The low abundance and activity of catalytic antibodies are major obstacles to their selection from the virtually unlimited repertoire of antibody binding sites. The requirement for new screening methodologies is further emphasized by the availability ofcombinatorial libraries, in which a functional polypeptide has to be selected out of millions of possibilities. We present a simple and sensitive screening approach (termed catELISA) based on immobilized substrates and immunodetection of the end product of the catalyzed reaction. (4), which are then screened to select those clones producing monoclonal antibodies that bind the hapten. The direct screening of culture supernatants of these hybridomas for antibody catalysis was heretofore not possible due to relatively high background reaction, the generally low catalytic efficiency of antibodies, and contaminating enzymes that catalyze the same reaction (5). Therefore, to detect catalytic activity, large quantities (usually from ascites fluid) of purified monoclonal antibodies are needed. Only a few, and occasionally none, of the dozens of clones that bind a hapten are catalytic; it is therefore widely recognized that these inefficient and labor-intensive procedures must be replaced by rapid and direct screening procedures (2,3,(6)(7)(8)27). Novel, nonhybridoma, methodologies, such as combinatorial variable-region cloning in phage (9, 10), were also used to generate antibodies. Yet, as noted (11), future applications of these methodologies for obtaining catalytic antibodies depend upon appropriate screening. Analysis of the unique problems involved in direct screening of hybridoma supernatants for antibody-mediated catalysis (5) led us to catELISA, an assay involving a substrateprotein conjugate immobilized on microtiter plates. Antibody-catalyzed conversion of any "solid-phase" substrate to a product is then detected by ordinary ELISA, using binding anti-product antibodies (Fig. 1) were able to rapidly screen thousands of hybridoma clones elicited against both a phosphonate TS analog and an amide substrate to detect catalytic cleavage of the corresponding p-nitrobenzyl ester. MATERIALS AND METHODSPreparation of Substrates and Hapten. All of the synthesized substances were purified to homogeneity (udged by thin-layer chromatography and NMR) by crystallization or silica column chromatography. Structures were confirmed by NMR and mass spectrum; satisfactory elemental analyses were obtained for all crystalline compounds. Esters la, lb, and le and amide 2 (Fig. 2) were prepared by the following procedures: (i) allowing the corresponding alcohol or amine to react with glutaric anhydride in the presence of a base (lb, ethanol, sodium ethoxide, reflux; la and le, p-nitrobenzyl alcohol or o-nitrobenzyl alcohol, 1,8-diazabicyclo[5.4.0]-undecane (DBU); 2, p-nitrobenzylamine; Et3N); (ii) coupling of t-butyl glycinate by using the acid chloride prepared with thionyl chloride; and (iii) removal of the t-butyl ester in the presence of trifluoroacetic acid. The methyl est...

Upon testing the ability of several strains of mice to elicit esterolytic antibodies after immunization with a

The x-ray structures of three esterase-like catalytic antibodies identified by screening for catalytic activity the entire hybridoma repertoire, elicited in response to a phosphonate transition state analog (TSA) hapten, were analyzed. The high resolution structures account for catalysis by transition state stabilization, and in all three antibodies a tyrosine residue participates in the oxyanion hole. Despite significant conformational differences in their combining sites, the three antibodies, which are the most efficient among those elicited, achieve catalysis in essentially the same mode, suggesting that evolution for binding to a single TSA followed by screening for catalysis lead to antibodies with structural convergence.

Tetranitromethane (TNM) chemically mutates the binding sites of antibodies so that the nitrated antibodies exhibit pH-dependent binding near physiological pH. Three monoclonal antibodies were selectively modified, each under different conditions, with the resultant loss of binding activity at pH > 8 which is recovered at pH < 6. Recovery and loss of binding are ascribed to the protonation and deprotonation, respectively, of the hydroxyl group of the resulting 3-nitrotyrosine side chain (pKa approximately 7) at the binding site of these antibodies. pH on-off dependency of binding activity, common to all TNM-modified antibodies studied by us so far, may find use in a variety of applications in which controlled modulation under mild conditions is required.

A number of monoclonal antibodies elicited against a nitrobenzyl (Nbzl)-phosphonate transition-state analogue (TSA), and which were selected for the hydrolysis of the corresponding Nbzl-ester, were also found to catalyze the hydrolysis of the analogous p-nitrophenyl(Np) ester with notable efficiency and specificity. The activity towards the Np-ester is higher in terms of rates (kca,; as expected from the higher intrinsic reactivity of Np-esters) ; however, the rate acceleration (k,,,/k,,,,,) is close to or lower than that observed with the Nbzl-ester. Unexpectedly, the affinity to the Np-ester substrate ( l / K M ) and therefore k,,/KM are significantly higher. The best example is antibody D2.4 having a k,,,/KM value of 64 SK' . M-' with the Nbzl-ester and 9400 s-' . M-' with the Np-ester. Moreover, due to a lower product inhibition by p-nitrophenol relative to p-nitrobenzyl alcohol, these antibodies exhibit more than 1000 turnovers with the Np-ester. The differential affinity of these antibodies to the Nbzl-phosphonate TSA versus the Nbzlester substrate (Ks/KTsA or K,/K,) correlates well with the observed rate enhancement (k,,,/k,,,,,). For the Np-ester, however, stabilisation of the transition state (as reflected by KJK,,, and by the catalytic proficiencies, kc,,lKMlk,,,,,) does not fully account for the catalytic power (k,,,lk,,,,,), indicating a more complex catalytic mechanism than simply transition-state stabilization.A comparison of the kinetic parameters of D2.4 with other Np-ester-hydrolyzing antibodies raised against Np-phosphonate haptens emphasizes the marked advantage of this antibody which was elicited against an Nbzl-phosphonate hapten. These results appear to be general: anti-(Nbzl-phosphonate TSA) antibodies obtained from other mouse strains and using different immunization protocols are also efficient Np-esterases. They demonstrate the use of an expanded TSA-hapten, where a spacer (a methylene group) mimics bonds that are partially cleaved in the transition state of the catalyzed reaction.