ANALYTICAL BIOCHEMISTRY 102, 167-170 (1980)

Homogeneous Enzyme Immunoassay for Proteins Employing /3-Galactosidase

IAN GIBBONS, CARL SKOLD, GERALD L. ROWLEY AND EDWIN F. ULLMAN

Syva Rescurch Institute, Pulo Alto. Cai(forniu 94304

Received August 6, 1979

A homogeneous enzyme immunoassay method for proteins is described. The assay com- ponents include a protein antigen-P-galactosidase conjugate, antiprotein antibody, and a synthetic macromolecular substrate. When antibody binds to the conjugate, enzyme activity is inhibited up to 95% because of steric exclusion of the substrate. Free-protein antigen com- petes for antibody, thus preventing inhibition. The extent of inhibition is related to the con- centration of protein analyte. The assay is simple to perform, highly sensitive, and is generally useful for proteins with wide structural variations.

The inhibition of the enzyme activity of enzyme-hapten conjugates by antihapten antibodies forms the basis of homogeneous enzyme immunoassays. The method is ex- ceptionally simple. It requires no separation step and has been widely employed for the measurement of drugs and hormones in serum and urine (1,2). One attempt has been reported to develop similar assays for macromolecular antigens based on anti- body-induced inhibition of protein antigen- enzyme conjugates (3). However, the method requires an inconvenient assay protocol and would be adversely affected by serum. We now wish to report a simple general procedure for the homogeneous enzyme immunoassay of serum proteins.

Homogeneous enzyme immunoassays have employed malate dehydrogenase, glucose - 6 - phosphate dehydrogenase, am- ylase, and lysozyme. The antibody-in- duced inhibition of the enzyme activity of hapten-dehydrogenase conjugates is due to conformational effects caused by intimate interaction of the enzyme and the bound antibody (4). By contrast, inhibition of protein antigen-dehydrogenase con- jugates by antibody is less efficient since

transmission of conformational effects is attenuated by the large protein antigen (S. Weber, unpublished work).

The mechanism of inhibition of hapten- lysozyme conjugates by antibodies has been postulated to depend on steric exclusion of the large glycopeptan substrate rather than conformational effects (1). This mechanism should also in principle allow inhibition of the enzyme activity of protein conjugates provided the substrate were sufficiently bulky. However, lysozyme cannot be used in a homogeneous enzyme immunoassay for serum proteins since serum interferes with its assay. As an alternative, P-galactosi- dase from Escherichia coli appeared to be nearly ideal as an enzyme label. This en- zyme is active at a pH at which there is low endogenous activity in serum, it retains its activity on conjugation to proteins, and there are exceptionally sensitive assay methods available for its detection.

MATERIALS AND METHODS

P-Galactosidase is specific for the galac- rose moiety of the substrate and the agly- cone can be widely varied (5). The synthesis

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168 GIBBONS ET AL.

20 I .

FIG. 1. Inhibition of human IgG//3-galactosidase conjugate activity by anti-human IgG. p-Galactosi- dase conjugate (9.7 nM) bearing 4.3 human IgG molecules per enzyme was incubated with rabbit anti- human y-chain antibodies (Dako) at room temperature for 3.5 h in 10 mM sodium phosphate, pH 7.0, con- taining 0.128 M NaCI, 5 mM NaN,, 1 mg/ml rabbit serum albumin, and 0.05 mM magnesium acetate. Ali- quots (50 ~1) were diluted for assay into 1 ml of 0.4 mM 40K dextran-linked substrate in the above buffer. The change in optical density at 420 nm was measured at 37°C for 30 s. Maximum activity was 0.13 absor- bance units/mm. The antibody titer was determined by adsorption with human IgG-labeled Sepharose beads.

of macromolecular substrates was, there- fore, approached by linking the commonly used o-nitrophenyl+-galactoside (ONPG)’ substrate through the o-nitrophenyl group to high-molecular-weight polymers. For this purpose dextrans (Pharmacia) having average molecular weights of lOK, 40K, 70K, and 2000K were carboxymethylated with sodium chloroacetate in 1.25 N NaOH to give about 0.2 groups/glucose residue. N,N’-Bis(3-aminopropyl)piperazine was coupled to the polymers using 1-ethyl-3- (3-dimethylaminopropyl)carbodiimide at pH 4.8, and the resulting aminodextrans were acylated with the N-hydroxy-suc- cinimide ester of 3-carboxy-6-nitrophenyl- /3-galactoside. Reaction conditions were chosen to ensure that there were few free primary amino groups remaining after completion of the reaction sequence. A de-

’ Abbreviations used: ONPG, o-nitrophenyl-p- galactoside: IgG, immunoglobulin G.

tailed description of the synthesis will be published elsewhere. Enzymatic hydrolysis of 0.4 mM solutions of the lOK, 40K, and 70K substrates (measured with respect to total ONPG residues) turned over, re- spectively, 17, 24, and 33% as rapidly as 0.4 mM unimolecular ONPG (K,, = 0.1 mM). Rates were measured on a Gilford Stasar spectrophotometer by aspirating the assay mixtures into the flow cell immedi- ately after mixing, and after a 10-s delay, reading the change in absorbance at 420 nm over a 30-s period. See Fig. 1 for assay conditions.

Human IgG was employed as a model protein antigen. Conjugates with pgalactosi- dase were prepared using [14C]succinylated IgG (nine succinyl groups per molecule) according to the method of Kitagawa and Aikawa (6). Reaction of IgG with the N- hydroxysuccinimide ester of m-( N-male- imido)benzoic acid introduced four male- imido substituents per molecule as deter- mined by addition of excess cysteine and back titration with Ellman’s reagent (7). The derivatized IgG was coupled with the enzyme through its thiol groups and sepa- rated from unconjugated enzyme and IgG by chromatography on Biogel A5M with 10 mM phosphate, pH 7,0.128 M NaCl, 1 mM Mg acetate. Recovery of enzyme activity was essentially quantitative. The composi- tion of the conjugates was determined from the ratio of radioactivity to enzyme activity.

RESULTS AND DISCUSSION

Up to 7.5 molecules of IgG could be at- tached to each P-galactosidase without sub- stantially changing the enzyme activity measured using unimolecular ONPG. By con- trast, the specific activities of the conjugates averaged about 30% higher than the native enzyme with the dextran-linked substrates. The origin of this increased activity is not yet firmly established but the increase is clearly opposite to what would be expected by reducing the steric accessibility of the

HOMOGENEOUS ENZYME IMMUNOASSAY FOR PROTEINS 169

macromolecular substrates to the enzyme conjugates.

When a conjugate with 4.3 IgGs per enzyme was incubated with excess rabbit anti-human IgG, the activities with 0.4 mM lOK, 40K, 70K, and 2000K substrates were inhibited by 42, 83, 87, and 95%, respec- tively, whereas the activity with respect to unimolecular ONPG was unaffected. Inhibition was caused by an antibody-in- duced increase in K,. Thus when a con- jugate with 2.8 IgGs per enzyme molecule was incubated with 9.7 molar equivalents of anti-human IgG per enzyme, the K, using the 40K substrate increased from 0.9 to 2.4 mM while the V,,, was unchanged. These phenomena are consistent with the expected effect of antibody to sterically decrease accessibility of macromolecular substrates to the enzyme active sites.

While the maximum inhibition of the con- jugates with excess antibody was dependent on the substrate molecular weight, the amount of antibody required to achieve inhibition was not similarly affected. Thus titration of a conjugate with antibody re- sulted in 50% of the maximum reduction in activity with any of the substrates when approximately two equivalents of anti- human IgG had been added (Fig. 1). Pre- incubation of the antibody with free human IgG before addition to the conjugates blocked the inhibition to a degree directly dependent on the IgG concentration used. The assay response in Fig. 2 was obtained after sequential addition of antibody and conjugate to the IgG followed in each case by a l-h incubation. In this assay less anti- body than required for full inhibition was employed to increase sensitivity. The assay was initiated by addition of substrate fol- lowed by a 30-s rate measurement. Alterna- tively, a single incubation of all three com- ponents prior to substrate addition could be used although the assay response was some- what reduced.

With this short measurement time a de- tectability limit of about 25 @ml in the

20

FIG. 2. Assay for human IgG. Samples containing human IgG were incubated for 1 h at room temperature with 13 pmol of rabbit anti-human y-chain antibodies in 50 /.LI of buffer. &Galactosidase conjugate (SO ~1 of a 19.4 nM solution) was added, the mixture incubated for an additional hour, and assayed. The reagents and assay conditions are described in Fig. I. The activity is given as a percentage of that with no antibody.

assay mixture was observed (see Fig. 2). In a study of 15 human serum samples using the former protocol, good correlation was obtained with measurements made by fluorescence excitation transfer immuno- assay(8)(v = 0.97,~ = 0.91x + 0.21,S.E.E. = 1.4 mg/ml, mean 12.1 mgfml). Good correlations with radial immunodiffusion assays have also been obtained using a different conjugate and a more stream- lined protocol (B. Gushaw, unpublished observations).

Interestingly, the maximum antibody-in- duced inhibition of enzyme conjugates was unaffected by the number of IgG molecules (M, 160,000) in the conjugate within the range of 2.5-7.5 IgG molecules/enzyme. Since the enzyme has four identical sub- units (of M, 116.000) (9), binding by anti- body to conjugates with low IgGienzyme ratios appears to simultaneously affect the activity of more than one site. The mecha- nism of this highly efficient inhibition has not yet been established but may be associated with formation of matrices.

The successful demonstration of similar assays for human IgM (M, - 900,000) and albumin (M, 65,000) (to be reported else-

170 GIBBONS ET AL.

where) indicates that this method is not unduly sensitive to antigen structural vari- ations. Although further clinical study is needed, the procedure appears ideally suited for clinical use since it is automatable and can be performed using simple clinical instrumentation.

REFERENCES

1. Rubenstein, K. E., Schneider, R. S., and Ullman, E. F. (1972) Biochem. Biophys. Res. Commun. 47, 846-851.

2. Greenwood, H. M., and Schneider, R. S. (1978) in Current Topics in Clinical Chemistry (Samuel Natelson, ed.), Vol. 3 pp. 455-473.

3. Morita, T. N., and Woodbum, M. J. (1978) Infecf. Immun. 21, 666-668.

4. Rowley, G. L., Rubenstein, K. E., Huisjen, J., and Ullman, E. F. (1975) /. Biol. Chem. 250, 3759-3766.

5. Wallenfels, K., and Weil, R. (1972)in The Enzymes (P. D. Boyer, ed.), Vol. 3, pp. 617-663, Academic Press, New York.

6. Kitagawa, T., and Aikawa, T. (1976) J. Biochem. Japan 19, 233-236.

7. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77.

8. Ullman, E. F., Schwarzberg, M., and Rubenstein, K. E. (1976) J. Biol. Chem. 251, 4172-4178.

9. Fowler, A. V., and Zabin, I. (1977) Proc. Nat. Acad. Sri. USA 74, 1507-1510.