Improving the copy numbers of antibody fragments expressed on the major coat protein of bacteriophage M13.

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.

Simultaneous detection of seven drugs of abuse by the Triage panel for drugs of abuse.

This novel, competitive immunoassay simultaneously detects seven drugs of abuse in urine. A urine sample is placed in contact with lyophilized reagents, the reaction mixture is allowed to come to equilibrium (10 min), and then the whole mixture is applied to a solid phase that contains various immobilized antibodies in discrete drug-class-specific zones. After a washing step, the operator visually examines each zone for the presence of a red bar. The method incorporates present threshold concentrations that are independent for each drug. In the absence of drug or in the presence of drug in quantities less than the threshold concentration, no colored bar is visible. Samples containing drug(s) at or above the threshold concentration cause a red bar to appear for the appropriate drug(s). Positive and negative procedural control zones are incorporated into each determination. The performance of the assay methodology matches that of instrumented immunoassay systems.

Internally referenced ImmunoConcentration assays.

We have developed a method for incorporating an internal reference zone in addition to the test zone in a single immunoConcentration device. We have illustrated the method by constructing an internally referenced assay for human choriogonadotropin (HCG) in serum. The internally referenced assay, which can be performed in about 5 min, provides a measured overall precision (CV) of approximately 15% at a concentration, in serum, of approximately 50 int. units/L. The test zone consists of an anti-HCG antibody adsorbed onto latex microspheres and entrapped within a porous support. The reference zone contains antibody against alkaline phosphatase (EC 3.1.3.1), which has also been adsorbed onto latex microspheres and trapped within the support. The use of an internal reference moderates the effects of variations in assay conditions on the interpretation of assay results.

ImmunoConcentration--a new format for solid-phase immunoassays.

A new format for solid-phase immunoassays has been developed in which a monoclonal antibody-coated membrane, incorporated into a cylindrical, disposable device, regulates sample and reagent delivery. We illustrate the method with a two-site, immunoenzymometric assay that can detect human choriogonadotropin at less than 50 int. units/L (4 micrograms/L) in urine and less than 25 int. units/L (2 micrograms/L) in serum and takes less than 5 min to perform. The solid-phase antibody is located in a circular area in the center of the membrane so that in the presence of the hormone, after addition of substrate, a blue enzyme product is generated in this circular area. The high ratio of surface area to volume within the microporous matrix of the membrane assures short diffusion distances and therefore rapid binding of liquid-phase reagents to the solid phase. Pseudo-first-order reaction kinetics describe the binding of antigen to immobilized antibody and the binding of enzyme-labeled antibody to immobilized antigen. The speed and simplicity of this format may facilitate testing for many analytes, both soluble and particulate, as well as serological testing.

Interaction of cytochrome c with reaction centers of Rhodopseudomonas sphaeroides R-26: localization of the binding site by chemical cross-linking and immunochemical studies.

The location of the cytochrome binding site on the reaction center of Rhodopseudomonas sphaeroides was studied by two different approaches. In one, cross-linking agents, principally dithiobis(propionimidate) and dimethyl suberimidate, were used to link cytochrome c and cytochrome c2 to reaction centers; in the other, the inhibition of electron transfer by antibodies against the subunits was investigated. Cytochrome c (horse) cross-linked to the L and M subunits, whereas cytochrome c2 (R. sphaeroides) cross-linked only to the L subunit. The cross-linked reaction center-cytochrome complexes were isolated by affinity chromatography. The rate of electron transfer in the cross-linked cytochrome c2 complex was the same as that in the un-cross-linked complex. However, when cytochrome c was used, the rate in the cross-linked complex was about 15 times slower than that in the un-cross-linked complex. Fab fragments of antibodies specific against the L and M subunits blocked electron transfer from both cytochrome c (horse) and cytochrome c2 (R. sphaeroides). Antibodies specific for the H subunit did not block either reaction. We conclude that the cytochrome binding site on the reaction center is close (approximately 10 A) to both the L and M subunits, possibly in a cleft between them.

Topography of reaction center subunits in the membrane of the photosynthetic bacterium, rhodopseudomonas sphaeroides.

The localization of the reaction center polypeptides (L, M, and H) in the membranes of both the wild-type, strain 2.4.1, and the carotenoidless mutant, R-26, of Rhodopseudomonas sphaeroides was determined by using affinity-purified antibodies specific for these proteins. Binding of the antibodies to reaction center subunits in spheroplasts was visualized in the electron microscope by immunoferritin labeling. The H and M subunits were labeled at both the cytoplasmic and the periplasmic surfaces of the membrane, whereas the L subunit was labeled only at the periplasmic surface of the membrane. Thus, the reaction center is asymmetrically oriented in the membrane with at least two subunits (H and M) spanning the membrane.