Selective cleavage of trityl protecting groups catalyzed by an antibody

Iverson, Brent L.; Cameron, Katherine E.; Jahangiri, Guiti K.; Pasternak, Dahna S.

doi:10.1021/ja00169a044

Cited by 40 publications

(8 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A total of 2 mg of surfactin (Calbiochem, San Diego, Calif.), dissolved in a 400-,ul mixture of phosphate-buffered saline (PBS) and dimethylformamide (PBS-dimethylformamide [3:1]), was mixed with 300 RI of Pierce Imject keyhole limpet hemocyanin (KLH)-100 pl of 1 M [1-ethyl-3-(dimethylamino)propyl]-carbodiimide-50 ,ul of 0.1 M N-hydroxyl-sulfosuccinimide (Pierce, Rockford, Ill.) at room temperature. The conjugation reaction was allowed to proceed for 20 min and subsequently quenched by the addition of 3.0 ml of 50 mM glycine (11). The conjugate was dialyzed against PBS overnight and used to immunize rabbits by subcutaneous injection with incomplete Freund's adjuvant (Sigma) on the basis of a standard schedule (2).…”

Section: Materuils and Methodsmentioning

confidence: 99%

Structural and immunological characterization of a biosurfactant produced by Bacillus licheniformis JF-2

Lin

Minton

Sharma

et al. 1994

Appl Environ Microbiol

150

View full text Add to dashboard Cite

show abstract

Section: Materuils and Methodsmentioning

confidence: 99%

Structural and immunological characterization of a biosurfactant produced by Bacillus licheniformis JF-2

Lin

Minton

Sharma

et al. 1994

Appl Environ Microbiol

150

View full text Add to dashboard Cite

show abstract

“…The variability of polyclonal antibody catalysis was investigated by immunizing five male New Zealand white rabbits from a heterozygous population with the triarylphosphonium hapten 1 (Scheme 1) conjugated to keyhole limpet haemocyanin (KLH). The positive charge on 1 is intended to resemble the positive charge developed during the acid-catalysed hydrolysis of trityl ether compounds such as substrate 2 [11,21]. Thus the resulting antibodies are expected to utilize primarily charge complementarity in the antibody-binding pocket to effect catalysis.…”

Section: Scheme 1 Structure Of the Phosphonium Hapten 1 And Trityl Etmentioning

confidence: 99%

“…The antibody-catalysed trityl hydrolysis reaction was chosen because it offers some important advantages for the present study. In particular there is no known enzyme analogue in serum to complicate catalytic assays, and a wellcharacterized monoclonal antibody is available for comparison [21].…”

Section: Scheme 1 Structure Of the Phosphonium Hapten 1 And Trityl Etmentioning

confidence: 99%

Polyclonal antibody catalytic variability

Stephens

Thomas

Stanton

et al. 1998

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

We have performed a systematic variability study of polyclonal antibody catalysis by using five rabbits immunized with the same hapten. Important results from this work are the following. (1) Similarities were observed in the catalytic polyclonal antibodies derived from all five rabbits. Four of the five rabbits produced polyclonal samples that were nearly the same in terms of catalytic activity, whereas the fifth rabbit, designated as rabbit 2, displayed a somewhat higher level of catalytic activity. The catalytic activities (as kcat/kuncat) of these polyclonal samples were similar to that from the best murine monoclonal antibody that had been previously elicited by the same hapten. (2) Titre was not an accurate indicator of polyclonal antibody catalytic activity. (3) A mathematical analysis to describe a distribution of Michaelis-Menten catalysts was performed to help interpret our results. (4) Kinetic analysis indicated that the binding parameters of the different samples were remarkably homogeneous, because one or two components were all that were required to fit the on-rate and off-rate data satisfactorily. Interestingly, the most active catalytic polyclonal sample, that from rabbit 2, displayed the slowest off-rate (so slow it could not be measured) and thus the highest overall affinity. (5) Catalytic analysis of eluted fractions of antibody from a substrate column indicated that each polyclonal sample was also relatively homogeneous in terms of catalytic parameters. The main conclusion of our study is that for this hapten-animal system, the overall catalytic immune response is relatively consistent at two levels. Consistent catalytic activity was observed between the polyclonal samples elicited in the different animals, and the elicited hapten-specific polyclonal antibodies were relatively homogeneous in terms of binding and catalytic parameters within each immunized animal. The observed similarities of the catalytic activity in the different animals is surprising, because the immune response is based on specific binding of antibodies to hapten. There is no known selective pressure to maintain consistent levels of catalytic activity. Our results can therefore be interpreted as providing evidence that for this hapten there is a fixed relationship between hapten structure and catalytic activity and/or consistent genetic factors that dominate the catalytic immune response.

show abstract

“…The results confirmed the catELISA selection. The identification of clones exhibiting relatively low rate accelerations (e.g., an- [15][16][17][18][19][20]. In spite of the presence of the nitrophenyl group (15), thep-nitrobenzyl alcohol product binds to antibody D2.3 with low enough affinity (Kd = 52 ,uM) to allow efficient turnover (Fig.…”

Section: Resultsmentioning

confidence: 99%

catELISA: a facile general route to catalytic antibodies.

Tawfik

Green

Chap

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

134

View full text Add to dashboard Cite

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...

show abstract

Selective cleavage of trityl protecting groups catalyzed by an antibody

Cited by 40 publications

References 0 publications

Structural and immunological characterization of a biosurfactant produced by Bacillus licheniformis JF-2

Structural and immunological characterization of a biosurfactant produced by Bacillus licheniformis JF-2

Polyclonal antibody catalytic variability

catELISA: a facile general route to catalytic antibodies.

Contact Info

Product

Resources

About