A Competitive Immunoassay for the Detection ofEsterolytic Activity of Antibodies and Enzymes1

Fabio Benedetti,*,2 Federico Berti,* Massimiliano Flego,* Marina Resmini,†,2,3 and Erica Bastiani‡*Dipartimento di Scienze Chimiche, Universita di Trieste, Via L. Giorgieri 1, 34127 Trieste, Italy;†Dipartimento di Chimica Organica e Industriale, Universita di Milano, Via L. Venezian 21,20133 Milano, Italy; and ‡Tecna, Area Science Park, Padriciano 99, 34012 Trieste, Italy

Received July 30, 1997

Screening of a large number of clones produced in afusion is often the bottleneck in the isolation of cata-lytic antibodies. The usual approach requires twosteps: clones are first selected for their high affinity tothe antigen, and then the good binders are tested fortheir catalytic activity. To simplify this selection pro-cess, a competitive enzyme-linked immunosorbent as-say (ELISA) has been developed that allows directscreening of the antibodies on the basis of their cata-lytic activity. In this assay, the product of the cata-lyzed reaction binds to an immobilized anti-productantibody in competition with a peroxidase–productconjugate. The screening assay has been developed forthe antibody-catalyzed hydrolysis of esters of p-amino-phenylacetic acid and has been tested on the porcineliver esterase (PLE)-catalyzed hydrolysis of the samesubstrates. This test allows the detection of productformation at the nanomolar level, while, in a typicalassay, the catalytic activity of PLE can be traced downto 200 fmol of enzyme. Under standard conditions forthe screening of hybridomas obtained from a fusion,the competitive ELISA allows detection of catalyticspecies with values of kcat > 5 3 1027 mol l21 s21 andkcat/kuncat > 50. While the assay has been designed forthe selection of catalytic antibodies, other potentialapplications of this methodology are in the screeningof libraries of engineered and designed enzymes and,in general, in the quantitative measurement of enzymeactivity. © 1998 Academic Press

Three different approaches to the design of enzymeswith novel catalytic properties have emerged in thepast decade. The first consists of the modification of anaturally occurring enzyme, either by chemical reac-tions (1, 2) or by genetic engineering (2, 3), in order toobtain proteins with new or improved catalytic proper-ties. A second approach to novel catalysts is based onthe evolutionarily guided generation and selection ofcatalytic species that are able to convert a rationallydesigned substrate (4). Finally, the immunosystem’sability to generate a virtually unlimited repertoire ofbinding sites can been exploited for the production ofcatalytic monoclonal antibodies for a wide variety ofchemical transformations (5, 6). A common feature ofthese approaches is the generation of large libraries ofmacromolecules in which the catalytic species must beisolated from the inactive ones. Therefore, simple andsensitive analytical methodologies to detect catalyticactivity are essential for the development of rapid andefficient selection strategies.

In the case of catalytic antibodies, in particular, theconventional strategy consists of the selection of IgGs,obtained by immunization (5) or from a combinatoriallibrary of genes (7, 8), that display high affinity for asuitable transition state analog; following this initialscreening, the good binders are then tested for catalyticactivity. This sequence of operations is mainly dictated bypractical reasons as it is easier, in general, to detectbinding than catalytic activity, particularly when the cat-alysts are present at low concentrations, as in the screen-ing of hybridoma cultures or of an antibody library. Itthus is convenient, from a large number of antibodies, toisolate those that are able to specifically bind the transi-tion state mimic and then to perform a kinetic assay onthis smaller subset, in order to detect catalysis. Thisapproach is not ideal for several reasons. The first is apractical issue in which a double screening is requiredthat could be avoided if a quick and sensitive assay for

1 Dedicated to the memory of Professor Giancarlo Jommi.2 To whom correspondence should be addressed. Fax: 139 40

6763903. E-mail: benedett@dschsun1.univ.trieste.it.3 Current address: Department of Biochemistry, Medical Sciences

Building, Queen Mary & Westfield College, University of London,Mile End Road, London E1 4NS, United Kingdom. Fax: 1 44 1819830531.

0003-2697/98 $25.00 67Copyright © 1998 by Academic PressAll rights of reproduction in any form reserved.

ANALYTICAL BIOCHEMISTRY 256, 67–73 (1998)ARTICLE NO. AB972482

catalytic activity were available. If such an assay can beapplied to the whole original set of antibodies, then thefirst selection of good binders becomes unnecessary. Theother limitations are of a more fundamental nature. Cat-alytic activity results from the ability of the catalyst (en-zyme or antibody) to selectively bind and stabilize thetransition state of a given reaction with respect to thereagents in their ground state (9–11). There is howeveran intrinsic limitation to our ability to accurately repro-duce the geometric and electronic features of a transitionstate in the stable analogs that are used as probes forstructural complementarity; a high specificity for the an-alog thus does not necessarily imply an equally highspecificity for the transition state. Moreover, if the anti-body’s binding site specifically recognizes some structuralfeatures of the analog present in both the transition stateand the ground state, selective stabilization of the formerwill be unlikely. A selection strategy based on bindingthus may lead to the isolation of good binders that are notnecessarily good catalysts, while at the same time reject-ing antibodies that might have serendipitously developeda catalytic activity not programmed by the structure ofthe hapten (11).

In this paper we present a kinetic assay for the directdetection of catalytic activity which is based on theimmunodetection of the product by a competitiveELISA4 test (Fig. 1a). A microplate is coated with anti-product antibodies via a secondary antibody; the reac-tion is normally carried out, in solution, in the presenceof the catalytic antibody, or enzyme, to be assayed; andthe reaction mixture is then incubated in the wellstogether with an enzyme conjugate of the product.Upon catalysis, the free product from the catalyzedreaction competes with this conjugate for binding tothe immobilized anti-product antibody, resulting in thedecrease of the absorbance signal obtained when achromogenic enzyme substrate is added to the well.

To prepare the methodology for the screening of a setof monoclonal antibodies raised against the transitionstate analog 4 (Fig. 2), the assay has been optimizedand tested on the uncatalyzed and enzyme-catalyzedhydrolysis of esters 1.

MATERIALS AND METHODS

Materials. BSA, HRP, gelatin from cold-water fishskin, EDC, Mes, TMB, Tris, dialysis tubes, all thereagents for organic synthesis, the enzymes, and horseliver acetone powder were purchased from Sigma–Al-drich Italia (Milano, Italy). Sephadex G25 was fromPharmacia Biotech (Milano, Italy). Maxisorp F8 mi-

crotitration plates were from Nunc (Denmark). Sheepanti-rabbit IgG was from Tecna (Trieste, Italy).

Reagents and products. p-Aminophenylacetic acid,protected as trifluoroacetamide (12), was activated asacyl chloride and coupled with 1-(a-naphthyl)-1-penta-nol and a-naphthylmethanol (13); the esters thus ob-tained were N-deprotected with sodium borohydride(14) and finally acylated at the amino group with glu-taric anhydride to give esters 1a and 1b, respectively.Similarly, product 2 was obtained from p-aminopheny-lacetic acid and glutaric anhydride.

Preparation of the hapten–protein conjugates. Theconjugates of product 2 with KLH, BSA, and HRP wereprepared as follows: 1.3 mg of EDC and 400 ml ofN,N-dimethylformamide were added to a solution ofthe protein (10 mg) and 2 (7.5 mg) in 1 ml 0.05 M Mesbuffer, at pH 4.5. The mixtures were stirred for 4 h andthen extensively dialyzed against PBS buffer and theconjugates were purified on Sephadex G25. A ratio ofabout 20 bound product molecules per enzyme mole-cule could be estimated from the ultraviolet spectra forboth the BSA and HRP conjugates. A TMB oxidationtest confirmed that the HRP conjugate retained thecatalytic activity of native HRP. The conjugates werestored at 220°C in PBS.

Preparation of the anti-product serum. A New Zea-land white rabbit was immunized with a KLH conju-gate of acid 2 (15). The conjugate (1 mg) in 1 ml 0.15 MNaCl was emulsified with Freund’s complete adjuvantand injected in multiple sites subcutaneously. Boosterimmunizations were subsequently administered after3 and 5 weeks using Freund’s incomplete adjuvant.One week after the second and third injections bloodsamples were taken from the ear vein and serum wasprepared by centrifugation. The titer of anti-2 antibod-ies was tested by noncompetitive ELISA using the BSAconjugate of acid 2 and compared to the titer of thepreimmune control serum. The antiserum thus ob-tained was used without any further purification.

ELISA. The microtitration plates were coated witha purified sheep anti-rabbit IgG serum diluted in 10mM carbonate buffer, at pH 9.6, to a final concentra-tion of 2.5 mg/ml. Each microwell was filled with 200 mlof this solution and the plates were incubated at 4°Covernight. The wells were washed with 0.05% Tween20 in PBS buffer and saturated with 3% powdered milkin PBS at 37°C for 2 h, and the washing cycle wasrepeated. ELISAs were performed in 0.1 M Tris–HClbuffer (pH 7.5) containing 1% gelatin from cold-waterfish skin. This buffer was used to prepare all the work-ing solutions of the anti-product serum (final dilution1:20,000) and of the product–HRP conjugate (final pro-tein concentration 0.3 mg/ml). The following additionswere made to the wells coated with the anti-rabbitserum: 100 ml of the solution to be assayed for thepresence of acid 2, 50 ml of the anti-product antibody

4 Abbreviations used: BSA, bovine serum albumin; HRP, horserad-ish peroxidase (EC 1.11.1.7); EDC, N-(3-dimethylaminopropyl)-N-ethyl-carbodiimide; PBS, phosphate buffer saline; TMB, 3,39,5,59-tetramethylbenzidine; KLH, keyhole limpet hemocyanin; PLE,esterase from porcine liver (EC 3.1.1.1); ELISA, enzyme-linked im-munosorbent assay.

68 BENEDETTI ET AL.

solution, and 50 ml of the product–HRP conjugate so-lution. The plates were incubated at 4°C overnight andwashed, and 100 ml of a solution of 0.1 mg/ml TMB in0.1 M citrate buffer (pH 4) containing 0.01% hydrogenperoxide was added to each well. The chromogenicreaction was stopped after 30–60 min at room temper-ature by adding 50 ml 2 M sulfuric acid. The opticaldensity at 450 nm was then read with a Spectra SLTmicroplate reader, and the concentration of product 2in the samples was obtained from calibration curvesfrom the same plate. The variability of the signal ob-tained in the absence of free 2 (maximum signal) wascalculated averaging over 10 independent experiments(i.e., 10 different microplates) the standard deviation(SD) relative to the readings of five wells. The detectionlimit for 2 was then evaluated as the minimum analytegiving a signal with a value two standard deviations

lower than the maximum signal. Intraassay precisionwas studied by running standards and samples in trip-licate, and interassay precision was calculated by cal-ibration curves obtained in 10 different assays.

Kinetics. The uncatalyzed hydrolysis of esters 1 (1mM) was followed at pH 11.7 in saturated aqueoussodium carbonate containing 5% acetonitrile. Aliquotsof the reaction mixture were collected at regular inter-vals and quenched by lowering the pH, and the mixturewas analyzed by HPLC (Merck Lichrospher 100-RP 18;MeOH, H2O, CF3COOH 82:17.5:0.5). Enzyme-cata-lyzed reactions were carried out in Tris buffer at pH 7.5and were stopped by removing the enzyme by ultrafil-tration prior to the ELISA. Lipases were used in abiphasic Tris–hexane system.

RESULTS AND DISCUSSION

Considerable effort has been made recently towardthe development of efficient and sensitive methods thatallow the direct selection of catalytic species among thelarge number of antibodies obtained from a fusion orfrom a library (16–23). Under the typical conditions forscreening, the concentration of the product from theantibody-catalyzed reaction is often beyond the detec-tion limits of spectrophotometric methods; the productis thus identified either with the aid of tags (16–19) orby ELISAs (20–22). In most cases (16–18, 20–22) theantibodies are selected for their ability to catalyze theconversion of an immobilized substrate: with this ap-proach, however, interferences from the matrix on thecatalyzed reaction cannot be excluded. An importantstep in the development of a general method for thedirect detection of catalytic activity was represented bythe catELISA technique (20, 21); in this assay (Fig. 1b),the substrate of the antibody-catalyzed reaction is con-jugated to a carrier protein and immobilized on thewells of an ELISA plate. Supernatants from hybridomacell cultures are then incubated in the wells: in thepresence of catalytic activity, the immobilized sub-strate is transformed into immobilized product. Thelatter is then detected with an anti-product antibodyand a secondary antibody conjugated to an enzyme.

We have recently started a project aimed at obtain-ing catalytic antibodies for the enantioselective hydro-lysis of esters such as 1a (Fig. 2), derived from steri-cally hindered secondary alcohols. To set up a protocolfor the selection of catalytic clones generated againstthe transition state analog 4, we tried to apply thecatELISA (21) to the detection of acid 2 which is pro-duced in the hydrolysis. In a preliminary experiment,we immobilized a BSA conjugate of this substrate on amicrotiter plate and carried out the uncatalyzed hydro-lysis at pH 11.7. Surprisingly, no hydrolysis of immo-bilized 1a could be detected when the plate was incu-bated with an antibody specific for the product 2, while,under the same conditions, hydrolysis of free 1a pro-

FIG. 1. Competitive (a) and noncompetitive catELISA (b). In theformer the free product (P), generated in the catalyzed reaction,competes with the enzyme-labeled product for binding to the prod-uct-specific antibody immobilized on the ELISA plate through asecondary antibody.

69DETECTION OF CATALYTIC ACTIVITY OF ANTIBODIES AND ENZYMES

ceeds with an observed rate constant kobs 5 1.16 3 1024

s21 (measured by HPLC). It is likely that the highlyhydrophobic environment surrounding the immobi-lized substrates interferes in this case with the reac-tion to be monitored. This prompted us to study analternative type of assay (Fig. 1a) that would avoidinterferences arising from the immobilization of thesubstrate while maintaining the high sensitivity ofcatELISA.

The initial step in the development of the assay wasthe preparation of the anti-product antibody. To thisend a KLH conjugate of acid 2 was used to obtain ananti-2 antiserum in rabbits. A binding assay carriedout on the BSA conjugate of acid 2, using nonimmu-nized rabbit serum as control, confirmed the high spec-ificity of this antiserum for reaction product 2.

The formation of the immunocomplex between theHRP–2 conjugate and the anti-2 serum was prelimi-narily optimized by performing a series of noncompet-itive ELISAs at different concentrations of both. Re-sults showed that the best conditions for the assaywere obtained when using the antiserum diluted1:20,000 and the HRP–2 conjugate at 0.3 mg/ml (50ml/well). Competitive inhibition of binding by free acid2 was then studied under identical experimental con-ditions: free 2 was incubated, at several concentra-tions, with fixed amounts of HRP–2 and anti-product

antibody. Inhibition of binding of the conjugate wasobtained at concentrations of acid 2 ranging from 200pM to 100 nM (Fig. 3); at these concentrations bindingwas completely inhibited, with the signal reduced tobackground. The presence of 1 nM free acid 2 is suffi-cient to give a 10% decrease in optical density andcan thus be taken as the detection limit of the assay(Fig. 3).

To minimize variability, the assay was designed sothat the reaction mixture to be tested, the product–enzyme conjugate, and the anti-product antibody areadded in a single step to the precoated wells (Fig. 1a).The protocol is thus considerably simplified with re-spect to the classical catELISA (Fig. 1b), requiring asingle washing cycle, after incubation. This is particu-larly important as washings are a major source ofvariability in ELISAs. Other measures taken to reducevariability include the presence of a high concentrationof proteins (from cold-water fish skin) in all the work-ing solutions in order to minimize aspecific binding andincubation at low temperature (4°C). As a result ofthese precautions, variability was very low, both in-traassay (4% on triplicates) and interassay (7% on cal-ibration curves).

A low cross-reactivity between products and reagentsis a strict requirement for a catELISA, aimed at specifi-cally detecting a reaction product. The cross-reactivity of

FIG. 2. Hydrolysis of naphthyl esters 1a and 1b to form the acid product 2. Compound 4 represents the transition state analog that wasconjugated to the KLH protein and used to generate the antibodies.

70 BENEDETTI ET AL.

the anti-2 serum for the substrates 1a and 1b was thusstudied, under the same conditions, in the 0.1–10 mMconcentration range (Fig. 3). Competition between thesecondary ester 1a and the HRP–2 conjugate was ob-served only at concentrations of ester approximately12,000-fold greater than those required for binding withacid 2 (cross-reactivity , 0.01%); no competition wasdetected with the primary ester 1b in the whole range(Fig. 3). Cross-reactivity was not investigated above 10mM because of the low solubility in water of ester 1a.Although solubility can be improved by the addition of anorganic solvent, no attempts were made to optimize theassay in the presence of a cosolvent.

The test was initially used in a kinetic experiment tomonitor the hydrolysis of substrate 1a (100 nM) at pH11.7 in the absence of any catalyst other than OH2.Aliquots of the reaction mixture were taken at fixedtime intervals and incubated in the ELISA plate asdescribed in the previous section. The experimentalpoints (Fig. 4), corresponding to over 50% of the reac-tion, are in excellent agreement with the theoreticalcurve calculated from the pseudo-first-order rate con-stant (kobs 5 1.16 3 1024 s21) determined by HPLC atthe same pH. It should be noted that with the ELISAtest the substrate concentration can be reduced by afactor of 104 with respect to the HPLC assay.

Having tested the analytical system on the spontane-ous hydrolysis of ester 1a, we then turned to study theenzyme-catalyzed reaction. We thus applied the compet-itive catELISA to the screening of several hydrolytic en-zymes at pH 7.5, where the background reaction is too

slow to be detected. Since no enzymatic hydrolysis ofeither ester 1a or 1b had been previously reported, weselected a set of enzymes commonly used in organic syn-thesis and characterized by a broad substrate specificity(24). None of the tested enzymes was able to acceleratethe hydrolysis of the sterically hindered secondary ester1a; however, when the assay was repeated for the hydro-lysis of the primary ester 1b (Table 1), formation of acid2 was clearly detected in the presence of PLE or pow-dered horse liver. The results were confirmed by HPLCon PLE-catalyzed hydrolysis.

The sensitivity of the test was then investigated atthe same pH. The concentration of substrate 1b wasset to 10 mM, the upper limit of the range in whichcross-reactivity had been previously investigated (Fig.3). This concentration is close to the Michaelis constantfor the same substrate; a value of 9 mM was found forKm in a preliminary experiment. Since concentrationsof acid 2 higher than 100 nM are over the range ofsensitivity of our ELISA test, at this substrate concen-tration no more than the first 1% of the reaction can befollowed. Ester 1b and PLE were thus incubated for 30min, with the concentration of enzyme ranging be-tween 16 pM and 130 nM. Figure 5 shows that theenzyme’s catalytic activity can be detected down to 4nM, corresponding to 200 fmol of catalytic species.Above this limit, and up to 130 nM enzyme, the per-centage of inhibition is linearly dependent on PLEconcentration. As we have already pointed out, thesolubility of ester 1a is a limiting factor in this reactionand for this reason the assay has not been optimized

FIG. 4. Hydrolysis of ester 1a (100 nM) in sodium carbonate at pH11.7 and 25°C. The solid line is calculated from the kobs obtained byHPLC (Merck Lichrospher 100-RP 18; MeOH, H2O, CF3COOH 82:17.5:0.5); the dots represent the experimental values obtained fromthe ELISA.

FIG. 3. Titration of product 2 (between 100 pM and 1 mM) andcross-reactivity of substrates 1a and 1b (between 0.1 and 10 mM) inthe competitive catELISA. A and A0 are the absorbances at 450 nmwith and without added free analyte. A/A0 thus corresponds to thefraction of bound HRP–2 conjugate. Error bars correspond to twostandard deviations.

71DETECTION OF CATALYTIC ACTIVITY OF ANTIBODIES AND ENZYMES

for substrate concentrations higher than 10 mM. How-ever, in the more general case, it would be possible tocarry out an assay in the presence of substrate inexcess with respect to Km, using ester 1 with a cosol-vent or a more soluble substrate producing the sameacid 2 by hydrolysis. Simple dilution of the reactionmixture would then be sufficient to bring back thesubstrate and product concentrations within the rangefor which the test has been optimized.

From the lower detection limit of the assay (1 nM) itcan be calculated that the slowest initial rate of hydro-lysis of ester 1 that allows detection of product 2 withina standard reaction time of 30 min is vi 5 1 3 1029 moll21/30 min ' 5 3 10213 mol l21 s21. The high sensitiv-ity of the test thus allows even a very slow reaction tobe detected after a relatively short incubation time.

Catalytic antibodies, in analogy with enzymes, fol-low Michaelis–Menten kinetics (25); therefore, the ini-tial rate of the catalyzed reaction vi,cat is

vi,cat 5@Ab# z kcat z @Si#

@Si# 1 Km, [1]

where [Ab] is the concentration of the antibody’s cata-lytic sites, kcat is the rate constant for the catalyzedreaction, [Si] is the initial substrate concentration, andKm is the antibody’s Michaelis constant. If [Si] @ Km,then the initial rate is independent from the substrateconcentration and equal to the maximum rate vmax:

vi,cat 5 vmax 5 kcat z @AB#. [2]

In a typical kinetic assay for the detection of catalyticactivity in a monoclonal antibody culture supernatant,the substrate concentration can be adjusted so as tomake the catalyzed reaction work at maximum rate,while 1 mM can be taken as the upper limit for theantibody’s binding site concentration (26); thus, intro-ducing in Eq. [2] the lower limit for vi previously cal-culated, it results that catalytic antibodies with a turn-over number as low as kcat 5 5 3 1027 s21 can bedetected by this competitive catELISA. At the same pH(7.5) at which the assay has been optimized, the ob-served pseudo-first-order rate constant for the sponta-neous hydrolysis of secondary ester 1a is kuncat 5 1 3

1028 s21 (M. Resmini, unpublished work). Therefore,any catalytic species with kcat/kuncat $ 50 can be iden-tified by this simple screening strategy.

While it is certainly true that, in general, a catalystwith kcat as low as 1027–1026 would be too weak to beof any practical use, there are reactions, such as, forexample, the hydrolysis of nonactivated amides, wherethe uncatalyzed process is so slow that this turnoverwould still correspond to a substantial acceleration. Inthese cases the high sensitivity of the assay would beparticularly valuable for selecting catalytic speciesthat might otherwise be undetected.

In conclusion, we have described a simple and sensitivecompetitive ELISA for the detection of catalytic activity,in which interferences with the solid support are mini-mized by carrying out the catalyzed reaction with the freesubstrate in solution. As an additional advantage, carry-ing out the reaction to be assayed in solution, rather thanon the immobilized substrate, allows the same test to be

FIG. 5. Catalytic activity in the PLE-catalyzed hydrolysis of ester1b (10 mM) at pH 7.5 and 25°C as a function of enzyme concentra-tion. A is the absorbance at 450 nm after 30 min incubation with theenzyme; A0 is the absorbance under the same conditions but withoutadded enzyme. Error bars correspond to two standard deviationscalculated from triplicate runs.

TABLE 1

Enzymatic Hydrolysis of Ester 1ba

Enzyme: NoneFAP 15lipase Lipase M

Candida albicanslipase LPL

Mucor mieheilipase PLE

Horseliverb

[2]c nM 0.4 0.3 0.2 0.5 ,0.2 0.2 32 61

a Conditions: [1b] 5 100 nM; pH 7.5; [Enzyme] 5 0.019 U/ml.b 1 mg/ml.c Concentration of acid 2 produced after 30 min incubation with the enzyme.

72 BENEDETTI ET AL.

applied to different substrates, and even different reac-tions, provided that the same product 2 is formed. Itshould also be possible to simultaneously test for cata-lytic activity on a pool of different substrates producingthe same product (e.g., different esters 1) and thus selectany antibody that is able to catalyze the conversion of atleast one substrate. The low detection limit of catalyticactivity (kcat 5 5 3 1027 mol l21 s21) should also allow toapply the assay to ‘‘difficult’’ reactions, such as, for exam-ple, the antibody-catalyzed hydrolysis of amides derivedfrom 2.

The assay described here was specifically designedfor the selection of catalytic antibodies; however, thereare other areas where this type of screening strategycould be applied. These include enzyme engineering (2,4), where libraries of proteins obtained by genetic en-gineering must be screened for catalytic activity, andmonitoring the effects of chemical modifications on cat-alytic activity (1). Finally, as a further example ofpossible applications, we are currently developing amethod for the determination of protein kinase inhib-itors in tissue extracts based on the quantitative mea-surement of enzyme activity by this modified cat-ELISA.

ACKNOWLEDGMENTS

This work was supported by CNR (Progetto Biotecnologie e Bio-strumentazioni). We thank Professor U. K. Pandit for critically read-ing the manuscript.

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