Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1995-08-22
1998-06-16
Patterson, Jr., Charles
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 71, 435 76, 4351885, G01N 33573
Patent
active
057668612
DESCRIPTION:
BRIEF SUMMARY
THIS APPLICATION IS A 371 OF PCT/US 94/00424 JAN. 12, 1994.
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to the direct screening of hybridoma supernatants or other potential sources of catalysts for catalytic activity, and more particularly to a method for screening or detection of non-enzyme catalytic polypeptides and proteins, including catalytic antibodies.
One of the important goals of biotechnology is the ability to produce catalysts for any desired chemical reaction. This has many far-reaching applications in chemistry, biology and medicine. Many approaches towards this goal have been explored including site-directed modification of existing enzymes, microbial induction of new enzymes, totally synthetic peptides, and synthetic organic substances. Although fascinating and illuminating with regard to catalysis in general, none of these routes have provided a reliable, generally applicable technique for obtaining "tailor-made" catalysts.
The ability of catalytic antibodies to be tailor-made to a predesigned substrate, combined with rate enhancement and turnover, has led to the most intriguing enzyme mimics described thus far (1-3). Catalytic antibodies are elicited against a hapten, typically, a stable synthetic analog of the transition state (TS) of the catalyzed reaction. The repertoire resulting after immunization is immortalized as hybridomas (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, 6-8). Novel, non-hybridoma, 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. Gong et al. (28) proposed recently a chromogenic assay for screening large antibody libraries but the approach is both limited to hydrolysis reactions and includes labile ester groups which give strong background signals and, as has been shown (5), this disadvantage practically restricts the utility of such approaches.
There are several variations in approaches to elicit catalytic antibodies but the overall technique is as follows: The reaction which is to be catalyzed is analyzed in terms of the mechanism believed to be involved; this entails a depiction of the species which must be stabilized for the reaction to be catalyzed. Typically, this species is an unisolable transient state (the transition state or a high energy intermediate) which lies along the reaction pathway from reactant to product. Next, one or more stable chemical substances which mimic the stereoelectronic structure of the transient state are designed and synthesized. These substances are then used as haptens to elicit monoclonal antibodies (MABs) and various tests are made on each of the many (1000 or more) resulting hybridomas in order to ascertain which should be further studied. This initial screening method generally involves an ELISA assay to those MABs that are most tightly bound to the hapten (or transition state analog, TSA). These selected hybridoma cultures are then expanded and larger amounts of antibody are produced in order to determine whether any of these antibodies can catalyze the desired reaction. There are many controls that are required to ensure that no adventitious catalysts (e.g. enzyme impurities) are responsible for the
REFERENCES:
Rezaee, M, et al. (1987) Int. J. Cancer 40, 823-829.
Proceedings of the National Academy of Sciences, vol. 90, issued 15 Jan., 93, Tawfik et al., "catElisa: A facile general route to catalytic antibodies", pp. 373-377, see entire article.
Ann. Rev. Biochem., vol. 61, issued 1992, Benkovic, "Catalytic Antibodies", pp. 29-54, especially pp. 44-45.
Analytic Biochemistry, vol. 202, issued 1992, Tawfik et al., "Detection of Catalytic Monoclonal Antibodies", pp. 35-39, see especially p. 39.
J. Am. Chem. Soc., vol. 114, issued 1992, Gong et al., "A Chromogenic Assay for Screening Large Antibody Libraries", pp. 1486-1487, see entire article .
Eshhar Zelig
Green Bernard S.
Tawfik Dan S.
Patterson, Jr. Charles
Yeda Research and Development Company Ltd.
Yissum Research and Development Co. of the Hebrew University of
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