Bacterial Expression of a Human Monoclonal Antibody-Alkaline Phosphatase Conjugate Specific for Entamoeba histolytica

ABSTRACT We previously produced human monoclonal antibody Fab fragments specific to Entamoeba histolytica in Escherichia coli. In order to use these Fab fragments for diagnostic purposes, an expression vector to produce a fusion protein of Fab and alkaline phosphatase (PhoA) in E. coli was designed and constructed. The E. coli PhoA gene was fused to the 3′ terminus of the gene encoding the heavy-chain Fd region. The kappa and Fd genes from a previously prepared antibody clone, CP33, which is specific for the 260-kDa lectin of E. histolytica, were used as human antibody genes. When the fusion protein of CP33 and PhoA was incubated with paraformaldehyde-fixed trophozoites of E. histolytica and developed with a substrate, the trophozoites appeared to be stained. These results demonstrate the feasibility of bacterial expression of a human monoclonal antibody-PhoA conjugate specific for E. histolytica and that the antibody can be used to detect E. histolytica antigen without the use of chemically conjugated secondary antibodies.

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Entamoeba histolytica: Bacterial Expression of a Human Monoclonal Antibody Which Inhibits in Vitro Adherence of Trophozoites

Experimental Parasitology ◽

10.1006/expr.2000.4546 ◽

2000 ◽

Vol 96 (1) ◽

pp. 52-56 ◽

Cited By ~ 7

Author(s):

Xun-Jia Cheng ◽

Seiji Ihara ◽

Masataka Takekoshi ◽

Hiroshi Tachibana

Keyword(s):

Monoclonal Antibody ◽

Entamoeba Histolytica ◽

Human Monoclonal Antibody ◽

Bacterial Expression

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Bacterial Expression of a Human Monoclonal Antibody That Inhibits In Vitro Adherence of Entamoeba histolytica Trophozoites

Archives of Medical Research ◽

10.1016/s0188-4409(00)00141-7 ◽

2000 ◽

Vol 31 (4) ◽

pp. S311-S312 ◽

Cited By ~ 1

Author(s):

Xun-Jia Cheng ◽

Katsuomi Watanabe ◽

Seiji Ihara ◽

Masataka Takekoshi ◽

Hiroshi Tachibana

Keyword(s):

Monoclonal Antibody ◽

Entamoeba Histolytica ◽

Human Monoclonal Antibody ◽

Bacterial Expression

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Preparation of Recombinant Human Monoclonal Antibody Fab Fragments Specific for Entamoeba histolytica

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.6.3.383-387.1999 ◽

1999 ◽

Vol 6 (3) ◽

pp. 383-387 ◽

Cited By ~ 12

Author(s):

Hiroshi Tachibana ◽

Xun-Jia Cheng ◽

Katsuomi Watanabe ◽

Masataka Takekoshi ◽

Fumiko Maeda ◽

...

Keyword(s):

Escherichia Coli ◽

Monoclonal Antibody ◽

Entamoeba Histolytica ◽

Heavy Chain ◽

Light Chain ◽

Dna Sequences ◽

Human Monoclonal Antibody ◽

Fab Fragment ◽

Fab Fragments ◽

Reference Strains

ABSTRACT Genes coding for human antibody Fab fragments specific forEntamoeba histolytica were cloned and expressed inEscherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+ RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced intoEscherichia coli. Three thousand colonies were screened for the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from fiveEscherichia coli clones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of the Escherichia colilysates to nine reference strains of E. histolytica were examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific for E. histolytica. The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis.

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A universal surrogate peptide to enable LC-MS/MS bioanalysis of a diversity of human monoclonal antibody and human Fc-fusion protein drug candidates in pre-clinical animal studies

Biomedical Chromatography ◽

10.1002/bmc.2759 ◽

2012 ◽

pp. n/a-n/a ◽

Cited By ~ 8

Author(s):

Michael T. Furlong ◽

Zheng Ouyang ◽

Steven Wu ◽

James Tamura ◽

Timothy Olah ◽

...

Keyword(s):

Monoclonal Antibody ◽

Fusion Protein ◽

Animal Studies ◽

Human Monoclonal Antibody ◽

Protein Drug ◽

Drug Candidates ◽

Fc Fusion Protein ◽

Surrogate Peptide

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Generation and Activity Evaluation of a Mouse-Human Immunoglobulin G1 Chimeric Antibody to Histoplasma capsulatum HSP60

10.20944/preprints202104.0101.v1 ◽

2021 ◽

Author(s):

Ágata Nogueira D'Áurea Moura ◽

Scott J. Garforth ◽

Leandro Buffoni Roque da Silva ◽

Darien Woodley ◽

Filipe Vieira Barbalho ◽

...

Keyword(s):

Monoclonal Antibody ◽

Histoplasma Capsulatum ◽

Physiological Stress ◽

Human Monoclonal Antibody ◽

Current Treatment ◽

Chimeric Antibody ◽

Human Antibody ◽

Chimeric Mouse ◽

Dimorphic Fungi

Heat shock proteins (Hsps) are highly conserved molecules that are constitutively expressed and upregulated in response to physiological stress conditions. These immunogenic chaperones can have essential functions in fungi, particularly in dimorphic pathogens. Histoplasma capsulatum and Paracoccidioides species are dimorphic fungi that are the causative agents of histoplasmosis and paracoccidioidomycosis, respectively, which are systemic mycoses with significant rates of morbidity and mortality. Current treatment consists of long-term antifungal agents, and there is an urgent need for new therapeutic approaches with higher efficacy, lower toxicity, better biodistribution and improved selectivity. We engineered an immunoglobulin G1 (IgG1) isotype chimeric mouse-human monoclonal antibody, titled ch-MAb 4E12, from the parental IgG2a MAb 4E12, a monoclonal antibody to H. capsulatum Hsp60 that is protective in experimental histoplasmosis and paracoccidioidomycosis models elicited by H. capsulatum var. capsulatum and Paracoccidioides lutzii, respectively. The ch-MAb 4E12 increased phagolysosomal fusion and enhanced the yeasts uptake by PMA differentiated human THP1 macrophage cells in vitro. At low concentrations, the chimeric antibody significantly reduced the pulmonary and splenic fungal burden compared to an irrelevant antibody or no treatment. These results are the first to show that a chimeric mouse-human antibody can modify infection caused by dimorphic fungi.

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