A new plasmid vector, pCRP, allowing the expression of human recombinant monoclonal antibody Fab fragments fused with a bacterial acid phosphatase has been constructed. pCRP can accept heavy- and light-chain cDNAs cloned from combinatorial antibody libraries displayed on filamentous phages with the pCombIII system and is able to direct expression to soluble Fabs in which the carboxy-terminus of the heavy chain is fused to the amino-terminus of the mature PhoN nonspecific acid phosphatase of Providencia stuartii. Using the pCRP vector, we expressed two different human recombinant Fabs cloned from combinatorial libraries (one anti-tetenus toxoid and the other anti-HIV-1 gp120) fused with the acid phosphatase. In both cases chimeric antibodies were obtained which retained the antigen-binding ability and the enzymatic activity. Similar Fab-enzyme fusions can be successfully used, even unpurified, in enzyme immunoassays.

Acid Phosphatase↗

PubMed1995

Cloning and characterization of human recombinant antibody Fab fragments specific for types 1 and 2 herpes simplex virus.

Twenty-six bacterial clones producing human recombinant Fab fragments specific for Herpes Simplex virus (HSV) antigens were obtained from an IgG1k human antibody combinatorial library displayed on filamentous phage, following panning against an HSV lysate. All the Fabs reacted against the HSV lysate in enzyme-linked immunosorbent assay and were able to recognize both type 1 and type 2 HSV in an indirect immunofluorescence assay (IFA). DNA sequencing of the heavy chain variable regions showed that these Fabs were different from those already described. One of these Fabs (Fab19) was purified and subjected to further characterization. Purified Fab19 was able to specifically recognize several different HSV-1 and HSV-2 strains (including 2 reference strains and 12 clinical isolates) in IFA. It was also able to neutralize the infectivity of both HSV-1 and HSV-2 strains, although the neutralizing activity was somewhat lower against HSV-2. In fact, 100% neutralization of infectivity was observed at a Fab concentration of 2 micrograms/100 TCD50 for the majority of HSV-1 strains, while a concentration of 8 micrograms/100 TCD50 was needed for 100% neutralization of all the HSV-2 strains tested. Owing to the above properties, Fab19 appears to be useful for diagnostic purposes and might also prove useful for in vivo immunoprophylaxis and therapy of HSV infections.

Animals↗

PubMed1995

A new plasmid cloning vector for direct detection of recombinant clones, based on inactivation of a bacterial acid phosphatase-encoding gene.

A new plasmid cloning vector for Escherichia coli (pPhoR) suitable for direct detection of recombinant clones has been constructed. The plasmid is a multicopy of a vector which carries a pUC-derived origin of replication and beta-lactamase gene, and the phoN acid phosphatase-encoding gene from Providencia stuartii. Foreign DNA fragments can be cloned into unique restriction sites located within the phoN gene causing a loss of the acid phosphatase activity which is normally overproduced by E. coli strains carrying pPhoR. Since PhoN production can be easily detected by a plate histochemical assay, recombinant clones carrying foreign DNA fragments inserted in the phoN gene can be easily detected as PhoN-negative clolonies on the above medium. The efficiency of the pPhoR-based cloning system for direct cloning of PCR amplimers of a variable region of the HIV-1 genome was comparable to that of conventional cloning systems for direct detection of recombinant based on beta-galactosidase inactivation. Advantage of the pPho-R-based system include reduced costs for histochemical assays and the possibility of being used with any E. coli host.

Acid Phosphatase↗

PubMed1994

Recombinant human Fab to glycoprotein D neutralizes infectivity and prevents cell-to-cell transmission of herpes simplex viruses 1 and 2 in vitro.

Herpes simplex viruses 1 and 2 (HSV-1 and -2) are associated with a number of conditions of varying severity, which are only partially responsive to current therapies. Human antibodies to the viruses offer a potential alternative. We describe here the generation of panels of human monoclonal Fab fragments to HSV-1 and -2 by panning a phage display combinatorial antibody library against whole lysates from the two viruses. Each lysate selected a largely distinct set of Fabs, although all of the Fabs were cross-reactive with both viruses. In a plaque-reduction assay, one Fab neutralized HSV-1 at 0.25 microgram/ml (50% reduction) and HSV-2 at 0.05 microgram/ml. This Fab also inhibited plaque formation when applied to virus-infected monolayers, completely abolishing HSV-2 plaque development at 25 micrograms/ml 72 hr postinfection, indicating the ability of the Fab to prevent cell-to-cell spread of virus. The Fab was shown to recognize viral glycoprotein D and to neutralize virus primarily by a postattachment mechanism. Recombinant Fabs may be useful for topical administration, although whole antibody will probably be required for systemic use.

Amino Acid Sequence↗

PubMed1994

Fractals without anomalous diffusion.

Explore the source record for details and available documents.

Journal Article↗

PubMed1993

Human monoclonal antibodies against a plethora of viral pathogens from single combinatorial libraries.

Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro.

Amino Acid Sequence↗

PubMed1992

Molecular cloning and characterization of DNA from human intestinal spirochetes.

HindIII cleaved human intestinal spirochete genomic DNA was cloned into the plasmid vector pBluescript. The inserts from three of these clones (pRB.C33, pRB.C47 and pRB.F11) successfully hybridized with DNA from the nine human intestinal spirochetes used in this study. The fourth insert (from clone pRB.A8) failed to hybridize with DNA from two of these isolates (HRM4 and HRM9), and, all four of the inserts hybridized with the M1 strain of swine intestinal spirochete Treponema innocens. Hybridization with DNA from the P18 strain of Treponema hyodysenteriae was observed only for the probe pRB.F11. No hybridization was detected with the human strain of Borrelia burgdorferi. With these probes we were thus able to demonstrate, in southern blot experiments, a restriction site polymorphism among the isolates of the human intestinal spirochetes we tested.

Adult↗

PubMed1990

Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2.

One of the regions responsible for the stable inheritance of the broad-host-range plasmid RK2 is contained within the PstI C fragment, located from coordinates 30.8 to 37.0 kb (P.N. Saurugger, O. Hrabak, H. Schwab, and R.M. Lafferty, J. Biotechnol. 4:333-343, 1986). Genetic analysis of this 6.2-kb region demonstrated that no function was present that stabilized by selectively killing plasmid-free segregants. The sequence from 36.0 to 37.0 kb mediated a twofold increase in plasmid copy number, but this region was not required for stabilization activity. The PstI C fragment was shown to encode a multimer resolution system from 33.1 to 35.3 kb. The resolution cis-acting site was mapped to 140 bp, sequenced, and observed to contain two directly repeated sequences of 6 and 7 bases and two perfect inverted repeats of 6 and 8 bases. The trans-acting factor(s) was mapped and functionally determined to encode a resolvase capable of catalyzing recombination at high frequency between cis-acting sites in either direct or inverted orientation. Multimer resolution alone did not account for complete plasmid stabilization by the PstI C fragment, since removal of regions adjacent to the 35.3-kb border of the minimal mrs locus dramatically reduced stabilization. The minimal region required for complete stabilization, from 32.8 to 35.9 kb, was capable of fully stabilizing plasmids independently of the replicon or the recA proficiency of the host. Stabilization activity was also fully expressed in several diverse gram-negative bacteria, whereas the F plasmid par locus functioned only in Escherichia coli. On the basis of these observations, we conclude that under the growth conditions used, the minimal stabilization locus encodes both an mrs activity and a stabilization activity that has the properties of a par locus.

Azotobacter↗

PubMed1988

Lyme disease in Italy: isolation of Borrelia burgdorferi from a patient.

A strain of Borrelia burgdorferi was isolated from a 48-year-old female patient suffering from a chronic form of polyarthritis. Significant titers of specific anti-Borrelia antibodies were not found. This is the first report of isolation of Borrelia burgdorferi from a patient in Italy.

Blotting, Western↗

PubMed1987

Expression of members of immunoglobulin gene family in somatic cell hybrids between human B and T cells.

Somatic cell hybrids were obtained between human T and B cells and tested for the expression of differentiated traits of both cell lineages. The T-cell parent SUP-T1 is CD3-, CD4+, CD1+, CD8+, is weakly positive for HLA class I determinants, and has an inversion of chromosome 14 due to a site-specific recombination event between an immunoglobulin heavy-chain variable gene and the joining segment of the T-cell receptor alpha chain. The B-cell parent, the 6-thioguanine- and ouabain-resistant mutant GM1500, is a lymphoblastoid cell line that secretes IgG2, kappa chains, and expresses B1, B532, and HLA class I and II antigens. All hybrids expressed characteristics of B cells (Ig+, B1+, B532+, EBNA+, HLA antigens), whereas only CD4 among the T-cell markers was expressed. The level of T-cell receptor beta-chain transcript was greatly reduced and no RNA of the chimeric T-cell receptor alpha-chain joining segment-immunoglobulin heavy-chain variable region was detected. Southern blot analysis indicated that absence of T-cell differentiation markers in the hybrids was not due to chromosomal loss. Rather, some B-cell-specific factor present in the hybrids may account for the suppression.

Antigens, Surface↗

PubMedDate not supplied

An improved phage display vector for antibody repertoire cloning by construction of combinatorial libraries.

Phagemid pComb3 is a widely used vector for molecular cloning of the antibody repertoire and for production of phage display libraries. However, in practical use, the utilization of this vector has some drawbacks. In this work we describe the construction of pComb3/TIG, an improved, easily manipulated vector for the cloning and display of antibody fragment libraries on the surface of filamentous phage. The two small "stuffer" fragments at the cloning sites were replaced with long DNA fragments, for easier differentiation of the correctly cut forms of the vector. Moreover, in pComb3/TIG the fragment at the heavy-chain-fragment cloning site contains an acid phosphatase-encoding gene. This feature allows the easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid instead of the heavy-chain fragment coding cDNA in a simple plate histochemical assay.

Antibodies↗

PubMedDate not supplied

Human monoclonal recombinant Fabs specific for HCV antigens obtained by repertoire cloning in phage display combinatorial vectors.

Molecular cloning of the antibody repertoire in phage display combinatorial vectors is a powerful method enabling the dissection of the immunoresponse against a given pathogen. In this paper we describe the construction of a combinatorial library displayed on phage surface, containing the antibody repertoire of a patient with high serological response against hepatitis C virus (HCV) antigens. Following selection of the library against solid-phase-bound antigen, sixteen human antibody Fab fragments able to bind to HCV-specific antigens were generated and studied for binding characteristics. The majority of them appeared to have specificity for the HCV c33 peptide. All the clones reacting with the c33 peptide shared the same heavy-chain CDR3 sequence. This is the first report of molecular cloning in a combinatorial phage display vector of the antibody repertoire of an anti-HCV-positive patient.

Amino Acid Sequence↗

PubMedDate not supplied

Probing the natural antibody repertoire by combinatorial cloning of IgM and IgD isotypes in phage display vectors.

It is well known that immunoglobulins with no identifiable immunogenic origin, called natural antibodies, are present in the sera of healthy individuals and their role as a defence against important pathogens has been proposed. Unfortunately, the studies are hampered by the fact that these immunoglobulins seem to have low affinity and to be polyreactive, and are commonly available in polyclonal preparations. Lately, new technologies for the production of monoclonal antibodies became available, and in particular the cloning of genes coding for antibody fragments in combinatorial phage display vectors provided a handy tool for the selection of human monoclonal antibodies. In this work, we describe the successful development of a technology for the molecular cloning of combinatorial phage display libraries containing genes coding exclusively for antibody fragment of the IgM or IgD phenotype. These libraries can be useful for molecular cloning of monoclonal antibodies of the IgM and IgD phenotype and can help elucidate the role played by natural antibodies in defence against infectious agents.

Antibodies, Monoclonal↗