Enzyme immunoassay techniques

Journal of Immunological Methods, 150 (1992) 5-21 © 1992 Elsevier Seienee Publishers B.Y. All rights reserved 0022-1759/92/$05.00

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11M 06325

Enzyme immunoassay techniques An overview T. Porstmann and S.T. Kiessig Department o[ Medical Immunology, Medical School (Charite), Humboldt Ilniuersity Berlin, Berlin, Germans (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 seale, simple adsorption of antigens and antibodies is still recommendable though affinity constants decrease bv 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-Iabelled reagents are detectable by fluorogenic product measuring with a sensitivity, which is 10-1000 times higher than using chrornogenic substrates, the sensitivity of the assays can be inereased only by faetor 2-10. Labelling enzymes cannot only be covalently bound to the antibody, but also via anti-enzyme antibodies. Pros and eons of the different methods of eoupling the enzyme z' 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 heterogeneaus 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. Ke» words: Assay principle; Bound-free separation: Marker enzyrne; Conjugates-substrate reaction; Assay parameter

Introduction Correspondcnce to: T. Porstmann. Department of Medieal lmmunology, Medieal School (Charitc), Humboldt University Bcrlin, Schumannstr, 20/21. P.O. Box ISO, 0-1040 Berlin, Gerrnnny rru. 37-2-2Hil.il4H4; Fax: 37-2·2Hil.(475).

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

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that of enzyme immunoassays (EIA) based on the reaction between antigen and antibody. Nevertheless, new variants are being steadily developed with the aim of (I) 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 labelIed ones. Most unlabelled techniques are based on secondary immune reactions, as, e.g., precipitation and agglutination. They are measured by light scattering or partide counting methods. LabelIed immunoassays are based on the primary immune reaction. There are two types, depending on the reaction principle: (l) type I 'reagent-observed'; (2) type 11 '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 11 assays have got a maximum theoretical sensitivity of about 10- 14 moljl (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- 16 moljl (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 dearly distinct from each other in order to permit epitope-different labelIed and unlabelled antibodies to bind without steric hindrance. Type I assays are thus unsuitable for hapten quantification. The different characteristics of type land type 11 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-OB· SERVED' AND TYPE II 'ANALYTE·OBSERVED' AS· SAYS (Frorn Nakamura, R.M., Voller, A. and Bidwell, D.E., 1986) Type I 'reagent-observed' (excess antibody) (l) Maximal sensitivity is attained as amount of antibody

approaches infinity, (2) Theoretical sensitivity of assay is one moleeule of analyte, (3) Cross-reaction antigens will be equipotent with exeess antibody system. (4) Antigen-antibody reaction is less influenced by substances such as salt and urea. (5) Assay time is relatively rapid with labelIed antibody procedures. Type II 'analyte observed' (excess analyte) (l) Maximal sensitivity is attained when antibody concentra-

tion approaehes 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 eonstant of the antibody. (4) Cross-reactive antigen will demonstrate a relative poteney dependent upon the rate of the equilibriurn eonstants of the analyte and cross-reactive antigen. (5) Assay reaction is slow since equilibrium must be reached.

- size, availability and labelling capacity of ana-

lytes, - sensitivity and speed of analyte determination. If the analyte is smalI, easy to isolate, synthesize and label, there are no special requirements on assay sensitivity, its determination in a type 11 assay using labelIed antigen will be obvious. H, 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 labelIed antibodies (Miles and Haies, 1968) will be the method of choice. To determine the extent of the immune reaction, the unbound labelIed re agent has to be either separa ted from the bound one (heterogeneous assay) Of the activity of the label has to be changed to the extent of the immune re action in a way to make separation of bound and free reactants (bf separation) superfluous (homogeneous assay),

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Separation of bound and free reactants (bf separation) The signal produced by the label is generally measured in the immune complex. Signals of free labelIed 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: (l) 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 autornation. The different principles of bf separation are listed in Table 11.

Solid phase techniques introduced by Wide and Porath (1966) have won through against the other methods due to the following advantages: Cl) 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, Dirnethyl sulfoxide (OMSO) and sodium dodecyl sulfate (SDS) inhibit solid-phase adsorption. However, unsoluble proteins (e.g., recombinant proteins) solubilized by SOS can be adsorbed by addition of 0.1-0.5 molyl K zHP0 4 to the wells, tubes or

Low IgG density

Intermediate IgG density

High IgG density

Overloaded IgG

o

2.5

5

10

20

40

Antibody concentration used tor adsorption {mg/I]

density, multilayer removed by washing

Fig. 1. Binding capacity of solid-phase antibodies in relation to antibody concentration used for adsorption determined with antibodies to al-fetoprotein and 125r-labelled AFP (left) and artist composition of solid-phase antibodies (right),

8 TABLE 11 PRINCIPLES AND METI-IODS OF BF SEPARATION (l) Fractionated precipitation - Polyethylene glycol 6000 (final cone. 15%) - Ammonium sulphate (final conc. 35-50%) - Cold ethanol (final cone. 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 I or purified insolubilized protein A (4) Solid-phase systems - Surface solid-phase systems (tubes, balls) - Particulate solid-phase media (cellulose, agarose beads magnetic particles)

balls, which precipitates SDS as potassium dodecyl sulfate. The precipitate is removed by washing the solid phase after protein adsorption. Adsorption is relatively independent of pR. In phosphate buffer, pR 7-8, the protein density reached is the same as in carbonate buffer at pR 9.5. The rate and extent of adsorption is only slightly influ. enced by temperature (McGinlay and Bardsley, 1989). For solid-phase adsorption, the diagnostically relevant antigen or the specific antibody should be as pure as possible since in protein mixtures proteins start to compete for free binding sites at a coating density of 150 ng/cm 2 protein (Pesce et al., 1977). In contrast to antibody adsorption, the antigen binding capacity and thus the measuring range of the assay can be enlarged by oriented solid-phase binding of anti-

-_. __._------_ ..... _._. __ ._-_.-.. -.--- ... __ .. _ - _ . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ,

Absorbeaoe 492 nm

2.0

1.5

Mon9

AFP

Mon38

1.0

0.5

2

20

200

AFP[pt/lI}

Mon38

AFP

Mon9

Fig, 2. Dose-response curves in an enzyme immunometric assay for AFP and dependency on the affinity eonstant of the monoelonal antibody used for solid-phase adsorptlon and for enzyme labelling:

Mon 9 unlabelled Mon 9-HRP labelIed Mon 9 solid-phase coated Mon 38 unlabelled Mon 38-HRP labelIed Mon 38 solid-phase coated

1.5XlO lO 1.8X 109 1 X 10 9 7.6x 10 6

1 X 107 L2x 10 5

9

II .....

}

150

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 weil, coated ball) (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 (Il sselmuiden et al. I 1989).

e--e-e i

~ 125

~

~ 100

\I'

~ 75

.....

"

50

..

,6.."

.'.

..

, "

",

25

0,5

1,5

2,5

3,5

4

4,5

IncubBtion Time {hJ

Fig, 3. Time courses of analyte binding to solid-phase insolubilized ligands in relation to surface area.... - - ..., ligand coated polystyrene bead with a surface area of - 1.25 cm2; . - - . , ligand coated latex microparticles with a surface area of -10 cm! using a final concentration of 0.125% solids, Reprinted from King et al. (1990 in: Immunodiagnosis of Cancer, p. 86, by courtesy of Marcel Dekker.)

bodies via the Fe portion. There are two procedures of choice: (l) precoating with protein A and reaction with the CH 2 and CH 3 domains of the antibodies; (2) antibody binding via carbohydrate moiety of the Fe 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/cm 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 p,g/ml IgG 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).

Enzymes and substrates The demands on marker enzymes (Table 111) 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 a1., 1985). However, due to the higher sensitivity of detection, fluoTABLE III (A) REQUIREMENTS ON ENZYMES AS MARKERS (I) High specific activity (turnover number) as free enzyme and after Jabelling (2) Availability of soluble, purified enzyrne at low cost and reproducible quality (3) High stability in free and conjugated farm under storage and assay conditions (4) Presence of reactive groups for covalent linkage (5) Simple and gentle labelling methods (6) Irrexpensive and stable non-toxic substrates with formation of stable chromogenic and zor fluorogenic products (B) ADDITIONAL REQUIREMENTS ON HOMOGENEOUS ASSAYS (1) High specific activity at optimum conditions for immune

(2) (3) (4) (5)

complex formation Absence of enzyrnatic or inhibitory activity in the sample Absence of substrates or products in the sampie Availability of selective inhibitors or inhibiting antibodies Availability of high molecular weight forms

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rimetry or luminometry permit a greater dilution of the product or a considerable reduction of the reaction volurne. Assays of a few microliters have thus become possible as ultramicromethods. Nonetheless, colorimetric product measuring is the most frequent detection method for EIA. Colorimetry has the following advantages: (1) visuaI evaluation in large-scale screenings (e.g., monoclonal antibodies 01' under field conditions);

(2) simple and relatively cheap photometers with extremely rapid measurings (2-5 s per microtitration plate); (3) long-lasting stability of the colored product after reaction stop. Requirements on chromogenic substrates are listed in Table VI. Kinetic measuring is primarily used in homogeneous EIAs. High specific enzyme activities and pronounced alterations of activity in the course of the immune reaction are necessary

TABLEIV (A) ENZYMES COMMONLY USED AS LABELS FOR HETEROGENEOUS EIA pH optimum

Enzyme

Source

Alkaline phosphatase

Calf intestine

9-10

ß-galactosidase

E. coli

6-8

Mol weight

Chromegenie substrates and measurement

1000

100000

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

600

540000

o-nitrophenyl-ß-D-galactopyranoside (oNPG) A = 420 nm

Spec, act. (U/mg at 37°C)

Chlorophenolic red-ß-Dgalactopyranoside (CPRG) A = 574 nm Peroxidase

Horseradish

5-7

4500

40000

H 202 j2,2 '-azino-di(3-ethylbenzthiazoline sulfonic acid-ö) (ABTS) A = 415 nm

° °

1-1 2

2 j3,3',5,5'-tetramethylbenzidine (TMB) A= 450 nm

1-1 2 2 / o-pheny1enediamine (oPD) A = 492 nrn Glucose oxidasej peroxidase

niger

Coupled enzyrne reaction glucose + chromogen for HRP

Urease peroxidase

Jack bean niger

Glucose + chromogen for HRP

Urease

Jack beans

Aspergillus

4-7

6.5-7.5

200

10000

186000

483000

Urea/bromcresol yellow A = 588 nm

(B) ENZYMES COMMONL Y USED AS LABELS FOR HOMOGENEOUS EIA

Enzyme

Source

pl-l optimum

Spec. act. (U /mg at 37°C)

Mol weight

Glucose 6-

NADP glucose 6-phosphate

phosphate

leuconostoc

dehydrogenase

mesenteroides

7.8

Egg white

4.5-5,5

Pig heart

8.5-9.5

Lysozyme

Chromegeniesubstrates and measurement A = 340 nm

400

104000 14500

Fragmentation of cell walls i Micrococcus lvsodeikticus) A = 450 nm

70000

NAD/malate A = 340 nm

Malate dehydro-

genase

1000

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TABLE V ENZYME ACTIVITIES AND DETECTION LIMITS OF NATIVE AND IgG COUPLED HORSERADISH PEROXIDASE (HRP), ALKALI NE PHOSPHATASE (AP) AND ß-GALACTOSIDASE USING CHROMOGENIC AND FLUOROGENIC SUBSTRATES Parameters Molar activity (rnolysx I X mol) Specific activity (mmolys X I X g) Detection limit of enzyme (rnolyl)

Chromogenic substrates

Fluorogenie substrates

HRP

AP

ß-Gal

HRP

AP

ß-Gal

2600 (ABTS)

850 (pNP)

354(oNPG)

196 (HPAA)

290 (MUP)

125 (MUG)

65 (ABTS) 8.5 (pNP) 10- 13 (ABTS) zx 10- 13 (pNP) 14 10- (oPD) 2xl0- 15 (TMB)

Spec. activity of conjugates (mmolys X I X g) 904 (ABTS) 16(ABTS) Detection limit of labelIed IgG 2 (oPD) (ngyrnl) 0.3 (TMB)

2.9 (pNP) 43 (pNP)

0.68 (oNPG) 4.9 (HPAA) 2.9 (MUP) 0.24 (MUG) 2 X 10- 13 (oNPD) s x 10- 14 (HPAA) 10- 15 (MUP) 5 X 1016 (MUG) 1O-1J (RG) 10- 15 (HPPA) 14 3 X 10- (CRPG)

0041 (oNPG) 350(oNPG)

0.9 (HPAA) 10 (HPAA) 0.3 (HPPA)

0.9 (MUP) 0,5 (MUP)

0.15 (MUG) 1.0 (MUG)

Key : ABTS 2,2'azino-diC3-ethylbenzthiazoline sulphonic acid-ö); oPD= o-phenylenediamine; TMB = 3,3',5,5'tetramethylbenzidine; pNP = p-nitrophenyl phosphate; oNPG = o-nitrophenyl-s-o-galactopyranoside; RG = resorufin-ß-ugalactopyranoside; CPRG = chlorophenolic red-ß-D-galaetopyranoside; HPAA = p-hydroxyphenylacetic acid; HPPA = 3-(phydroxyphenyl) propionie acid; MUP = 4-methylumbelliferyl phosphate; MUG = 4-methylumbellferyl-ß-D-galactopyranoside.

to guarantee sensitive assays with short substrate reaetion times. Two-point measurings after reaetion stop are used in heterogeneous EIAs testing large panels of sampIes and whenever high sensitivity is required. Substrate reaetion times are between 10 and 30 min and are terminated by addition of stopping reagents (in most eases acids or alkaline solutions) which often lead to a bathoehromic or hypsoehromic shift of the absorption maximum

TABLE VI REQUIREMENT ON CHROMOGENIC SUBSTRATES (I) Water soluble, odorless, colorless, non-rnu tagenie. non-

toxie (2) High molar extinction coefficient of formed product with a hroud absorbance maxirnurn between 400 und 600 nm 0) High binding constant Ior thc enzyme (IOIV Km value) (4) High stability nf substrate under storage conditions and of Iormcd pruduct uftcr reuction stop (e.g., non-light sensitivity) (5) Linearity belween color intensity and enzyrne concentration over a wide rungc (6) Absence in the sample especially in homogeneous assays

and an increased absorption coefficient of the formed produet (Porstmann, B. et al., 1981; Porstmann, T. et al. , 1981). The marker enzyme of choice for heterogeneous EIAs is horseradish peroxidase (HRP)_ Both chromogenic (substrate: H 2 0 2 and 3,3',5,5'-tetramethylbenzidine) and fluorogenie product measuring (substrate: H 2 0 2 and 3-{4-hydroxypenyJ) propionic acid) produced a higher sensitivity in the two-site EIA than using alkaline phosphatase (AP) and ß~galactosidase (ß-GaD as marker enzymes under identical conditions of immune reaction (Porstmann, B. et al., 1985). The sensitivity of the assay can be increased several times if AP and ß-Gal (but also HRP at 10°C and below) are used as marker enzymes by proloriging the substrate reaction. This is, however, of no interest for laboratory routine, where there are, in addition, often problems with the background, The signal of the initial product can be amplified even more by enzymatic cycling systems. The sensitivity of a two-site assay can be enhanced by factor 40 in comparison with p-nitrophenyl phosphate as the classical substrate by a redox cycle of alcohol dehydrogenasej diaphorase and excessive

12 TABLE VII REQUIREMENTS ON LABELLING PROCEDURES (1) Simple and rapid perforrnance

(2) Reproducible composition of conjugate molecules (constant molar ratio of enzyrne and reagent), homogeneous conjugate moleeules witb respect to moleeular mass (3) High yield of labelled reagent, low yield of homopolyrners of enzyme and re agent (4) Lew-grade inactivation of reagent and enzyme (5) Adjustable labelling grade of reagent moleeules (6) Simple proeedures to separate the labe lied from the unlabelled reagent and the free enzyrne, (7) Lang-term stability without lass 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 horno- 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 actioated enzyme and reagent (Tijssen, 1985). Short coupling times require high concentrations of reactive enzyme and re agent (> 10 mgy'rnl), 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 Kawaai, 1974) it is at least 80%. If the reaction conditions, IgG concentrations and molar HRP/IgG ra tios are identical, only 30-40% of the IgG molecules are labelIed 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, 8., 1979). Reagents are usually labelIed with AP or ß-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 enzyrne-labelled 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 a1. , 1981). Conjugate purification procedures are based on: 0) different solubility behavior of enzymelabelIed antibodies and free enzyme (ammonium sulfate precipitation); (2) different charges of enzyme-Iabelled and free reagents and free enzyme Gon-exchange chromatography); 0) different molecular sizes of enzyme-Iabelled 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' EIA procedures, in which the marker enzyme is bound via an anti-enzyme antibody (Lenz, 1976). This enzymey 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 apreformed enzymey anti-enzyrne antibody complex is bound via an anti-IgG antibody to the analyte-antibody complex (Sternberger et al.,

13

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 (Ternynck et al., 1983).

(2) Chimera antibody technique Analyte-specific antibodies are covalently bound to enzyme-specific ones by glutaraldehyde 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' metbod using

Enzymes covalently bound

highly active enzyme (Porstmann, B. et al., 1985), it has not spread. It is, however, an alternative whenever direct Iabelling 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 IgO, biotin Zlg'G ratios between 10: 1 and 50: 1 are recommended for coupling, Highly biotinylated IgG 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-Iabelled 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). Two-site enzyme immunoassays for antigen detec-

tion The easiest variant is the direct two-site assay, If it is performed as a one-step assay with simul-

Enzymes immunologically bound

II

y.

--

w

direct assay

w

indirect assay

anti-hapten antibodies

enzyrneanti-enzyme

antibody chimera

bispecific antibodies

complexes

Fig. 4. Immune complexes formed by the different variants of enzyme immunometric assays using covalently and immunologically bound marker enzyme.

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taneous incubation of sampie, solid-phase antibody and labelIed antibody, the high-dose hook effect has to be taken into consideration (Nomura et al., 1983). When absorbance decreases despite increasing analyte concentration, the type I assay has changed into a type II assay. There is growing competition for reaction with the solid-phase antibody between the free analyte and the one to which the free enzyme-Iabelled antibody has bound (Porstmann, T. et a1., 1983). The following aspects should be considered in the development of one-step two-site EIAs: (I) 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, e.g., 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 sampie to be tested in two dilutions differing from each other by at least factor 100. lf the tag antibody cannot be labelIed or if the sensitivity is 01' priarity over the speed of the EIA, the tag antibody should be detected in the indirect EIA by a labelIed 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 01' the analyte-specific antibody are used as solid-phase antibody and the labelIed anti-species antibody reacts specifically with the Fe portion of the cornplete tag antibody; (2) the tag antibody is labelIed with a hapten (e.g., FITC or FDNB) and if its binding to the analyte is detected by an enzyme-labelled anti-FITC or anti-FDNB antibody. The indireet 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 IgA in the presence of free subunits, the solid-phase antibody being directed against the first polypeptide chain and the enzyrne-labelled antibody against the second one (Hamaguchi et al., 1982; Wada et al., 1982).

Homogeneous enzyme 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 eomplexes (e.g., by addition 01' anti-species antibodies); (4) substrates of high moleeular weight (UlJman 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 I and type II assays (Fig. 5), depending on the reaction principle used. However, only competitive EIAs for analytes of low molecular weight have spread in routine diagnosis.

Competuioe binding assays In antigen-labelJed techniques the hapten is either directly labelIed with active enzyme (direct modulation) or bound to a modulator of enzyme activity (enzyrne inhibitor or inactive pre-stage of the enzyrne) (indirect modulation), If the antibody binds to the enzyme-labelied hapten, the enzyme aetivity is bloeked and product formation is directly correlated to the hapten eoncentration of the sampie. Malate dehydrogenase with a high moleeular weight substrate (Rowley et al., 1975) and glucose-6-phospha te dehydrogenase are used as marker enzymes. The latter aets in coupled reaetions in which thc

15

formed NADH reduces either a ferric salt to a colored ferrous dipyridyl complex Amax = 525 nm) or nitro blue tetrazolium (NBT) by means of diaphorase as auxiliary enzyme. The absorption coefficient of NBTH at AS80nm is thre.e times higher than of NADH at A340nm (Dona, 1985). The enzyme multiplied immunoassay technique (EMIT) was also adapted to macromolecules using high molecular weight dextran bound nitrophenyl-ß-galactoside as fluorogenic substrate. In an IgG EIA the extent of inhibition of enzyme activity after reaction with anti-IgG 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 IgG 100-200 ,ug/I) did not make this variant of homogeneous assay an alternative of heterogeneous EIAs.

In the liposome-Iabelled immunoassay, enzyme (peroxidase) is released by complement-mediated lysis of antigen- or hapten-coated liposomes (Haga et al., 1981) after antibody binding, which then converts the substrate in solution. Since labelIed liposomes compete with the analyte in serum for antibody binding, product formation is conversely related to the concentration of the analyte in the sampIe (rev. by Nakamura et al., 1986). It is, yet, difficult to standardize the size of liposomes and to deterrnine the content of encapsulated enzyme as weil as to label a reproducibly constant number of antigens or haptens in order to render all liposomes equally weil susceptible to complement. In substrate-Iabelled immunoassays using substrate-Iabelled hapten, immune complex formation protects the substrate ß-galactosyl umbelliferone from the enzyme ß-galactosidase and pre-

Homogeneous enzyme immunoassays I

Non-competitive binding assays

Competitive binding assays

I

I Substrate labelied

Antigen labelIed

technique

technique

I

I

Hybrid antlbody

Enzyme InhibItory

Enzyme

Assoclated

immunoassay

homogeneous

enhancemenl

enzyme sensitive

Immunoassay

Immunoassay

technlque

I

I

I

I Direct rnodulatlon

Illndirect modulation

I I I

I

I

I

Substrate labelIed

Enzyme multiple

Enzyme modulator-

Apoenzyme

Cloned enzyme

Enzyme

nucrescence

Immunoessay

mediated

reactlvation

donor

channeling

tecnnlque

Immunoassay

immunaassay

Immunoassay

Immunoassay

l,mm""~,~ -----

---- - ------

.".,._._---_.__ ._----

Fig, 5. Classification of homogeneous enzyrne immunoassuys.

__._--

..•..

16

vents the development of fluarescent umbelliferone (Burd et al., 1977). Since substrate-labelled analyte competes with sarnple analyte for antibody binding, fluorescence intensity produced by enzyme-mediated hydrolysation is directly proportional to the analyte concentration. Substrate-labelled immunoassays are characterized by a relative substrate deficiency and are thus less sensitive than EMITs working with excessive substrate. In enzyrne "modulator labelled immunoassays the affinity of the modulator to the enzyme deeides upon the sensitivity of the assay. Inhibitors are inactivated after the antibody has bound to the labelIed 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 donar 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 ß-galactosidase. Prosthetic groups, coenzymes and NH 2 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 sampIe. The prineiple of enzyme-channelling immunoassay is based on a coupled enzyme reaction which is accelerated when the two enzymes get very elose to each other by immune reaction. Hapten and enzyme 1 (hexokinase) are coimmobilized on agarose beads and sampIe hapten competes with agarose-coupled hapten for binding to antibody labelIed with enzyme 2 (glucose-ö-phosphate dehydrogenase), Binding of enzyme 2labelled antibody to insolubilized hapten approaches both enzymes to start the coupled reaction (Litrnan et al., 1980). The activity of the second enzyme is inversely proportional to the

antigen concentration in the sampie. This assay variant can also be used for deterrnination of macromolecules, e.g., IgG.

Non-competitioe binding assays In the non-cornpetitive assay variant, the enzyme channelling immunoassay uses epitope-different antibodies, one labelIed with enzyme land 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 preeipitation 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 hornogeneous hybrid antibody immunoassays. After binding to the analyte, the enzyme-inhibiting antigen binding site of the bispecific 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 redueing the flexibility of the hybrid antibodies in the hinge region by chemical modification. Unlike in antigen-Iabelled technique, the enzyrnes o-amylase or dextranase are covalently coupled to analyte-specific antibodies in the CI1zyme-labelled one. In the enzyme inhibitory homogeneous immunoassay antigen binding inhibits access of highly molecular substrates to the active center of the enzyme. Product Iormation is indirectly proportional to antigen concentration in the sampie. To enhance steric hindrance and tn eliminate interferences due to rheumatoid factors, Fab instead of complete IgG has been bound to the enzyme. Proteins, such as AFP und fcr-. ritin, were still detectable in this assay in conccn-. trations of 10 ng.zrnl. In the enzyme enhancement immunoassay , loading effects are used to approach enzyrne-. labelIed antibodies and substrate. Antibodies la-. belled with ß-galactosidase und succinylated antibody bind to the macromolecular antigen with

17

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 01' in free form. Peroxidase in HRP-labelled free antibodies is more rapidly denatured in higher H 2 0 2 concentrations (35 mM) 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 eoneentration. Pseudoperoxidase activity of hemoglobin in hemolytic sampIes results in overestimation of antigen concentration (rev, by Ashihara, 1990). The potentially higher suseeptibility of hornogeneous EIAs to serum constituents requires a higher dilution of sarnples, 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 maeromolecules.

Immunoassays for specific antibodies Detection of specifie antibodies first deseribed

by Engvall and Perl mann (1971) using the enzyme-linked immunosorbent assay (EUSA) is much easier than their quantifieation, since they aet 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 EUSA, eompetitive ELISA and capture bridge EUSA the deteetion of the antibodies' idiotype is of primary importance. If, however, the isotype of specifie antibodies is of interest for infeetion serology (e.g., deteetion of prirnary infection), the capture ELISA should be used as direet 01' indircet variant.

In the sandwich 01' antiglobulin ELISA, speeifie antibodies reaet, independently of the isotype, with the insolubilized antigen. They are detected by enzyme-Iabelled species-specific antibodies 01' by labelIed protein A (Surolia and Pain, 1981). Since protein A weakly reacts to some IgG isotypes (e.g., human IgG3, mouse IgG1) and the detection of antibodies by high-affinity speciesspecifie 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 diagnostically relevant epitopes on the solid-phase, Therefore, the diagnostically relevant antigen should be as pure as possible (Kenny and Dunsmoar, 1983). Purified recombinant proteins 01' synthetic peptides will increasingly displace viral 01' 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 (Döpel et a1., 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 sampie. Since affinity may vary a lot, false negative determinations are possible in competitive antibody ELiSAs rather than in sandwich ELISA. Competitive .ELISA, unlike sandwich ELISA, permits both simultaneous and sequential incubation of sample and labelled antibody. Sequential ineubation is reeommendable for detecting lowaffinity antibodies. Capture bridge ELISAs use the bivalence of the antibodies to be tested by conneeting enzyme-labelled antigen with solid-phase insolubilized antigen. In simultaneous incubation of sampie and labelled antigen a high dosis hook effeet may oecur in high antibody concentration, as in the one-stcp two-site assay. In sequential incubation, very low antibody eoncentrations may be underdetermined due to bivalent binding to isolubilized antigen moleeules available in relative excess and thus lead to false negative results. In the eapture ELISA anti-species antibodies direeted to the different heavy chains are adsorbed to solid-phase. Antibodies of the respcctive isotype from the sampie are captured in the

18

w

w

sandwich

competitive

direct

indirect

capture

ELiSA

ELiSA

capture

capture

bridge

ELiSA

ELiSA

ELiSA

Fig. 6. Immune complexes formed by the different assay variants to detect specific antibodies.

first step. In the second step labelIed antigen is added to eheck if there are antibodies of the respective specificity among the isotype captured. The ELISA is preferably used to deteet IgM antibodies of the desired speeificity. 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 labelIed antigen is added (Van Loon et al., 1983). Its binding to speeifie antibody is deteeted, Iike in the two-site EIA, using enzyme-Iabelled antibodies directed to the antigen. IgM rheumatoid faetors may be disturbing in this variant (Duermeyer et al., 1979). The immune complexes formed in the different variants of ELISAs for speeifie antibodies are demonstrated in Fig. 6. Immunoassay parameters After a test has been developed for laboratory seale, which permits a good differentiation in the

coneentration range relevant for the analyte using defined standard material (rnaximum P/ N ratio L 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) praetieability; (6) specifieity and sensitivity (Singer et al., 1987). Aecuracy is the degree of agreement betwecn the analyte eoneentration determined in the assay and the real coneentration in the material to be tested. The best way to determine it is to cornpare it to other weIl established methods of eoncentration determination based on other proccdures. Accuracy is influenced by: (l) specificity of the antibodies; (2) quality of the standard; (3) disruptive faetors of the immune and enzyrnc reaetion;

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 sampie. For antigen assays, a confidence interval of 99.8% is recommended, which corresponds to a cut-off value = Xtrueblanks + 2,8 X SD zero-dose sample. In antibody assays, a cut-off value = Xtrue blanks + 5 X 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 biologicalor medical consequences of a misclassification. Sensitivity is defined as: true positives

- - - - - - - - - . - x 100% true positives + false negatives Specificity is defined as: true negatives

- - - - - - - - - , - , - x 100% true negatives + false positives 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:

x analyte 1 + 2.8 SD ~ x analyte 2 -

2.8 SO

The measurement range of the assays depends on two pararneters:

(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 01' 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 SO and the coefficient of variation CV (%) = SD Ix ' 100. Because of the heteroscedastic error in the assay in determining different analyte concentrations, imprecision profiles have to be established over the who!e range of the standard curve. The measuring range of an assay shou!d 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 SO 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 interna! quality control (within !aboratory) to check the reproducibility of assay handling as weil as an external quality contro! (between Iaboratories) to test the fundamenta!s of the assay, as, e.g., accuracy, specificity, sensitivity.

x,

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21

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