BACKGROUND: Antibody fragments have been expressed on the major coat protein of filamentous phage using a gene VIII expression system, but with low copy numbers (averaging 0.2 Fab/phage). OBJECTIVES: As a general strategy to increase copy number, the phage vector was optimized by site directed mutagenesis. STUDY DESIGN: One mutation was the introduction of a random six amino acid tether between the heavy chain and pseudo gene VIII to form a phage library. The other mutation was the removal of two cysteine residues which form a disulfide bond between the heavy and light chains. An assay was developed to measure Fab concentrations and used to calculate the average number of copies displayed on phage. RESULTS: Phage libraries containing random tethers were panned, and clones containing a proline rich motif were extracted. Removing the interchain disulfide had a greater effect on copy number and soluble Fab concentrations in the periplasmic space of the bacterial cultures. CONCLUSION: A tenfold increase in the copy number was achieved using the optimized vector. Incorporation of these vector mutations may be a general strategy for optimizing Fab display on the major coat protein of bacteriophage M13.

Bacteriophage M13↗

PubMed1995

A study of reversibly inactivated lipases for use in a morphine-triggered naltrexone delivery system.

A key component of an implant that can be triggered by external morphine to release naltrexone is an inactivated enzyme that can be activated by morphine and which can then rapidly remove a protective coating surrounding a bioerodible polymer containing dispersed naltrexone. In this article we describe a lipase that has been conjugated with O3-carboxymethylmorphine, morphine-beta-3-glucuronide and O3-carboxypropylmorphine. The enzyme conjugate was then inactivated by complexation with affinity-purified goat polyclonal antimorphine antibodies. Antibody lipase interactions were measured by pH Stat and ELISA techniques. Affinity constants of the antibodies determined by radioimmunoassay using tritium-labeled morphine were 4.10 x 10(6), 3.18 x 10(6) and 3.38 x 10(7), respectively. While a concentration of 10(-5)M morphine was required to restore lipase activity, it is likely that a combination of correct morphine tether and correct affinity-purified antibody can increase sensitivity to the desired 10(-8)10(-9)M morphine level. Thus, a functioning device can almost certainly be constructed. However, it is unlikely that reactivation times of 1-2 h necessary for clinical usefulness in treatment of narcotic addiction can be achieved.

Animals↗

PubMed1995

Biocompatibility and in vivo morphine diffusion into a placebo morphine-triggered naltrexone delivery device in rabbits.

Two key factors in developing a clinically useful triggered naltrexone delivery system are device biocompatibility and ability of morphine to diffuse from the blood into the device in concentrations useful to trigger delivery. Two types of devices were implanted subcutaneously into rabbits and their biocompatibility was investigated. One device consisted of the outer semipermeable regenerated cellulose acetate tubing, closed at both ends with double knots and filled with poly(N-vinyl pyrrolidone) as osmotic filler. The other device was a placebo device that contained within the regenerated cellulose acetate tubing, also closed at both ends with double knots, all device components except naltrexone. Both devices were biocompatible. When the regenerated cellulose acetate device filled with osmotic filler was implanted in rabbits which were subsequently dosed at day 7 and day 14 post-implant with 150 mg kg(-1) morphine sulphate, the concentration of morphine in the device was only about 10-fold less than that measured in rabbit blood. Thus, enough morphine diffuses into the device to make triggering in a real-life situation feasible.

Animals↗

PubMed1991

Approaches to the design of semisynthetic metal-dependent catalytic antibodies.

The binding site of the Fab fragment of antibody MOPC315 was chemically modified with cyclam (1,4,8,11-tetraazacyclotetradecane) and 1,10-phenanthroline. In the presence of the metal ions cobalt(II), copper(II), gallium(III), iron(II), nickel(II), zinc(II), these semisynthetic antibodies did not catalyse the hydrolysis of amides or phosphate ester substrates, or the oxidation of a 1-hydroxy-2-alkene or of 3,4-dihydrobenzamide substrates. Hydrolysis of a coumarin ester substrate was catalysed by the Fab derivatized with a cyclam moiety with a kcat of 0.063 min-1 and Km of 11.7 microM; the presence of metal ions was not required for catalysis.

Antibodies↗

PubMed1989

Design of catalytic antibodies.

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Affinity Labels↗

PubMed1988

Introduction of nucleophiles and spectroscopic probes into antibody combining sites.

A general chemical strategy has been developed whereby antibody combining sites can be selectively derivatized with natural or synthetic molecules, such as catalytic groups, drugs, metals, or reporter molecules. Cleavable affinity labels were used to selectively introduce a thiol into the combining site of the immunoglobulin A MOPC 315. This thiol acted both as a nucleophile to accelerate ester thiolysis 60,000-fold and as a handle for selectively derivatizing the antibody with additional functional groups. For example, derivatization of the antibody with a fluorophore made possible a direct spectroscopic assay of antibody-ligand complexation. This chemistry should not only extend our ability to exploit antibody specificity in chemical catalysis, diagnostics, and therapeutics, but may also prove generally applicable to the functional modification of other proteins for which detailed structural information is unavailable.

Affinity Labels↗