AU4213289A

AU4213289A - Method of catalyzing stereochemical reactions

Info

Publication number: AU4213289A
Application number: AU42132/89A
Authority: AU
Inventors: Richard J. Massey; Michael J. Powell; Richard C. Titmas
Original assignee: IGEN Inc
Current assignee: IGEN Inc
Priority date: 1988-08-26
Filing date: 1989-08-25
Publication date: 1990-03-23
Also published as: JPH04501656A; EP0431060A1; IL91397A0; EP0431060A4; AU4195693A; WO1990002192A1; AU657864B2; ZA896408B

Description

METHOD OF CATALYZING STEREOCHEMICAL REACTIONS

This application is a continuation-in-part of copending U.S. Application Ser. No. 674,253, filed

November 27, 1984, which is a continuation-in-part of U.S. Application Ser. No. 556,016, filed November 29, 1983, the contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to the use of monoclonal antibodies to catalyze stereochemical

reactions. Monclonal antibodies are immunoglobulins produced by hybridoma cells. A monoclonal antibody reacts with a single antigenic determinant and provides greater specificity than a conventional, serum-derived antibody. Furthermore, screening a large number of monoclonal antibodies makes it possible to select an individual antibody with desired specificity, avidity, and isotype. Hybridoma cell lines provide a constant, inexpensive source of chemically identical antibodies and preparations of such antibodies can be easily

standardized. Methods for producing monoclonal

antibodies are well known to those of ordinary skill in the art, e.g., Koprowski, H., et al., U.S. Patent

No. 4,196,265, issued April 1, 1980.

Antigen recognition by a monoclonal antibody is attributable to a specific combining site in the N-terminal region of the immunoglobulin (Ig) molecule. Ig molecules are thought to react with antigens via the same types of short range forces characteristic of all

protein-protein interactions. Antigen-antibody

interactions are highly specific because of the

complementary three-dimensional shapes of the antibody's combining site and of the corresponding antigenic

determinant or epitope. Such complementary shapes permit the molecules to approach each other closely and to interact over a substantial surface area. The

specificity of antibody=antigen interactions is evidenced by the fact that changes in the configuration of the antigenic determinant result in marked decreases in the binding constant of the antigen to the antibody. The binding constant of an antibody for its antigen is generally much higher than that of an enzyme for its substrate.

Uses of monoclonal antibodies are known. One such use is in diagnostic methods, e.g., David, G., and Greene, H., U.S. Patent No. 4,376,110, issued March 8, 1983.

Monoclonal antibodies have also been used to recover materials by immunoadsorption chromatography, e.g., Milstein, C., 1980, Scientific American 243:66, 70. However, it has not been suggested that

monoclonal antibodies can be used to catalyze chemical reactions. Indeed, the field of catalysis has developed independently from the field of immunology. The only reported attempt at using antibodies as catalysts, of which applicants are aware resulted only in insignificant rate enhancement of the desired reaction. G.P. Royer, 1980, Advances in Catalysis 29:197-227.

During the course of a chemical reaction, the reactants undergo a series of transitions passing through different states until the products are reached. In molecular terms, these transitions through intermediate states reflect changes in bond lengths, angles, etc. The transition from reactants to products may be viewed as involving formation of an intermediate which decomposes to produce the products. The overall rate of the

reaction can be expressed in terms of the equilibrium constant characterizing the equilibrium between the reactants, the intermediate, and the products.

Catalysis can be regarded as a stabilization of the intermediate with respect to the state of the

reactants. A catalyst is a substance that increases the rate of the reaction and is recovered substantially unchanged chemically at the end of the reaction.

Although the catalyst is not consumed, it is generally agreed that the catalyst participates in the reaction.

Despite the commercial importance of catalysis, major limitations are associated with both simple

chemical catalysis and enzymatic catalysis. Chemically catalyzed processes often do not produce high yields of desired products. Such processes often result in the production of impurities from side reactions.

Furthermore, chemical catalysts are not known for many important chemical reactions. Other limitations include the relatively high cost of catalysts; the requirement for chemical activation; lack of utility under

atmospheric conditions or in the presence of small amounts of water; and flammability or explosivity in the presence of atmospheric oxygen. Enzymatic catalysis depends on the existence and discovery of naturally occurring enzymes with the appropriate specificity and catalytic function needed to perform a particular

reaction. Enzymes are unknown for many chemical

reactions.

The present invention overcomes these limitations by providing a novel approach to catalysis. The invention provides a method for the preparation and use of monoclonal antibodies as convenient, readily obtainable, and inexpensive catalysts having a degree of specificity and efficiency of action not previously achievable in the catalytic arts.

SUMMARY OF THE INVENTION

The present invention relates to a method involving monoclonal antibodies for increasing the rate of a chemical reaction involving conversion of at least on e reactant to at least one product.

In the practice of this invention, the reactant(s) is (are) contacted with an appropriate

monoclonal antibody under conditions suitable for the formation of a complex between the monoclonal antibody ant the reactant(s). The complexed reactant(s) is (are) converted to the product(s), and the product(s) released from the complex.

In one embodiment, this invention is useful in increasing the rate of chemical reactions which can also be catalyzed by enzymes such as oxidoreductases,

transferases, hydrolases, lyases, isomerases, and

ligases. In another embodiment, this invention is useful in increasing the rate of chemical reactions for which no catalytic enzymes are known. Such reactions include, among others, oxidations, reductions, additions,

condensations, eliminations, substitutions, cleavages, and rearrangements.

In accordance with this invention, the rate of the chemical reaction may be increased by more than a hundredfold and preferably more than ten thousandfold.

Conditions suitable for antibody-reactant complex formation are provided by a solution phase or emulsion reaction system including a protic solvent, preferably water, maintained at a pH value between about 6.0 and about 8.0, preferably between about 6.0 and about 7.5, and maintained at a temperature from about 4°C to about 50°C, preferably between about 20°C and about 45°C. The ionic strength μ = ½∑c-z² _i, where c is the

concentration and z is the charge of an ionic solute. It should be maintained at a value below 2.0 moles/liter, preferably between about 0.1 and 1.5 moles/liter. The method of this invention may be carried out under reduced or elevated pressure, but preferably is practiced at ambient pressure.

A monoclonal antibody appropriate for use in the practice of this invention is characterized by K> 1, where K = k_r/k_p, k_r is the affinity constant of the monoclonal antibody for the reactant and k is the affinity constant of the monoclonal antibody for the product. The monoclonal antibody is further

characterized by an r₁ > r_o, where r₁ is the rate of formation of the complex between the antibody and the reactant and where r_o is the rate of the chemical

reaction in the absence of monoclonal antibody, by an r₂ > r_o where r₂ is the rate of the conversion of the complexed reactant to the complexed product and by an r₃ > r_o, where r₃ is the rate of release of the product from the complex.

Methods for preparing appropriate monoclonal antibodies are also disclosed. In one embodiment, hybridoma cells prepared by modifications of methods well known to those of ordinary skill in the art are screened for the ability to produce appropriate monoclonal

antibodies.

In another embodiment, anti-idiotype monoclonal antibodies are prepared for known enzyme-substrate systems. The anti-idiotype monoclonal antibodies can be used to increase the rate of conversion of the substrate to the product and are not subject to allosteric control. DETAILED DESCRIPTION OF THE INVENTION

As indicated above, the present invention provides methods for increasing the rate of a chemical reaction involving conversion of at least on reactant to at least one product. In the practice of this invention, the reactant(s) is (are) contacted with at least one appropriate monoclonal antibody under suitable conditions permitting the formation of a complex between the

monoclonal antibody and the reactant(s), conversion of the reactant(s) to the product(s) and release of the product(s) from the complex.

The monoclonal antibodies useful in the present invention are prepared by modification of the technique disclosed by Koprowski et al. in U.S. Patent No.

4,196,265, issued April 1, 1980, which is hereby

incorporated by reference. The details of that process are well known to those of ordinary skill in the art. In one embodiment of this invention, a series of monoclonal antibodies directed to the reactant are prepared under suitable conditions. This involves first immunizing BALB/C mice with an appropriate antigen. The antigen may be the desired reactant; the desired reactant bound to a peptide or other carrier molecule; a reaction

intermediate or an analog of the reactant, the product, or a reaction intermediate. "Analog," as the term is used herein, encompasses isomers, homologs, or other compounds sufficiently resembling the reactant in terms of chemical structure such that an antibody raised against the analog may participate in an immunological reaction with the reactant but will not necessarily catalyze a reaction of the analog. For example, if the reaction to be catalyzed is the cleavage of o-nitrophenyl-ß-D-galactoside, the antigen may be the analog dinitrophenol bound to a carrier, e.g., keyhole limpet hemocyanin, or the antigen may be the reactant o- nitrophenyl-ß-D-galactoside.

As another example, if the reaction to be catalyzed is the condensation of two molecules of aminolevulinic acid to yield porphobilinogen:

the antigen may be the analog 3-glycyl-4-hydroxy-4- methol-1,5-hepatanedoic acid:

Antibody-producing lymphocytes are then removed from the spleens of the immunized mice and hybridized with myeloma cells such as SP2/0 cells to produce hybridoma cells. These hybridoma cells are then plated in the wells of microtiter plates. The series of monoclonal antibodies being produced by the hybridoma cells is screened under appropriate conditions to identify monoclonal antibodies which catalyze the desired reaction under appropriate conditions. Screening may be conveniently accomplished by treating a standardized solution of the reactant with an aliquot of medium withdrawn from a microtiter well and measuring the presence of the desired product by

conventional instrumental methods. This measurement may be readily conducted, for example, by spectrophotometric methods or by gas-liquid or high pressure liquid

chromatography. By comparison with standardized samples of the desired product or reactant, rates of reaction may be quantified. In this manner, wells containing

hybridoma cells producing catalytic monoclonal antibodies are identified. The selected hybridoma cells are then cultured to yield colonies.

These colonies may be further propagated in vitro or in vivo systems. In the latter case, mice such as syngeneic BALB/C mice are inoculated intraperitoneally with the selected hybridoma cells and produce tumors, generally within two or three weeks. These tumors are accompanied by the production of ascites fluid which contains the desired monoclonal antibodies. The

monoclonal antibodies are then separately recovered from the ascites fluid by conventional methods such as

ultrafiltration, ultracentrifugation, dialysis, and immunoaffinity chromatography.

The monoclonal antibodies of this invention can be characterized by the following equations:

(1) K = k_r/k_p > 1

(2) r₁ > r_o

(3) r₂ > r_o

(4) r₃ > r_o

In equation (1), K is defined as the ratio of the

affinity constant of the monoclonal antibody for the reactant, k_r, to the affinity constant for the monoclonal antibody to the product, k_p. The equation reflects the fact that the monoclonal antibody has a stronger binding affinity to the reactant than it does to the product.

Thus, as a consequence of chemical modification of the reactant to form the product, the binding affinity of the monoclonal antibody for the complexed molecule decreases, and the molecule, the product, is released from the complex, thereby regenerating the free monoclonal

antibody catalyst. Preferably, K is greater than 10 ².

Equations (2), (3), and (4) describe the kinetic characteristics of the monoclonal antibodies useful in this invention.

Equation (2) states that 4₁, defined as the rate of formation of the complex between the antibody and the reactant, must be greater than r_o where r_o is the rate of the chemical reaction in the absence of

monoclonal antibody. Equation (3) states that r₂, defined as the rate of conversion of the complexed reactant to the complexed product, must be greater than r_o. Equation (4) states that r₃, defined as the rate of release of the product from the complex, must be greater than r_o. Those skilled in the art will recognize that the rates r_o, r₁, r₂, and r₃ may conveniently be

determined directly or indirectly by known methods.

As a result of these characteristics, the monoclonal antibodies of this invention can effect a rate acceleration in chemical reactions preferably by more than a factor of 10² and even more preferably by more than a factor of 10⁴.

In accordance with this invention, the separately recovered monoclonal antibodies are contacted with the reactant under suitable conditions permitting the formation of a complex between the monoclonal

antibody and the reactant. Those of ordinary skill in the art will appreciate that the conditions suitable for complex formation may vary depending on the particular reactant and monoclonal antibody under consideration.

Accordingly, the methods of this invention may be

practiced under a variety of reaction conditions, as long as the monoclonal antibodies are not prevented from complexing with the reactant(s) or otherwise rendered inactive. More specifically, suitable conditions for complex formation encompass solution phase and emulsion reaction systems including a protic solvent, preferably water, maintained at a pH value between about 6.0 and about 8.0, preferably between about 6.0 and about 7.5, and at a temperature from about 4°C to about 50°C, preferably from about 20°C to about 45°C. The ionic strength, μ = ½ ∑c_iz² _i, where c is the concentration and z is the electronic charge of an ionic solute, should be maintained at a value below about 2.0 moles/liter, preferably between 0.1 and 1.5 moles/liter. The method of this invention may be carried out at reduced or elevated pressure, but preferably is practiced at ambient pressure. In addition to solution phase and emulsion reaction systems, suitable conditions also include the use of support materials to which the monoclonal antibody is attached. Such support materials are well known to those of ordinary skill in the art as are methods for attaching monoclonal antibodies to them.

The method of this invention is widely useful to increase the rate of any chemical reaction. This method is applicable, for example, to chemical reactions involving the conversion of one reactant to one product. Such reactions include the conversion of an α-amino acid, and can be illustrated by the conversion of indole pyruvic acid to L-tryptophan. Another example is

provided by the conversion of a cyclic polylnucleotide to a linear polynucleotide, the term "polynucleotide" being used herein to include both poly- and oligonucleotides.

The method of this invention is also applicable to chemical reactions of more complex stoichiometry. The rate of reaction involving the conversion of two

reactants to one product, for instance, can also be increased in accordance with this invention. An example of such a reaction is the conversion of two molecules of amino-levulinic acid into one molecule of

porphobilinogen.

The method is also useful for reactions

involving the conversion of one reactant into two

products. Such reactions may be illustrated by the conversion of a β-D-galactoside into D-galactose and a second product, as well as by the cleavage of a

polynucleotide, polypeptide, or polysaccharide into two fragments derived respectively therefrom. As used herein, the terms "polypeptide" and "polysaccharide" include poly- and oligopeptides and poly- and

oligosaccharides, respectively.

The method has further utility in increasing the rate of chemical reactions involving the conversion of one reactant into multiple products. Such reactions include, among others, the conversion of polynucleotides, polypeptides, and polysaccharides into fragments derived respectively therefrom. In another embodiment of this invention, a reactant is contacted with more than one monoclonal antibody, each of which is directed to a different determinant on the reactant. Thus, where the reactant is a polynucleotide and the monoclonal antibodies are directed to different nucleotide sequences within the polynucleotide, specific polynucleotide fragments may be cleaved from the reactant.

The method is also useful in increasing the rate of reactions involving the conversion of two

reactants into two products. Such reactions include the exchange of functional groups between one reactant and a second reactant to yield two new products, e.g.,

transesterification.

The preceding enumeration of stoichiometries is not meant to be exclusive; rather it is intended to indicate the wide scope of utility of the present

invention and indeed that this method is not limited by the stoichiometry of the reaction under consideration.

As is evident also by the preceding discussion and illustrative examples, the method of this invention is useful over the wide variety of chemical reactions including oxidations, reductions, additions,

condensations, eliminations, substitutions, cleavages, and rearrangements, among others.

These examples also illustrate the high degree of catalytic specificity characteristic of this

invention. In the practice of this invention, for instance, monoclonal antibodies may be prepared which interact with a polynucleotide only at a specific

nucleotide sequence or with a peptide only at a specific amino acid sequence.

The method of this invention may be used to increase the rate of reactions which may also be

catalyzed by an enzyme. For example, the enzyme may be an oxidoreductase, such as alcohol dehydrogenase, glucose oxidase, xanthine oxidase, dihydrouracil dehydrogenase. or L-amino acid oxidase; a transferase such as

guanidinoacetate methyl transferase, serine hydroxymethyl transferase, or aspartate aminotransferase; a hydrolase such as acetylcholesterase, glucose-6-phosphatase, or a phosphodiesterase; a lyase such as pyruvate

decarboxylase, aldolase or histidine ammonia-lyase; an isomerase such as ribulose phosphate epimerase, or a ligase such as tyrosyl-tRNA synthase or acetyl CoA carboxylase.

As indicated previously, this method may be used to increase the rate of conversion of two molecules of aminolevulinic acid to one molecule of

porphobilinogen, a reaction catalyzed in nature by the enzyme aminolevulinic acid dehydratase; to increase the rate of conversion of a cyclic polynucleotide to a linear polynucleotide or of a linear polynucleotide to two or more fragments thereof, reactions involving cleavage of a specific phosphodiester bond in the polynucleotide catalyzed in nature by phosphodiesterase (restriction) enzymes; to increase the rate of conversion of an α-keto acid such as indole pyruvic acid to an α-amino acid such as L-tryptophan, a reaction involving transfer of an amino group from a reactant to a product catalyzed in nature by a transaminase enzyme; and to increase the rate of conversions of a β-D-galactoside to D-galactose and a second product, a reaction involving cleavage of a β-D-galactosidase.

In another embodiment of this invention, monoclonal antibodies directed to an antigen which is a known substrate for an enzyme are prepared and used to increase the rate of conversion of the substrate to the product. This method is useful, for example, in

increasing the rate of conversion of o-nitrophenyl-β-D-galactoside, a known substrate for the enzyme β-D-galactosidase, to o-nitrophenol and D-galactose. In this method, a series of monoclonal antibodies to the enzyme are prepared by inoculating BALB/C mice with the enzyme and proceeding according to the general technique

described above. The series of antibodies so produced is screened under suitable conditions to identify a first monoclonal antibody which binds to the active site of the enzyme. Such a monoclonal antibody may be identified by screening for antibodies which under appropriate

conditions inhibit binding of the antigen (substrate) to the enzyme. This screening process may be conveniently carried out by conventional methods of measuring enzyme binding activity, e.g., radioimmunoassay (RIA). This first monoclonal antibody so identified is separately recovered according to the general technique and is used to inoculate fresh BALB/C mice. By following the general technique, a series of monoclonal antibodies to the first monoclonal antibody is produced. The antibodies so produced are termed "anti-idiotype" monoclonal

antibodies. The series of anti-idiotype monoclonal antibodies is then screened according to the general method to identify anti-idiotype monoclonal antibodies which bind the antigen (substrate) under suitable

conditions and convert it to the product. By "suitable conditions" are meant conditions within the parameters described above for antibody-reactant complex formation. An anti-idiotype monoclonal antibody so produced and separately recovered may be used in accordance with this invention to increase the rate of conversion of substrate to product.

Using such a monoclonal antibody in place of the enzyme in this embodiment of the invention is

especially advantageous where the enzyme is allosteric. Allosteric enzymes are enzymes which are stimulated or inhibited by a modulator molecule which may be the substrate, the product, or some other molecule. As a result, the kinetic behavior of allosteric enzymes is greatly altered by variations in the concentration of the modulator(s). A relatively simple example of allosteric behavior may be illustrated by an enzyme which is subject to feedback inhibition. In such a case, the catalytic efficiency of the enzyme decreases as the concentration of an immediate or subsequent product increases. Use of such enzymes in many applications is thus limited and requires continuous removal of product. In accordance with this invention, use of the appropriate anti-idiotype monoclonal antibody which is not subject to allosteric control in place of the enzyme can thus overcome the problems and limitations of allosterism.

It is also contemplated that the method of this invention can be used to increase the rate of reactions which can also be catalyzed by nonproteinaceous organic molecules, hereinafter termed cofactors, such as

pyridoxal phosphate, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, adenosine triphosphate, thiamine pyrosphosphate, flavin mononucleotide, biotin,

tetrahydrofolic acid, coenzyme B12 and coenzyme A.

Reactions which can be catalyzed by pyridoxal phosphate, for instance, include the inter-conversion of α-keto acids and α-amino acids. This and other reactions catalyzed by cofactors alone are relatively slow and nonselective. To overcome the problems encountered in using the cofactor alone, a monoclonal antibody may be prepared in accordance with this invention that combines the relatively inefficient catalytic capabilities of a cofactor alone with the highly specific and efficient advantages of the monoclonal antibody. To prepare such a monoclonal antibody, mice are inoculated with the

cofactor bound to the reactant or to an analog of the reactant or product, and the general technique of

Koprowski described above is followed. A series of hybridoma cells is then prepared according to the general method and screened for the production of monoclonal antibodies which can complex with free cofactor and reactant, increase the rate of the chemical reaction, and release the product. Such a monoclonal antibody directed against indole pyruvic-acid-pyridoxamine phosphate imine, for example, selectively increases the rate of conversion of indole pyruvic acid to the amino acid tryptophan. In the practice of this embodiment of the invention, the appropriate cofactor is added to the reaction mixture preferably in an amount at least equimolar to that of the monoclonal antibody.

In addition, the ability to rationally design industrial catalysts with high specificity and turnover numbers has been greatly sought after and can now be realized with the advent of monoclonal antibody

catalysts. These monoclonal antibody catalysts exhibit chemical reaction accelerations of several million over background. The rational design and isolation of these monoclonal antibody catalysts is achieved by immunizing mice with appropriate compounds such as reactant, an analog of the reactant or isomer of the reactant, or an analog that resembles the transition state, or a

stereoisomer of the transition state analog, for the desired reaction, followed by the routine techniques of monoclonal antibody production.

The following examples are set forth to

illustrate specific embodiments of the invention.

Materials and Methods

In the examples below, the chemical and biological reagents were obtained from commercial sources as follows: o-nitrophenyl-β-D-galactosidase and buffers were obtained from Sigma Chemical Co., Saint Louis,

Missouri; dinitrophenol (DNP) and dinitrobenzene

sulfonate were obtained Eastman Kodak Co., Rochester, New York; goat anti-mouse immunoglobulin labeled with

horseradish peroxide and 2,2'-azino-di(3-ethylbenzthiazoline sulfonic acid) (ABTS) were obtained from KPL Laboratories, Inc., Gaithersburg, Maryland;

microtiter plates (Immulon II®) were obtained from

Dynatech, Alexandria, Virginia; kanomycin was obtained from GIBCO Laboratories, Grand Island, New York; fetal calf serum and keyhole limpet hemocyanin and other proteins can be obtained from Calbiochem-Behring,

San Diego, California; cell growth media and supplements can be obtained from M.A. Bioproducts, Walkersville, Maryland; Sp20 myeloma cells (ATCC CRL 1581) were

obtained from the American Type Culture Collection,

Rockville, Maryland; other reagents, e.g., 9-nitrophenyl-β-D-galactoside, 5-aminolevulinic acid, hydrogen

peroxide, phenol, magnesium sulfate, sodium bicarbonate, indole-3-pyruvic acid, pyridoxal 5-phosphate molecular sieves and morpho CDI can be obtained from Aldrich

Chemical Co., Saint Louis, Missouri. BALB/C mice were obtained from the National Cancer Institute, Frederick Research Facility, Frederick, Maryland. Adjuvants were obtained from Sigma. Mouse mammary tumor virus RNA may be extracted by conventional methods from a commercially available mouse mammary tumor virus, e.g., MTV ATCC VR-731 (American Type Culture Collection). The analog 3-glycyl-4-hydroxy-4-methyl-1,5-heptanedioic acid may be prepared by conventional synthetic methods, e.g., by base catalyzed condensation suitably protected molecules of aminolevulinic acid and levulinic acid (Aldrich) followed by deprotection and HPLC purification.

Example 1

Immunization of mice with o-nitrophenyl-β-D-αalactoside

One group of female BALB/C mice (Group 1 in Table 1) at 7 weeks of age were inoculated intravenously with 10 mg of o-nitrophenyl-β-D-galactoside (ONPG) and intraperitoneally with 12 mg of ONPG on day 0. The ONPG was dissolved in 0.1M phosphate buffer at pH 7.3 at a concentration of 25 mg/ml and warmed to 37°C. On day 33, the mice were inoculated intraperitoneally with 12.5 mg of ONPG in incomplete Freund's adjuvant. The ONPG phosphate buffer solution was mixed with an equal volume of incomplete Freund;'s adjuvant and emulsified prior to inoculation. A blood sample was obtained from each mouse on day 54. The serum was separated from the blood sample by centrifugation and stored at 4°C.

Example 2

Immunization of mice with dinitrophenol-keyhole

limpet hemocvanin conjugate

Mice inoculated as in Example 1 were inoculated intraperitoneally on day 91 with dinitrophenol (DNP) coupled to keyhole limpet hemocyanin (KLH) and emulsified in incomplete Freund's adjuvant. The inoculum contained 10 mg of protein as determined by the method of Bradford, 1976, Anal . Biochem . 72:248. The dinitrophenol was coupled to KLH by the method of Little and Eisen, 1967, Meth . Immunol . Immunochem . 1 : 12. The DNP-KLH inoculation was repeated on day 101. The inoculum was prepared as described for the inoculum used on day 91. A blood sample was obtained from each mouse on day 105 and the serum separated by centrifugation and stored at 4°C.

Example 3

Immunization of mice with o-nitrophenyl-β-D-galactoside

BALB/C mice (Group 2 in Table 1) were inoculated intraperitoneally with 50 mg or 100 mg of ONPG emulsified in complete Freund's adjuvant on day 0, intravenously, with 10 mg of ONPG in 0.1M phosphate buffer (pH 7.3) on day 30, and intraperitoneally with 12.5 mg of ONPG in incomplete Freund's adjuvant (25mg/ml) on day 63. The mice were bled 9 days later, serum was separated by centrifugation and stored at 4°C.

Example 4

Immunization of mice with dinitrophenolkeyhole limpet hemocyanin conjugate

Mice inoculated as in Example 3 were then inoculated, intraperitoneally, with 10 mg of DNP-KLH emulsified in incomplete Freund's adjuvant on days 121 and 131, and bled on day 135. Serum was separated by centrifugation and stored at 4°C. Example 5

Evaluation of mouse sera

A. Preparation of microtiter plate wells

Fifty (50) microliters of a solution containing ONPG in carbonate buffer (1 mg/ml) was added to each well of a polystyrene microtiter plate. After 18 hrs. at 4°C, the solution was removed and the wells washed 4 times with phosphate buffered saline containing 0.05% Tween-2- (PBS-Tween). The ONPG-coated wells were then blocked by inoculating the wells with PBS-Tween containing 1% bovine serum albumin (BSA) for 120 min. at 37°C.

B. ONPG-binding assay

Sera collected in Examples 1 and 3 and serum from mice which had not been immunized were diluted to various degrees with PBS-Tween containing 1% BSA.

Aliquots of the solution so prepared were added to ONPG-coated wells prepared as described above and incubated at 37°C for 120 min. The solutions were then. removed and the wells washed 4 times with PBS-Tween. The presence of serum antibodies binding to ONPG was detected by the method of Engvall and Perlman, 1971, Immunochem . 8: 871, using an anti-mouse goat immunoglobulin conjugated with horseradish peroxidase. After unbound anti-mouse

antibody was removed from the wells by washing,

2,2'-azino-di(3-ethylbenzthiazoline sulfonic acid) (ABTS) and hydrogen peroxide were added to each well and left in contact with the well for 15 to 20 min. Colored product was detected in the wells that were contacted with 1:10 to 1:320 dilutions of serum from mice immunized with ONPG. Of the sera collected in Example 3, that obtained from mice initially inoculated with 100 mg of ONPG had a titer greater than 1:320, which was at least twofold greater than the titer of serum obtained from mice initially inoculated with 50 mg of ONPG. Serum from mice not immunized with ONPG did not produce colored product in this assay. These results demonstrated that serum from mice immunized with DNPG contained antibodies that bound ONPG.

C. Assay for activity in catalyzing

the cleavage of ONPG

The catalytic activity of antibodies which react with ONPG was determined in the following way.

Fifty (50) microliters of diluted mouse serum obtained in

Examples 1 and 3 as described above was contacted for 18 hours at 23°C with 50 microliters of ONPG in PBS-Tween buffer containing 1% BSA. Similarly, 50 mg of the enzyme β-D-galactosidase in 50 microliters of PBS-Tween-BSA buffer was contacted with the ONPG solution. Catalytic activity resulting in the formation of β-D-galactose and o-nitrophenol, which has a yellow color, was not detected with any of the serum samples. As expected, the enzyme β-D-galactosidase had catalytic activity.

Serum collected in Examples 2 and 4 from mice which had received additional inoculations with DNP coupled to KLH was then assayed. The serum was tested for the presence of antibodies that bind ONPG by the method described above. It was shown that serum from the immunized mice contained anti-ONPG antibodies. Serum at a dilution of 1:5,120 yielded a positive reaction for the presence of anti-ONPG antibodies. This demonstrated that additional immunizations with an analog coupled to KLH had resulted in an increased concentration of anti-ONPG antibodies in the serum. No reactions were seen using serum from mice that had not been immunized.

The catalytic activity of the serum antibodies in the serum collected in Examples 2 and 4 was tested as described above.

The results are shown in Table 1 and demonstrate that catalytic activity was detected in serum samples from immunized mice. TABLE 1

Catalytic Activity of Mouse Sera and β-D-Galactosidase

Absorbance at 405 nm

Serum Dilutions 10 min. 18 hrs. ^ Absorbance²

Group 1

ONPG antisera

1:10 .019 .023 .004

1:20 .005 .003 .000

1:40 .008 .010 .002

4

Group 2

ONPG antisera

1:10 .006 .031 .025

1:20 .004 .042 .038

1:40 .006 .009 .003

Normal mouse sera

1:10 .003 .009 .006

1:20 .000 .000 .000

1:40 .005 .007 .002 β-D-galactosidase

50.0 ng .229 .362 .133

5.0 ng .029 .496 .467

0.5 ng .000 .112 .112 1 Corrected for absorbance of serum.

2 Difference in value, at 10 min. and 18 hrs.

3 Sera obtained in Example 2.

4 Sera obtained in Example 4.

Example 6

Preparation of spleen cells for fusion (hybridization) by immunization with o-nitrophenyl-β-D-galactoside

Antibody-producing mice immunized as in

Example 4 and assayed as in Example 5 are sacrificed and their spleens removed. The spleens of ten (10) mice are gently teased and passed through a fine nylon screen to yield a lymphocyte (spleen cell) suspension. The

suspension is washed three (3) times in serum-free

RPMI-1640. Example 7

Preparation of myeloma cells from fusion (hybridization)

Myeloma cells derived from the SP2/0 line are grown in HB101 medium supplemented with 2% fetal bovine serum, penicillin, and streptomycin (complete HB101).

SP2/0 cells are subcultured daily for three days before use in cell fusions and are seeded at densities not exceeding 10 cells/ml. The SP2/0 cells are washed once in RPMI-1640 before fusion.

Example 8

Preparation of hybridoma cells

A suspension of lymphocytes prepared as in Example 6 is mixed in a 4:1 ratio with a suspension of SP2/0 myeloma cells prepared according to Example 7.

The cells are pelleted and a polyethylene glycol (PEG) 1450 (Eastman-Kodak, Rochester, New York) solution (containing 50% PEG 25/vol in RPMI-1640) is then added dropwise to the cell pellets at a ratio of 1 ml of PEG to 1.6x10⁵ lymphocytes. After cell fusion with the PEG solution, the cell suspension is centrifuged at 200xg for 5 min., the supernatant is removed and the cells are gently suspended in complete HB101 at a final

concentration of 10⁷ cells per ml. This final cell suspension is then dispensed in 100 μl volumes in wells of a 96-well microtiter plate and cultured at 37°C.

After 24 hours, 100 μl of HAT medium (complete HB101 supplemented with 1 x 10^-4 M hypoxanthine, 4.0 x 10^-4 M aminoptenn, and 1.6 x 10 ^-5 M thymidme) is then added to each well. Cells are fed every 2 to 3 days by aspirating approximately 100 μl of medium from each well and adding

100 μl of fresh HAT medium.

Extensive death of the parental myeloma cells and lymphocytes is observed during week 1 of culture in

HAT medium.

Ten to fifteen days after incubation, cell growth in the HAT medium indicative of successful

hybridization is observed. Example 9

Screening the hybridoma cells producing

catalytic monoclonal antibodies

Microtiter wells containing hybridoma cells prepared according to Example 8 which produce antibodies capable of catalyzing the cleavage of o-nitrophenyl-β-D-galactoside into o-nitrophenol and D-galactose are assayed as follows: a second 96-well microtiter plate

(the assay plate) is prepared containing a 0.05 M

solution of o-nitrophenyl-β-D-galactoside in each well and maintained at 37°C. a 100 μl aliquot of the contents of each hybridoma-containing well of the first plate (hybridoma plate) is withdrawn and transferred to a corresponding well of the assay plate. Preferably, the presence of o-nitrophenol is measured

spectrophotometrically. Alternatively, five (5) minutes after each transfer, a 50 μl aliquot of the assay plate well is analyzed by HPLC for the presence of one or both of the products. Each assay-plate well found to contain o-nitrophenol and D-galactose is identified and the corresponding hybridoma plate well is marked.

Example 10

Culturing hybridoma cells

A portion of each catalytic hybridoma cell suspension identified in Example-9 is seeded in each well of a new microtiter plate. The plating efficiency of the hybrid cells is 50% (i.e., 50% of the seeded cells multiply to form colonies). With this procedure, 80-100% of the wells yield colonies of hybrid cells within two (2) weeks. The hybridoma cells are again tested for catalytic antibody production by the method described in Example 9. Hybridoma cells which continue to produce catalytic antibodies are again clone using thymocyte feeder cells, but at densities of one hybrid cell per three wells. The procedure is repeated whenever less than 90% of the clones from a specific set are making antibodies. Example 11

Catalytic monoclonal antibodies in vivo

Intraperitoneal inoculation of 10 hybrid cells selected according to Example 9 into snygeneic BALB/C mice induces palpable tumors in more than 90% of the inoculated mice within 2 to 3 weeks. These tumors are accompanied by the production of ascites fluids (0.5 to 3.0 ml per mouse). The immunoglobulin concentration in ascites fluids and sera of hybridoma-bearing mice is determined by a radial immunodiffusion assay. The concentrations of monoclonal antibodies in the serum and ascites fluid of an individual mouse are roughly

equivalent, each containing 5 to 50 mg of antibody per ml. The monoclonal antibody capable of catalyzing the cleavage of o-nitrophenyl-β-D-galactoside is then

harvested from the serum or ascites fluid by conventional methods such as gel filtration or ultrafiltration.

Example 12

Use of a monoclonal antibody to catalyze

the cleavage of o-nitrophenyl-β-D-galactoside

To a solution containing 30.12 g (100 mmol) o-nitrophenyl-β-D-galactoside in 1000 ml distilled water buffered at pH 6.8 with 0.5 M phosphate buffer and maintained at 37°C is added 10 mg of monoclonal

antibodies prepared according to Example 6. The reaction mixture is gently agitated for 2.0 hours. The monoclonal antibodies are then recovered from the reaction mixture by ultrafiltration. The filtrate is then cooled to 10°C and treated with 9.2 g (110 mmol) sodium bicarbonate.

The D-galactose is recovered by extracting the filtrate with three 100 ml portions of diethyl ether. The ether portions are combined, washed once with 1.0 N sodium bicarbonate, dried over magnesium sulfate, filtered and concentrated under reduced pressure to yield D-galactose. The aqueous portion is then combined with the sodium bicarbonate wash and acidified to pH 3 by the addition of 5 N hydrochloric acid. The acidified aqueous portion is then extracted three times with ether. The etheral extracts are combined, dried over magnesium sulfate, filtered and concentrated at reduced pressure to yield 9-nitrophenol. The o-nitrophenol and D-galactose may be further purified by HPLC or by recrystallization.

Example 13

Preparation of catalytic monoclonal antibodies

for porphobilinogen (PBC) production

Spleen cells for hybridization are prepared according to the method of Example 6, except that the

BALB/C mice are immunized with 3-glycyl-4-hydroxy-4-methyl-1,5-heptanedioic acid. Myeloma cells are prepared according to Example 7. The spleen cells and the myeloma cells are then fused to yield hybridoma cells according to the method of Example 8. The hybridoma cells are then screened by a modification of the method of Example 9 in which the assay substrate is aminolevulinic acid (0.05M) and the assay tests for the appearance of an HPLC-peak corresponding to PBG. The hybridoma cells so identified are cultured according to the method of Example 10 and are obtained from mice according to the method of

Example 11.

Example 14

Use of monoclonal antibodies to catalyze

the production of PBG

to a solution containing 13.1 g (100 mmol) aminolevulinic acid in 1000 ml of distilled water

buffered at pH 6.8 with 0.5M phosphate buffer and

maintained at 37°C is added 30 mg of the monoclonal antibodies prepared according to Example 13. The

reaction mixture is gently agitated for 2.0 hours. The monoclonal antibodies are then recovered from the

reaction mixture by ultrafiltration. The reaction mixture is lyophilized, and the residue is

chromatographed to yield purified PBG. Example 15

Preparation of catalytic monoclonal antibodies

for L-tryptophan production

A Schiff base is prepared by mixing 2.03 g (10 mmol) indole-3-pyruvic acid, 2.65 g (10 mmol)

pyridoxamine phosphate and 3 g of dry 4A molecular sieves in dry methanol under a nitrogen atmosphere. The

reaction mixture is gently agitated overnight, filtered and concentrated under reduced pressure to yield the Schiff base. Spleen cells are prepared by the method of Example 6 except that the BALB/C mice are immunized with the Schiff base. The spleen cells so obtained are fused according to the method of Example 8 with myeloma cells prepared according to Example 7. The hybridoma cells are then screened by a modification of the method of

Example 9 in which the substrate is a mixture of indole- 3-pyruvic acid (0.05M) and pyridoxamine-5-phosphate

(p.05M) and the assay tests for the appearance of an HPLC peak corresponding to L-tryptophan. The hybridoma cells so identified are cultured according to the method of

Example 10 and are obtained from mice by the method of

Example 11.

Example 16

Use of monoclonal antibodies to catalyze

the production of L-tryptophan

To a solution containing 20.3 g (100 mmol) of indole-3-pyruvic acid and 26.5 g (100 mmol) of

pyridoxamine-5-phosphate in 1000 ml of distilled water buffered at pH 6.5 with 0.5M phosphate buffer and

maintained at 37°C is added 50 mg of the monoclonal antibodies prepared according to Example 15. The

reaction mixture is gently agitated for 2 hours. The monoclonal antibodies are then recovered by

ultrafiltration. Dialysis of the reaction mixture followed by lyophilization yields the product

L-tryptophan. Example 17

Preparation of catalytic monoclonal

antibodies capable of cleaving RNA

at a specific nucleotide sequence

A. Preparation of the antigen

To a solution of bovine serum albumin (BSA)

(50 mg) dissolved in cold water (8 ml) and titrated to pH

6.5 with 0.1 N sodium hydroxide, is added 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (morpho CDI) followed immediately by mouse mammary tumor virus 35S RNA (50 mg). The reaction mixture is allowed to warm to room temperature and is stored for 18 hours with periodic gentle agitation. The reaction mixture is then dialyzed against four changes of 0.05M ammonium bicarbonate followed by four changes of water. The

RNA-protein (BSA) conjugate is then lyophilized and weighed into vials for storage under nitrogen at -77°C. Alternatively, keyhole limpet hemocyanin (KLH), ovalbumiή (OA) and rabbit serum albumin (RA) may be used in place of BSA. All of these proteins are obtainable from

Calbiochem.

B. Preparation of the monoclonal antibodies

Spleen cells for hybridization are prepared according to the method of Example 6, except that the BALB/C mice are immunized with the BSA-bound 35S RNA prepared in A above. Myeloma cells are prepared

according to Example 7. The spleen cells and the myeloma cells are then fused to yield hybridoma cells according to the method of Example 8.

The hybridoma cells so obtained are screened by incubating aliquots of the microtiter well contents with 35S RNA in 0.5M phosphate buffer (pH 6.1) containing 0.9% NaCl at 37°C for varying lengths of time. The RNA is then purified by phenol extraction. The number of fragments generated by antibody cleavage is determined by 2-dimensional polyacrylamide gel electrophoresis and the nucleotide sequence at each cleavage site is resolved. Both determinations are made according to the methods described by Schwartz et al., 1983, Cell 32: 853-869. By comparing the fragments obtained from RNA cleavage induced by the contents of each microtiter well with the Eco R1-induced fragments, hybridoma cells are selected which produce monoclonal antibodies capable of catalyzing

RNA cleavage only at Eco R1 cleavage sites. The

hybridoma cells so identified are cultured according to the method of Example 10 and are obtained from mice by the method of Example 11.

Example 18

Use of monoclonal antibodies to catalyze

RNA cleavage at Eco R1 sites

Mouse mammary tumor virus 35S RNA (50 mg) is added to 100 ml of distilled water buffered at pH 6.1 with 0.5M phosphate buffer containing 0.9% NaCl and maintained at 37°C. Monoclonal antibodies (5 mg)

prepared according to Example 17 are added to the

reaction mixture which is then incubated for 30 minutes with gentle agitation. The RNA is then purified by phenol extraction and the fragments purified by

polyacrylamide gel electrophoresis.

Example 19

Anti-idiotype monoclonal antibodies to β-D-galactosidase A. Preparation of monoclonal antibodies

to the enzyme active site

BALB/C mice are immunized with the enzyme β-D-galactosidase. Myeloma cells are prepared according to Example 7. The spleen cells and the myeloma cells are then fused to yield hybridoma cells according to the method of Example 8. The hybridoma cells thus obtained are screened for production of monoclonal antibodies which bind to the active site of the enzyme. Screening is conveniently conducted by RIA assay of the competitive inhibition of the microtiter well contents against β-D- galactosidase and radio-labeled o-nitrophenyl-β-D-galactoside. Hybridoma cells so selected are then cultured according to the method of Example 10 and obtained in larger quantity from mice according to the method of Example 11.

B. Preparation of the anti-idiotype monoclonal antibody

Spleen cells for hybridization are prepared according to the method of Example 6, except that the BALB/C mice are immunized with the monoclonal antibodies prepared and selected according to Example 7. The spleen cells and the myeloma cells are fused to yield hybridoma cells according to the method of Example 8. The

hybridoma cells thus obtained are first screened

according to the method of Example 9. Hybridoma cells selected on the basis of the preliminary screening are then screened for allosterism. This is accomplished by measuring the presence of one of products according to Example 9, but at periodic time intervals. From the data so obtained, a reaction rate may be calculated. By repeating the assay in the presence of varying amounts of the reactant and again with varying amounts of the product not being measured, changes in the kinetic behavior of the antibody can be detected. In this manner, anti-idiotype monoclonal antibodies exhibiting allosteric control may be eliminated. The hybridoma cells producing non-allosteric anti-idiotype monoclonal antibodies are cultured according to the method of

Example 10 and obtained by propagation in mice according to the method of Example 11.

C. use of anti-idiotype monoclonal antibodies

The anti-idiotype monoclonal antibodies obtained in B may be used according to the method of Example 12. Example 20

Resolution of stereoisomers by monoclonal

antibody catalysts with esterase activity

The following example outlines an approach for resolving a racemic mixture of compound A, Figure 2. In this approach, the stereoisomers are separated by

acylation of the phenolic hydroxyl group of A, followed by deacylation of a single enantiomer by a catalytic antibody. This approach draws upon the extensive

literature precedents concerning ester hydrolyses by antibody catalysts and will allow rapid separation of the stereoisomers by exploiting the different properties of phenolic esters and hydroxyl groups in solvent extraction techniques.

When achiral substrate I (Figure 1) is reduced in a symmetrical environment with a symmetrical reagent (an achiral reaction), a 50/50 mixture of the two

enantiomeric forms, 2-(S) and 2-(R), of the product results. The stereochemical situation is depicted in Figure 1.

The right and left faces of symmetrical

structure I (Figure 1) are distinguishable to a chiral reagent and so give rise to unequal amounts of the products from the two modes of attack. The difference may vary from essentially zero to 100%. A single

enantiomer of A is isolated from the racemic mixture by selective deesterification of the phenolic hydroxyl of compound A (Figure 2).

A catalytic antibody to hapten VI is isolated, which resembles the transition state for the hydrolysis of the underived R-enantiomer. It is used to selectively hydrolyze the R-enantiσmer of III to the phenol IV. The remaining ester of the desirable S-enantiomers is stable and so separation of the two enantiomers requires

isolation of phenol IV from ester V. This separation can be done easily using conventional separation techniques such as chromatography, extraction, etc. After separation, the desired enantiomer V is chemically hydrolyzed by base to its phenol.

Hybridoma screening

For screening for catalytic antibodies, the substrate X can be used. Substrate X (Figure 3) is added to each antibody-producing cell-line sample and the presence of catalytic activity will be measured by fluorescence of the 7-hydroxy-4-methylcoumarin that is generated. Strong fluorescence over time will indicate that a hybridoma cell line producing catalytic antibodies has been identified. As a result of using X for

screening, the R group in VI will become XI m order that the immunogen and the screening molecules resemble each other. The carboxyl group in XI is used to link structure VI to a carrier protein and is used for

immunization.

Example 21

Immunogen designed to elicit an antibody

that can catalyze a stereoselective reaction

This immunogen will elicit antibodies that catalyze stereoselective cleavage of the chiral nitrophenyl ester substrate (2) :

to release P-nitrophenol which can be assayed

spectrophotometrically.

Stereoselective cleavage of chiral esters is important in the synthesis of optically pure

pharmaceuticals, e.g., propranolol. Example 22

Immunogen designed to catalyze

a stereoselective cyclization reaction

This will elicit antibodies that catalyze cyclization of the chiral substrate (4) :

to generate the lactone (5) :

Example 23

Immunization with enantiomeric transition state

analogs and selection of antibodies that carry out

chiral reactions on only one isomer

Immunogen similar to Example 20, but instead of using the pure enantiomer, a racemic mixture (6) is used as the immunogen:

and the chiral substrates (7) and (8) used to screen for stereoselective catalytic antibodies:

Example 24

Bioluminescence screening assay

for stereoselective hydrolysis of ester substrates

Hydrolysis of the ester linkage catalyzed by an enantiomeric antibody catalyst specific for the R or S isomer at the chiral center bearing group R will release D-luciferin which is assayed by firefly luciferase and ATP to provide a sensitive nonradioactive means of detecting catalytic antibodies specific for chiral substrates in hybridoma cell supernatants.

Example 25

A stereospecific cyclization catalyzed by an antibody

Amide derivatives of the phosphonate ester 2[2- phenoxy-2-oxo-6(aminoethyl)-1,2-oxophosphorine were prepared as shown in below.

where Ph = phenyl, and i-Pro = isopropyl.

The corresponding ester 1, with chirality at the carboinol atom, was prepared as the O-protected trimethylsilyl derivative:

where Ph = phenyl, Me = methyl, and Ac = acetyl.

The synthesis of the intermediate 2 was stereospecific and yielded only on diastereomer. An immunogenic conjugate was prepared by reaction of the phosphonate 4 with a carrier protein (keyhole limpet hemocyanin).

Monoclonal antibodies were obtained by standard protocols using lymphocytes from mice immunized with 4 linked to a protein carrier. Antibodies were screened for hydrolytic activity (25mm phosphate, pH 7.0, 25°C) by monitoring substrate depletion with high performance liquid 'chromatography. The rates of phenol release from the ester 1 in the presence of catalytic antibody 1 were determined spectrophotometrically. The initial rates as a function of substrate concentration followed Michaelis-Newton kinetics. Over this concentration range, there was no apparent substrate inhibition. The k_cat/k_uncat observed with catalytic antibody 1 were up to 167-fold rate acceleration. The catalytic activity was a property of the monoclonal antibody because: (1) neither the hydrolytically more labile coumarin ester corresponding to 1 nor the phenyl-t-hydroxypentanoate, which labels the important acetamidomethyl recognition element were substrates; (2) a second monoclonal antibody that bound 4 did not catalyze the liberation of phenol from 1; and (3) the reaction of monoclonal antibody 1 was competitively inhibited linearly by the additions of the transition state analog, the N-acetyl derivative of 2.

The cyclization of 1 by monoclonal antibody 1 stopped at approximately 50% of the initial ester

concentration. When more substrate solution was added to the initial reaction solution, a second depletion of approximately 50% of the added substrate was observed,

The stereospecific cyclization of 1 to 5

was confirmed by the NMR studies.

Claims

What is claimed is:

1. A method for increasing the rate of a chemical reaction involving conversion of at least one reactant to at least one product which comprises

contacting the reactant with at least one appropriate monoclonal antibody to the reactant under suitable conditions permitting formation of a complex between the monoclonal antibody and the reactant, conversion of the reactant to the product and release of the product from the complex, the monoclonal antibody being characterized by a K > 1, where K = k_r/k_p, k_r is the affinity constant of the monoclonal antibody for the reactant and k_p is the affinity constant of the monoclonal antibody for the product, by an r₁ > r_o, where r₁ is the rate of formation of the complex between the antibody and the reactant and where r is the rate of the chemical reaction in the absence of monoclonal antibody, by an r₂ > r_o, where r₂ is the rate of the conversion of the complexed reactant to the complexed product and by an r₃ > r_o, where r₃ is the rate of release of the product from the complex.

2. A method for catalyzing a stereochemical reaction wherein at least one reactant is converted to at least one product comprising the step of: contacting the reactant with at least one monoclonal antibody capable of catalytically increasing the rate of conversion of reactant to product in said stereochemical reaction, said contact being performed under conditions wherein a complex is formed between said monoclonal antibody and said reactant, the said reactant is catalytically

converted to said product, and the said product is released from said complex.

3. A method as recited in claim 2 wherein one reactant is converted to one or more products.

4. A method according to claim 3 wherein the reactant is a polysaccharide and the products are

saccharides derived therefrom.

5. A method according to claim 3 wherein the reactant is a polynucleotide and the products are

nucleotides derived therefrom.

6. A method according to claim 3 wherein the reactant is a β-galactoside and at least one of the two products is galactose.

7. A method as recited in claim 2 wherein two reactants are converted into one or more products.

8. A method as recited in claim 2 wherein the reaction is a reaction which is also capable of being catalyzed by a nonproteinaceous organic molecule.

9. A method as recited in claim 8 wherein said nonproteinaceous organic molecule is a cofactor and an effective amount of said cofactor is present in the reaction.

10. A method as recited in claim 9 wherein the cofactor is pyridoxal phosphate.

11. A method as recited in claim 2 wherein the reaction is a reaction which is also capable of being catalyzed by an enzyme.

12. A method as recited in claim 11 wherein an effective amount of an enzyme is present in the reaction.

13. A method as recited in claim 12 wherein the reaction involves cleavage σf a phosphodiester bond in a polyribonucleotide and wherein the enzyme is a restriction enzyme.

14. A method as recited in claim 12 wherein the reaction involves cleavage of a phosphodiester bond in a polyribonucleotide and wherein the enzyme is a restriction enzyme.

15. A method as recited in claim 12 wherein the reaction involves cleavage of a galactosyl linkage and wherein the enzyme is β-galactosidase.

16. A method as recited in claim 12 wherein the reactant is a cyclic polynucleotide and the product is a linear polynucleotide.

17. A method as recited in claim 2 wherein the reactant is complexed with more than one monoclonal antibody, each of which is directed to a different determinant on the reactant.

18. A method as recited in claim 17 wherein the reactant is a polynucleotide and the monoclonal antibodies are directed to different nucleotide sequences within the polynucleotide.

19. A method as recited in claim 2 wherein the rate of reaction in the presence of the monoclonal antibody is more than 100 times the rate in the absence of the monoclonal antibody.

20. A method as recited in claim 2 wherein the reaction is carried out in an aqueous solution at a pH between 6.0 and 8.0, at ambient pressure, at a

temperature of 4°C to 50°C and at an ionic strength of less than 2.0 moles/liter.

21. A method for catalyzing a stereochemical reaction wherein at least one reactant is converted to at least one product comprising the step of: contacting the reactant with at least one monoclonal antibody capable of catalytically increasing the rate of conversion of reactant to product in said stereochemical reaction, said contact being performed under conditions wherein a complex is formed between said monoclonal antibody and said reactant, the said reactant is catalytically

converted to said product, and the said product is released from said complex, said monoclonal antibody being characterized by a K > 1, where K = k_r/k_p, k_r is the affinity constant of the monoclonal antibody for the reactant and k is the affinity constant of the

monoclonal antibody for the product, by an r₁ > r_o, where r₁ is the rate of formation of the complex between the antibody and the reactant and where r_o is the rate of the chemical reaction in the absence of monoclonal antibody, by an r₂ > r_o, where r₂ is the rate of the conversion of the complexed reactant to the complexed product and by an r₃ > r_o, where r₃ is the rate of release of the product from the complex.

22. A method as recited in claim 21 wherein said stereochemical reaction is one which is also capable of being catalyzed by a cofactor or by an enzyme and wherein a cofactor or enzyme is present in said reaction.

23. A method for catalyzing a stereochemical reaction wherein at least one reactant is converted to at least one product comprising the step of: contacting the reactant with at least one monoclonal antibody capable of catalytically increasing the rate of conversion of reactant to product in said stereochemical reaction, said contact being performed under conditions wherein a complex is formed between said monoclonal antibody and said reactant, the said reactant is catalytically

converted to said product, and the said product is released from said complex, said monoclonal antibody having been produced by a process comprising the steps of:

(a) generating a plurality of monoclonal antibodies to an antigen selected from the group consisting of the stereoisomers of:

(i) the reactant,

(ii) the reactant bound to a carrier molecule, (iii) a reaction intermediate,

(iv) an analog of the reaction intermediate,

(v) an analog of the reactant, and

(vi) a reaction product;

(b) screening said plurality of monoclonal

antibodies to identify a monoclonal antibody which catalyzes the desired reaction; and

(c) using the so-identified monoclonal antibody

from step (b) as a catalyst.

24. A method as recited in claim 23 wherein said antigen is an analog of the reactant.

25. A method as recited in claim 23 wherein said stereochemical reaction is one which is also capable of being catalyzed by a cofactor or by an enzyme and a cofactor or enzyme is present in said reaction.

26. A method as recited in claim 23 wherein the monoclonal antibody identified in step (b) is

produced in quantity by culturing a plurality of

hybridoma cells each of which produces said monoclonal antibody.

27. A method for catalyzing a stereochemical reaction wherein at least one reactant is converted to at least one product comprising the step of: contacting the reactant with at least one monoclonal antibody capable of catalytically increasing the rate of conversion of reactant to product in said stereochemical reaction, said contact being performed under conditions wherein a complex is formed between said monoclonal antibody and said reactant, the said reactant is catalytically

(a) immunizing an animal with a stereoisomer analog of the reactant, and thereby generating

antibody-producing lymphocytes in said animal; (b) removing said antibody-producing lymphocytes from said animal;

(c) fusing said antibody-producing lymphocytes with myeloma cells and thereby producing a plurality of hybridoma cells each producing monoclonal antibodies;

(d) screening said plurality of monoclonal

(e) producing a quantity of the monoclonal antibody identified in step(d) by culturing a plurality of hybridoma cells, each of which produces said monoclonal antibody.

28. A method for catalyzing a stereochemical reaction which is known to be catalyzed by an enzyme and wherein at least one reactant is converted to at least one product comprising the step of: contacting the reactant with at least one monoclonal antibody capable of catalytically increasing the rate of conversion of reactant to product in said stereochemical reaction, said contact being performed under conditions wherein a complex is formed between said monoclonal antibody and said reactant, the said reactant is catalytically

converted to said product, and the said product is released from said complex, said monoclonal antibody having been prepared by a process comprising the steps of:

(a) generating a plurality of monoclonal antibodies to said enzyme;

(b) screening said plurality of monoclonal

antibodies to identify a first monoclonal antibody which inhibits binding of the reactant to the enzyme;

(c) recovering the said first monoclonal antibody; (d) generating a plurality of anti-idiotype

monoclonal antibodies to the said first

antibody recovered in step (c);

(e) screening said plurality of anti-idiotype

monoclonal antibodies generated in step (d) to identify a second monoclonal antibody which binds the reactant and catalyzes the desired stereochemical reaction; and

(f) producing a quantity of the monoclonal antibody identified in step (e) by culturing a plurality of hybridoma cells, each of which produces said monoclonal antibody.

29. A method for catalyzing a stereochemical reaction wherein a stereoisomer contained within a mixture of stereoisomers is converted to a product comprising the step of: contacting the mixture with at least one monoclonal antibody capable of catalytically increasing the rate of conversion of reactant to

stereoisomer in said stereochemical reaction, said contact being performed under conditions wherein a complex is formed between said monoclonal antibody and said stereoisomer, the said stereoisomer is catalytically converted to said product, and the said product is released from said complex.

30. A method for catalyzing a stereochemical reaction wherein at least one reactant is converted to at least one product comprising the step of: contacting the reactant with at least one monoclonal antibody capable of catalytically increasing the rate of conversion of reactant to product in said stereochemical reaction, said contact being performed under conditions wherein a complex is formed between said monoclonal antibody and said reactant, the said reactant is catalytically

(a) generating a plurality of monoclonal antibodies to an antigen which is a mixture of

stereoisomers, said mixture containing a species selected from the group consisting of a stereoisomer of:

(i) the reactant,

(ii) the reactant bound to a carrier molecule, (iii) a reaction intermediate,

(iv) an analog of the reaction intermediate, (v) an analog of the reactant, and

(vi) a reaction product;

(b) screening said plurality of monoclonal

antibodies to identify a monoclonal antibody which catalyzes the desired stereochemical reaction; and

(c) using the so-identified monoclonal antibody

from step (b) as a catalyst.

31. A method for preparing a catalytic monoclonal antibody for a stereochemical reaction

comprising the steps of:

stereoisomers, said mixture containing a species selected from the group consisting of a stereoisomer of

(i) the reactant,

(ii) the reactant bound to a carrier molecule, (iii) a reaction intermediate, (iv) an analog of the reaction intermediate,

(v) an analog of the reactant, and

(vi) a reaction product;

(b) screening said plurality of monoclonal

(c) using the so-identified monoclonal antibody from step (b) as a catalyst.