) preeoating with protein A and reaction with the CI-12 and CH 3 domains of the antibodies; (2) antibody binding via carbohydrate moiety of the Fc portion after periodate oxidation to NH 2 groups at the solid phase. Solid-phase binding reduces the affinity constant of the antibody by one order of magnitude (Arends, 1971). The binding constant continues to decrease with increasing loading density of antibodies due to steric hindrance (Fig. 1). The solid phase has only a limited capacity for binding proteins. If the antibody concentration exceeds 1 p . g / c m 2 IgG, unstable bi- and polylayers are formed, from which analyte molecules binding to the second layer are removed as antigen-antibody complexes by bf separation. Therefore, it should 'be considered that: (1) antibody concentration should not exceed 10 /zg/ml lgG for solid-phase adsorption; (2) in two-site assays always the antibody with the higher affinity constant should be used for solid-phase adsorption (Fig. 2). The solid-phase considerably influences sensitivity and duration of the assay. The larger the relative surface and the shorter the ways of diffusion, the quicker the equilibrium between bound and unbound reagents will be reached (Fig. 3).

E n z y m e s and substrates

The demands on marker enzymes (Table l i d make only a limited number of enzymes admissi¢ ble for EIAs (Table IV). Basically, the enzyme should permit fluorimetric, luminometric or colorimetric measurement of the products formed. Although peroxidase and alkaline phosphatase can be detected as free enzymes by fluorescent and luminescent products with a higher sensitivity than by colorimetry (Table V), the detection limit in the type I assay can be increased by fluorogenic product measuring only by factor 2-10 at maximum (Porstmann, B. et al., 1985). However, due to the higher sensitivity of detection, fluoTABLE !11 (A) REQUIREMENTS ON ENZYMES AS MARKERS (1) High specific activity (turnover number) as free enzyme and after labelling (2) Availability of soluble, purified enzyme at low cost and reproducible quality (3) High stability in free and conjugated form under storage and assay conditions (4) Presenceof reactive groups for covalent linkage (5) Simple and gentle labellingmethods (6) Inexpensiveand stable non-toxicsubstrates with formation of stable chromogenicand/or fluorogenicproducts (B) ADDITIONAL REQUIREMENTS ON HOMOGENEOUS ASSAYS (1) High specificactivity at optimum conditionsfor immune complex formation (2) Absenceof enzymaticor inhibitoryactivityin the sample (3) Absenceof substratesor products in the sample (4) Availabilityof selective inhibitorsor inhibitingantibodies (5) Availabilityof high molecularweight forms

rimetry or luminometry permit a greater dilution of the product or a considerable reduction of the reaction volume. Assays of a few microliters have thus become possible as ultramicromethods. Nonetheless, eolorimetric product m e a s u r i n g is the most frequent detection method for EIA. Colorimetry has the following advantages: (1) visual evaluation in large-scale screenings (e.g., monoclonal antibodies or u n d e r field conditions);

(2) simple and relatively cheap p h o t o m e t e r s with extremely rapid measurings ( ' ; - 5 s per microtitration plate); (3) long-lasting stability of the colored product after reaction stop. R e q u i r e m e n t s on chromogenic substrates are listed in Table VI. Kinetic m e a s u r i n g is primarily used in h o m o g e n e o u s EIAs. High specific enzyme activities and p r o n o u n c e d alterations of activity in the course of the i m m u n e reaction are necessary

TABLE IV (A) ENZYMES COMMONLY USED AS LABELS FOR HETEROGENEOUS EIA Enzyme

Source

pH optimum

Alkaline phosphatase

Calf intestine

9-10

/3-galactosidase

E. coli

6-8

Spec. act. (U/mg at 37°C)

Mol weight

Chromogenic substrates and measurement

1000

100000

p-nitrophenyl-phosphate A= 405 nm (pNP)

600

540000

o-nitrophenyl-fl-o-galactopyranoside (oNPG) A= 420 nm Chlorophenolic red-fl-Dgalactopyranoside (CPRG) = 574 nm

Peroxidase

Horseradish

5-7

45(111

40000

H 202/2,2'-azino.di(3.ethyl. benzthiazoline sulfonic acid-6) (ABTS) A= 415 nm H 202/3,3',5,5'-tetramethylbenzidine (TMB) A= 450 nm H 202/o-phenylenediamine (oPD) A= 492 nm

Glucose oxidase/ peroxidase

Aspergillus niger

Urease peroxidase

Jack bean niger

Urease

Jack beans

4-7

200

186000

Coupledenzyme reaction glucose + chromogen for IIRP Glucose + chromogen for HRP

6.5-7.5

I0000

483000

Urea/bromcresol yellow x = 588 nm

(BI ENZYMES COMMONLY USED AS LABELS FOR HOMOGENEOUS EIA Enzyme

Source

pH optimum

Glucose 6phosphate dehydrogenase

leuconostoc mesenteroides 7.8

Lysoz)'me

Egg white

4.5-5.5

Malate dehydrogenase

Pig heart

8.5-9.5

Spec. act. (U/mg at 37°C)

Mol weight

Chromogenic substrates and measurement NADP glucose 6-phosphate A= 340 nm

400

1000

104000 14500

Fragmentation of cell walls (Micrococcus lysodeikti~us) a = 450 nm

70000

NAD/malate A= 340 nm

II ]'ABLE V ENZYME ACTIVITIES AND DETECTION LIMITS OF NATIVE AND IgG COUPLED HORSERADISH PEROXIDASE (HRP), ALKALINE PHOSPHATASE lAP) AND /3-GALACTOSIDASE USING CHROMOGEN1C AND FLUOROGENIC SUBSTRATES Parameters Molar activity (mol/s x l x tool) Specific activily (mmol/s x l x g) Detection limit of enzyme (tool/I)

Chromogenie substrates HRP AP

/3-Gal

Fluorogenic substrates HRP AP

/3-Gal

2600(ABTS)

354 (oNPG)

196 (HPAA)

125 (MUG)

8511(pNP)

291)(MUP)

65 (ABTS) 8.5 (pNP) I).68 (oNPG) 4.9 (HPAA) 2.0 (MUP) 0.24 (MUG) 10- t3 (ABTS) 2×I0+t3(pNP) 2×I0-13(oNPD) 5×10 14(HPAA) 10Is(MUP) 5×IIIn'(MUG) 10-14 (oPD) 10 - ~-~(RG) It) - 15(HPPA) 2× 10-15 (TMB) 3× 10 -14 (CRPG)

Spee. activity of conjugates (mmol/s×l × g) 9.4 (ABTS) Detection limit 16 (ABTS) of labelled IgG 2 (oPD) (ng/ml) 0.3 (TMB)

2.9 (pNP) 43 (pNP)

0.41 (oNPG) 350 (oNPG)

0.9 (HPAA) Ill (HPAA) O.3 (HPPA)

0.9 (MUP) tl.5 (MUP)

0.15 (MUG) 1.0 (MUG)

Key: ABTS

= 2,2'azino-di(3-ethylbenzthiazoline sulphonic acid-6); oPD = o-phenylenediamine; TMB = 3.3'.5,5'tetramethylbenzidine; pNP = p-nitrophenyl phosphate; oNPG = o-r~itrophenyl-/3-D-galactopyranoside: RG = resorufin-/3-Dgalaetopyranoside; CPRG =chlorophenolic red-/3-D-galactopyranoslde: HPAA = p-hydroxyphenylacetic acid; HPPA = 34phydroxyphenyl) propionic acid; MUP = 4-methylumbelliferyl phosphate: MUG = 4-rqethylumbellferyl-/3-D-galactopyrannside.

to g u a r a n t e e sensitive assays with short s u b s t r a t e reaction times. T w o - p o i n t m e a s u r i n g s after reaction stop are u s e d in h e t e r o g e n e o u s E1As testing large p a n e l s of s a m p l e s a n d w h e n e v e r high sensitivity is required. S u b s t r a t e reaction t i m e s are b e t w e e n 10 a n d 30 m i n a n d are t e r m i n a t e d by addition of s t o p p i n g r e a g e n t s (in m o s t c a s e s acids or alkaline solutions) w h i c h o f t e n lead to a b a t h o c h r o m i c or h y p s o c h r o m i c shift of t h e absorption m a x i m u m

TABLE VI REQUIREMENT ON CHROMOGENIC SUBSTRATES (1) Water soluble, odorless, colorless, non-matagenic, nontoxic (2) High molar extinction coefficient of formed product v'~.n a broad absorbance maximum between 400 and 61111nm (3) High binding constant for the enzyme (low K m value) (4) High stability of substrate under storage conditions and of formed product after reaction stop (e.g., non-light sensitivity) (5) Linearity between color intensity and enzyme concentration over a wide range (6) Absence in the sample especially in homogeneous assays

a n d an increased absorption coefficient of t h e f o r m e d p r o d u c t ( P o r s t m a n n , B. et al., 1981; P o r s t m a n n , T. et al., 1981). T h e m a r k e r e n z y m e of choice for h e t e r o g e n e o u s E I A s is h o r s e r a d i s h peroxidase ( H R P ) . Both c h r o m o g e n i c (substrate: H 2 0 2 a n d 3,3',5,5'-tetramethylbenzidine) a n d fluorogenic product m e a s u r i n g (substrate: H a 0 z a n d 3-(4-hydroxypenyl) propionic acid) p r o d u c e d a h i g h e r sensitivity in t h e two-site E I A t h a n using alkaline p h o s p h a t a s e (AP) a n d /3-galactosidase (/3-Gal) as m a r k e r e n z y m e s u n d e r identical conditions of i m m u n e reaction ( P o r s t m a n n , B. et al., 1985). T h e sensitivity of t h e assay can be inc r e a s e d several times if A P a n d /3-Gal (but also H R P at 10°C a n d below) are used as m a r k e r e n z y m e s by prolonging the s u b s t r a t e reaction. T h i s is, however, o f n o interest for laboratory routine, w h e r e t h e r e are, in addition, often problems with the background. T h e signal of the initial p r o d u c t can be amplified even m o r e by enzymatic cycling systems. T h e sensitivity of a two-site assay can be e n h a n c e d by factor 40 in c o m p a r i s o n with p - n i t r o p h e n y l phosp h a t e as t h e classical substrate by a redox cycle of alcohol d e h y d r o g e n a s e / d i a p h o r a s e a n d excessive

12 TABLE Vll REQUIREMENTSON LABELLINGPROCEDURES (!) Simpleand rapid performance (2) Reproducible composition of conjugate molecules (constant molar ratio of enzyme and reagent), homogeneous conjugate moleculeswith respect to molecular mass (3) High yield of labelled reagent, low yield of homopolymers of enzyme and reagent (4) Low-gradeinactivationof reagent and enzyme (5) Adjustable labellinggrade of reagent molecules (6) Simpleprocedures to separate the labelled from the unlabelled reagent and the free enzyme. (7) Long-term stability without loss of immunological and enzymatical activity.

ethanol, in which the red colored formazan is formed from colorless INT and which is started by AP used as marker enzyme and NADP used as initial substrate (Johannsson et al., 1985). However, dosage of conjugate and substrate and bf separation have to be done with great care in order to prevent unspecific reactions.

Enzyme labelling and conjugate purification Enzymes are covalently bound to the reagents either directly by reactive groups to both or after introducing reactive groups (e.g., thiol or maleimid groups) indirectly via homo- or heterobifunctional agents in two-step procedures (Ishikawa et al., 1983). Requirements on optimal conjugation are listed in Table VII. In general, the labelling reaction follows the mass law of action and the conjugate yield depends on the concentration of activated enzyme and reagent (Tijssen, 1985). Short coupling times require high concentrations of reactive enzyme and reagent ( > 10 mg/ml). In the two-step method using glutaraldehyde (GA) (Avrameas and Ternynck, 1971) only 10% of the HRP molecules are activated, in the periodate (PI) method (Nakane and Kawaoi, 1974) it is at least 80%. If the reaction conditions, IgG concentrations and molar H R P / l g G ratios are identical, only 30-40% of the IgG molecules are labelled in GA coupling, against 90-95% in periodate coupling. The degree of substitution of reagent by

enzyme in PI coupling is 2-3 times higher than in GA coupling. The enzyme activity in the conjugates, however, decreased only by 5-10% in GA coupling, but in PI coupling down by 40-50% (Porstmann, T. and Porstmann, B., 1979). Reagents are usually labelled with AP or fl-Gal using the GA method (Avrameas, 1968). The most frequent coupling method for peroxidase is the PI method improved by Wilson and Nakane (1978). Most labelling procedures, except for the hinge region coupling method (Ishikawa et al. 1983), result in heterogeneous complexes regarding the molecular weight. In immunoassay free enzyme, especially when polymer, leads to a decreased P/N ratio due to an increased background (N). Unlabelled reagent molecules compete with enzymeqabelled ones for analyte binding and thus reduce the signal emitted from the immune complex (P). The removal of unlabelled molecules from the conjugate mixture is the main step towards increasing the sensitivity of the assay (Porstmann, T. et al., 1981). Conjugate purification procedures are based on: (l) different solubility behavior of enzymelabelled antibodies and free enzyme (ammonium sulfate precipitation); (2) different charges of enzyme-labelled and free reagents and free enzyme (ion-exchange chromatography); (3) different molecular sizes of enzyme-labelled and unlabelled reagents and free enzyme (gel filtration, dialysis); (4) different structures, e.g., carbohydrate residues of reagents (antibodies) and enzyme (HRP) (ligand chromatography on Protein Aand ConA-Sepharose). In addition to EIAs with covalent linkages between enzymes and reagents there are also 'unlabelled' E l A procedures, in which the marker enzyme is bound via an anti-enzyme antibody (Lenz, 1976). This enzyme/anti-enzyme complex must be linked to the immune complex in which the analyte is bound. There are, in general, three ways of doing this:

(1) Bridge antibody technique Comparably to immunohistochemistry a preformed enzyme/anti-enzyme antibody complex is bound via an anti-lgG antibody to the analyte-antibody complex (Sternberger et al.,

1970). Analyte-specific and anti-enzyme antibodies have to be produced in the same species. Complexes with monoclonal anti-enzyme antibodies, formed in excessive enzyme, produce the best sensitivity in EIA (Tcrnynck et al., 1983).

highly active enzyme (Porstmann, B. ct al., 1985), it has not spread, it is, however, an alternative whenever direct labelling of antibodies decreases the binding constant of much more than one order of magnitude. Instead of labelling the enzyme, the reagent can be biotinylated, in biotinylation of IgG, biotin/igG ratios between 10:1 and 5(1:1 arc recommended for coupling. Highly biotinylatcd lgG molecules make tests more sensitive but tend to increase the background as a result of unspecific binding. The biotinylated reagent is detected either by streptavidin-labelled HRP or AP, or by biotinylated enzyme linked to the biotinylated reagent via avidin, acting as bridge (soluble avidine-biotin complexes, ABC) (Hsu et al., 1981).

(2) Chimera antibody technique Analyte-specific antibodies are covalently bound to enzyme-specific ones by glutaraldchyde and complexed to enzyme with molar excess (Guesdon et al., 1983). (3) Bispecific antibody technique Bispecific monoclonal antibodies produced by the fusion of a hybridoma secreting monoclonal antibodies to the analyte with a hybridoma producing monoclonal anti-enzyme antibodies equimolarly bind analyte and marker enzyme (Karawajew et al., 1988). Though the 'unlabelled' method, due to the specificity of the anti-enzyme antibody, permits use of very crude enzyme preparations with the same sensitivity as the 'labelled' method using

Two-site enzyme immunoassays for antigen detection The easiest variant is the direct two-site assay. if it is performed as a one-step assay with simul-

Enzymes covalently bound

Enzymes immunologically bound

I

direct assay

Ir

indirect assay

anti-hapten antibodies

I

enzymeanti-enzyme complexes

antibody chimera

bispecific antibodies

Fig. 4. Immune complexesformed by the different variants of enzyme immunometricassays using cowdcnllyand immunologically bound marker cnzyme.

tancou,~ incubation of sample, solid-phase antibody and labelled antibody, the high-dose hook effect has to be taken into consideration (Nomura ctal., 1983). When absorbance decreases despite increasing analyte concentration, the type ! assay has changed into a type 11 assay. There is growing competition for reaction with the solid-phase antibody between the free analyte and the one to which the free enzyme-labelled antibody has bound (Porstmann, T. et al., 1983). The following aspects should be considered in the development of one-step two-site EIAs: (1) the solid-phase antibodies should have the highest possible antigen binding capacity (e.g., antibody-coated microparticles); (2) the biological variation of the analyte under normal and pathological conditions should not exceed a certain range. If the concentration is changed by more than three orders of magnitude, as, eg., in acute phase proteins, sample and conjugate should be incubated sequentially (two-step EIA). Because of the hook effect it is generally recommendable to use the sample to be tested in two dilutions differing from each other by at least factor 100. If the tag antibody cannot be labelled or if the sensitivity is of priority over the speed of the EIA, the tag antibody should be detected in the indirect EIA by a labelled anti-species antibody. To avoid cross-reaction with the solid-phase antibody, capture and tag antibodies have to be raised in different species (two-species assay). However it is recommendable to produce the anti-species antibody in the same species as the analyte specific capture antibody. A two-species assay can be circumvented, if: (1) only Fab' or F(ab') 2 of the analyte-specific antibody are used as solid-phase antibody and the labelled anti-species antibody reacts specifically with the Fc portion of the complete tag antibody; (2) the tag antibody is labelled with a hapten (e.g., FITC or FDNB) and if its binding to the analyte is detected by an enzyme-labelled anti-FlTC or anti-FDNB antibody. The indirect two-site EIA is at least 2-5 times more sensitive than the direct EIA (Porstmann, T, et al., 1982).

The immune complexes formed in the different variants of two-site EIAs with covalently and immunologically bound marker enzymes are shown in Fig. 4. Only two-site EIAs permit determination of complex proteins, as, e.g., proteohormones or secretory lgA in the presence of free subunits, the solid-phase antibody being directed against the first polypeptide chain and the enzyme-labelled antibody against the second one (Hamaguchi et al., 1982; Wada et al., 1982).

Homogeneousenzyme immunoassays The sensitivity of homogeneous EIAs primarily depends on the changes of the signal to be measured. Such changes are attained during or after the immune reaction by: (1) high-affinity antibodies; (2) low molecular analytes; (3) formation of large immune complexes (e.g., by addition of anti-species antibodies); (4) substrates of high molecular weight (Ullman and Maggio, 1981). The small size of haptens acting as analytes insures an intimate interaction between antibody and enzyme or its modulator in the formed immune complexes much better than large antigens. Homogeneous EIAs can also be divided into type 1 and type !I assays (Fig. 5), depending on the reaction principle used. However, only competitive EIAs for analytes of low molecular weight have spread in routine diagnosis.

Competitit'e binding assays In antigen-labelled techniques the hapten is either directly labelled with active enzyme (direct modulation) or bound to a modulator of enzyme activity (enzyme inhibitor or inactive pre-stage of the enzyme) (indirect modulation). If the antibody binds to the enzyme-labelled hapten, the enzyme activity is blocked and product formation is directly correlated to the hapten concentration of the sample. Malate dehydrogenase with a high molecular weight substrate (Rowley et al., 1975) and glucose-6-phosphate dehydrogenase are used as marker enzymes. The latter acts in coupled reactions in which the

formed NADH reduces either a ferric salt to a colored ferrous dipyridyl complex Am,X= 525 nm) er nitro blue tetrazolium (NBT) by means of diaphorase as auxiliary enzyme. The absorption coefficient of NBTH at Ass.n m is three times higher than of NADH at A34.. m (Dona, 1985). The enzyme multiplied immunoassay technique (EMIT) was also adapted to macromolecules using high molecular weight dextran bound nitrophenyl-/3-galactoside as fluorogenic substratc. In an IgG EIA the extent of inhibition of enzyme activity after reaction with anti-lgG depends on the molecular weight of the substrate (no inhibition with oNPG) which is indicative for steric hindrance of substrate access to the enzyme (Gibbons et al., 1980). The low sensitivity (detection limit for lgG 100-200 /zg/I) did not make this variant of homogeneous assay an alternative of heterogeneous EIAs.

in the liposomc-labelled immunoassay, enzyme (peroxidase) is released by cemplement-mcdiatcd lysis of antigen- or hapten-coated liposomcs (Haga et al., 1981) after antibody binding, which then converts thc substrate in solution. Since labelled liposomcs compete with the analyte in serum for antibody binding, product formation is conversely related to the concentration of the analyte in the sample (rev. by Nakamura et al., 1986). It is, yet, difficult to standardize the size of liposomcs and to determine the content of encapsulated enzyme as well as to label a reproducibly constant number of antigens or haptens in order to render all liposomes equally well susceptible to complement. In substrate-labelled immunoassays using substrate-labellcd hapten, immune complex formation protects the substrate fl-galactosyl umbelliferonc from the enzyme /3-galactosidase and pre-

Homogeneous enzyme immunoassays F Non-competitive binding assays

Competitive binding assays

I

Substrate labelled technique

Antigen labelled

Direct modulation

Indirect modulation

I

i

Hybridantibody immunoassay

LEn. . . bi,o E°me' homogeneous immuneassay

technique

i

L

i

enhance enhancement

immuno immunoassay

I

l Substratelabelled fluorescence

immunoa.ssay

I

I

I

immunoassay technique

mediated immunoassay

, reactivalion ][ immunoassay

donor ,mmunoassay

Fig. 5. Classification of homogeneous enzyme immunoassays.

enzymesensitNe II technique

J

Enzyme channeling immunoassay

I

vents the development of fluorescent umbelliferone (Burd et al., 1977). Since substrate-labelled analyte competes with sample analyte for antibody binding, fluorescence intensity produced by enzyme-mediated hydrolysation is directly proportional to the analyte c o n c e n t r a t i o n . Substrate-labelled immunoassays are characterized by a relative substrate deficiency and are thus less sensitive than EMITs working with excessive substrate. In enzyme'modulator labelled immunoassays the affinity of the modulator to the enzyme decides upon the sensitivity of the assay. Inhibitors are inactivated after the antibody has bound to the labelled analyte so that enzyme activity is kept in the sample in the absence of analyte. In addition to enzyme inhibitors, as, e.g., methotrexate as inhibitor of dihydrofolate reductase (Place et al., 1983), also activity inhibiting antibodies, e.g., anti-peroxidase (Ngo and Lenhoff, 1980)or the avidin-biotin system (Ngo et al., 1981) have been used for enzyme modulation. In apoenzyme reactivation immunoassay (Morris et al., 1981), cofactor-labelled immunoassay (Carrico et al., 1976) and cloned enzyme donor immunoassay (Henderson et al., 1986) inactive enzymes or enzyme subunits are reactivated by the prosthetic group FAD in the case of glucose oxidase or by coenzyme, as NAD, for lactate dehydrogenase or completed by NH 2 terminal peptides for /3-galactosidase. Prosthetic groups, coenzymes and NH2 terminal peptides as hapten labels are prevented from enzyme reactivation after antibody hapten reaction. The product formed by the reactivated enzyme is directly proportional to the hapten concentration in the sample. The principle of enzyme-channelling immunoassay is based on a coupled enzyme reaction which is accelerated when the two enzymes get very close to each other by immune reaction. Hapten and enzyme 1 (hexokinase) are coimmobilized on agarose beads and sample hapten competes with agarose-coupled hapten for binding to antibody labelled with enzyme 2 (glucose-6-phosphate dehydrogenase). Binding of enzyme 2labelled antibody to insolubilized hapten approaches both enzymes to start the coupled reaction (Litman et al., 1980). The activity of the second enzyme is inversely proportional to the

antigen concentration in the sample. This assay variant can also be used for determination of macromolecules, e.g., lgG.

Non-competitive binding assays In the non-competitive assay variant, the enzyme channelling immunoassay uses epitope-different antibodies, one labelled with enzyme I and the other one with enzyme 2. All assays based on proximal linkage, however, have got, as the heterogeneous one-step two-site assay, a high dose hook effect, but not as a result of changed assay principles, but rather comparable to reduction of agglutination and precipitation in excessive antigen by binding enzyme 1- and enzyme 2-labelled antibodies to different antigen molecules (Ashihara, 1990). Bispecific antibodies, in which the analytespecific part of the antibody is combined with the enzyme-inhibiting part, have been used for homogeneous hybrid antibody immunoassays. After binding to the analyte, the enzyme-inhibiting antigen binding site of the bispeciflc antibody is blocked so that the enzyme is not inhibited anymore. Enzyme activity is thus directly proportional to the antigen concentration (Ashihara, 1990). Sensitivity and measuring range of the assay can be increased by reducing the flexibility of the hybrid antibodies in the hinge region by chemical modification. Unlike in antigen-labelled technique, the enzymes a-amylase or dextranase are covalently coupled to analyte-specific antibodies in the enzyme-labelled one. In the enzyme inhibitory homogeneous immunoassay antigen binding inhibits access of highly molecular substrates to the active center of the enzyme. Product formation is indirectly proportional to antigen concentration in the sample. To enhance steric hindrance and to eliminate interferences due to rheumatoid factors, Fab instead of complete lgG has been bound to the enzyme. Proteins, such as AFP und ferritin, were still detectable in this assay in concentrations of 10 ng/ml. In the enzyme enhancement immunoassay, loading effects are used to approach enzymelabelled antibodies and substrate. Antibodies labelled with/3-galactosidase und succinylated antibody bind to the macromolecular antigen with

negative load, which attracts the cationic substrate. The enzyme activity is linearily related to antigen concentrations. Because of a high dose hook effect, the test is, for the same reasons as the assays based on proximal linkage, only suitable for parameters with a moderate biological variance (Nishizono et al., 1988). In the associated enzyme-sensitive technique, the differing susceptibility of the enzyme to the substrate 'is used in the immune complex or in free form. Peroxidase in HRP-labelled free antibodies is more rapidly denatured in higher H20 2 concentrations (35 raM) than in HRP-labelled antibodies involved in the immune complexes. The residual enzyme activity of about 8-10% is nearly twice as high as in monomeric HRP antibody form. Product formation is directly proportional to antigen concentration. Pseudoperoxidase activity of hemoglobin in hemolytic samples results in overestimation of antigen concentration (rev. by Ashihara, 1990). The potentially higher susceptibility of homogeneous EIAs to serum constituents requires a higher dilution of samples. Thus, they do not reach the potential sensitivity of heterogeneous enzyme immunometric assays and will not be, at least not in the near future, an alternative in highly sensitive determination of macromoleeules.

lmmunoassays for specific antibodies Detection of specific antibodies first described by Engyall and Perlmann (1971) using the enzyme-linked immunosorbent assay (ELISA) is mucla easier than their quantification, since they act both as binder and as analyte in the assay. The extent of immune complex formation is thus not only dependent upon the amount of antibodies to determine, but also on their affinity. In sandwich ELISA, competitive ELISA and capture bridge ELISA the detection of the antibodies' idiotype is of primary importance. If, however, the isotype of specific antibodies is of interest for infection serology (e.g., detection of primary infection), the capture ELISA should be used as direct or indirect variant.

in the sandwich or antiglobulin ELISA, specific antibodies react, independently of the isotype, with the insolubilized antigen. They are detected by enzyme-labelled species-specific antibodies or by labelled protein A (Surolia and Pain, 1981). Since protein A weakly reacts to some IgG isotypes (e.g., human lgG3, mouse igGl) and the detection of antibodies by high-affinity speciesspecific antibodies is generally more sensitive, labelled protein A has not won through for sandwich ELISAs. Independently of the indicator, the sensitivity of antibody detection depends on the density of diagnosttcally relevant epitopes on the solid-phase. Therefore, the diagnostically relevant antigen should be as pure as possible (Kenny and Dunsmoor, 1983). Purified recombinant proteins or synthetic peptides will increasingly displace viral or bacterial lysate antigens since they do not only enhance the sensitivity, but also the specificity of the assay because of the higher epitope density attainable (D~ipel et al., 1991). Competitive ELISAs detect specific antibodies independently of their isotype by competition with enzyme-labelled specific antibodies for antigen binding. The sensitivity of the assay, however, depends on the affinity of the antibody in the sample. Since affinity may vary a lot, false negative determinations are possible in competitive antibody EL1SAs rather than in sandwich EL!SA. Competitive ELISA, unlike sandwich ELISA, permits both simultaneous and sequential incubation of sample and labelled antibody. Sequential incubation is recommendable for detecting lowaffinity antibodies. Capture bridge ELISAs use the bivalence of the antibodies to be tested by connecting enzyme-labelled antigen with solid-phase insolubilized antigen. In simultaneous incubation of sample and labelled antigen a high dosis hook effect may occur in high antibody concentration, as in the one-step two-site assay. In sequential incubation, very low antibody concentrations may be underdetermined due to bivalent binding to isolubilized antigen molecules available in relative excess and thus lead to false negative results. In the capture ELISA anti-species antibodies directed to the different heavy chains are adsorbed to solid-phase. Antibodies of the respective isotype from the sample are captured in the

14.1

UJ

Iii

ILl

sandwich

competitive

direct

indirect

capture

ELISA

ELISA

capture

capture

bridge

ELISA

ELISA

ELISA

Fig. 6. Immune complexesformed by the different assayvariants to detect specificantibodies.

first step. In the second step labelled antigen is added to check if there are antibodies of the respective specificity among the isotype captured. The ELISA is preferably used to detect IgM antibodies of the desired specificity. Only this assay variant excludes competition for antigen binding between IgG and IgM antibodies of the same specificity. In the indirect capture ELISA unlabelled instead of labelled antigen is added (Van Loon et al., 1983). Its binding to specific antibody is detected, like in the two-site EIA, using enzyme-labelled antibodies directed to the antigen. IgM rheumatoid factors may be disturbing in this variant (Duermeyer et al., 1979). The immune complexes formed in the different variants of ELISAs for specific antibodies are demonstrated in Fig. 6.

Immunoassay parameters After a test has been developed for laboratory scale, which permits a good differentiation in the

concentration range relevant for the analyte using defined standard material (maximum P/N ratio > 15), the assay has to be tested for the following parameters, prior to using it in practice: (1) accuracy; (2) detection limit and analytical sensitivity; (3) imprecision; (4) measurement range; (5) practicability; (6) specificity and sensitivity (Singer et al., 1987). Accuracy is the degree of agreement between the analyte concentration determined in the assay and the real concentration in the material to be tested. The best way to determine it is to compare it to other well established methods of concentration determination based on other procedures. Accuracy is influenced by: (1) specificity of the antibodies; (2) quality of the standard; (3) disruptive factors of the immune and enzyme reaction;

19 (4) systemic errors in the assay procedure (Porstmann, T. and Kiessig, 1991). The detection limit of the assay corresponds to the lowest concentration of antigen exceeding the zero-dose precision. The detection limit mainly depends on the confidence interval, which is chosen from the arithmetic mean of replicates of the zero-dose sample. For antigen assays, a confidence interval of 99.8% is recommended, which corresponds to a cut-off value = xtru~bl~,k~+ 2.8 × SD zero-dose sample. In antibody assays, a cut-off value =-~t~uchl~,k~+ 5 × SD zero-dose sample is recommended in order to increase specificity. The selection of cut-off value should be done very carefully to minimize misclassifications. It finally depends on the sensitivity and specificity required for determining a given parameter, ie, on the biological or medical consequences of a misclassification. Sensitivity is defineti as: true positives true positives + false negatives

× 100%

Specificity is defined as: true negatives true negatives + false positives

x 100%

The reliability of positivity and negativity, defined as predictive value, strongly depends on the prevalence of the event provoking the parameter to be measured (e.g., tumor antigens, anti-HIV antibodies). The analytical sensitivity is defined as the responsiveness of the assay to changes in the concentration of the analyte. It describes the smallest difference, which is still safely determined as being different, it depends on the steepness of the dose-response curve and on the imprecision of analyte concentration determination in this concentration range. The minimum difference in concentrations, which are still determined as different is calculated as: .~ analyte 1 + 2.8 SD < 2 analyte 2 - 2.8 SD The measurement range of the assays depends on two parameters:

(1) the linearity which is the range of the assay, in which recovery is linearily proportional to the amount of added analyte with a slope = 1. The analyte concentrations obtained from five fold determinations (y axis) are plotted versus the theoretical ones (x axis); (2) the imprecision which characterizes the error with which the different analyte concentrations in the assay are determined. To determine the intra-batch or within-run imprecision, different analyte concentrations are analyzed 20 times in a single batch. To determine the between-day or between-run imprecision the different analyte concentrations are analyzed in two-fold determinations in 20 different analytical batches. Analyte concentrations are referred to in order to determine .L SD and the coefficient of variation CV ( % ) = S D / 2 " 100. Because of the heteroscedastic error in the assay in determining different analyte concentrations, imprecision profiles have to be established over the whole range of the standard curve. The measuring range of an assay should be limited to analyte concentrations which are determined with an intra-batch imprecision, CV < 10%, and a between-day imprecision, CV < 15%. As a rule, imprecision is admissible if it results from an SD which does not exceed half the intra-individual biological variation of the analyte. For a satisfactory EIA the between-day imprecision should not exceed the two- to three-fold within-run assay imprecision. The use of assays in laboratory routine requires an internal quality control (within laboratory) to check the reproducibility of assay handling as well as an external quality control (between laboratories) to test the fundamentals of the assay, as, e.g., accuracy, specificity, sensitivity.

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munoassay for alphafetoprotein (AFP). Clin. Chim. Acta 135, 13. Rowley, G.L., Rubenstein, K.E.. Huisjen, J. and Unman, E.F. (1975) Mechanism by which antibodies inhibit haptenmalate dehydrogenase conjugates. J. Biol, Chem. 250. 3759. Singer, R., Clarke, S.F., Crafter, V.A., Gray, B.C., Kilshaw, D., Randen, J.A.. Robinson, J.L. and Whitc, J.M. (1987) Selection and evaluation of laboratory instrumentation in clinical chemistry: II Guidlines for selection and evaluation. Med. Lab. Sci. 44, 6. Sternberger, L.A., Hardy, P.H., Cuculis, J.J. and Meyer, H.G. (1970) The unlabeled antibody enzyme method of immunohistochemistry. J. Histochem. Cytochem. 18, 315. Surolia, A. and Pain, D. (1981) Preparation of protein-A enzyme monoconjugate and its use as a reagent in enzyme immunoassay. In: Methods Enzymol. 73, 176. Ternynck, T., Grcgoire, J. and Avrameas, S. (19~3) Enzyme anti-enzyme monoclonal antibody soluble immune, complexes (EMAC): their use in quantitative immunoenzymatic assays. J. lmmunol. Methods 58, 109. Tijssen, P. (1985) Practice and Theory of Enzyme Immunoassay. Elsevier, Amsterdam, ch. 11, p. 224. Ullman, E.F. and Maggio, E.T. (1981) Principles of homogeneous enzyme-immunoassay. In: E.T. Maggio (Ed.), Enzyme-lmmunoassay. CRC Press, Boca Raton, FL., p. 105. Van Loon, A.M., van der Logt, J.TM., Heessen, F.W.A. and van der Veen, J. (1983) Enzyme-linked immunosorbent assay that uses labeled antigen for detection of immunoglobulin M and A antibodies in toxoplasmosis: Comparison with indirect immunofluorescence and doublesandwich enzyme-linked immunosorbent assay. J. Clin. MicrobioL 17, 997. Wada, H.G., Damisch, R.J., Baxter, S.R., Marcia, F.M., Fraser, R.C., Linn, J., Brownmiller, LJ. and Lankford, J.C. (1982) Enzyme immunoassay of the glycoprotein tropic hormones - choriogonadotropin, lutropin, thyrotropin with solid-phase monoclonal antibody for the a subunit and enzyme coupled monoclonal antibody specific for the fl-subunit. Clin. Chem. 28, 1862. Wide, L. and Porath, J. (1966) Radioimmunoassays of proteins with the use of Sephadex-coupled antibodies. Biochim. Biophys. Acta 130, 257. Wilson, M.B. and Nakane, P.K. (1978) Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In: W. Knapp, K. Holubar and G. Wick (Eds.), Immunofluorescence Related Staining Techniques. Elsevier, Amsterdam, p. 215.