Preparation of Recombinant Human Monoclonal Antibody Fab Fragments Specific forEntamoeba histolytica

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.

Production of an Anti-Severe Acute Respiratory Syndrome (SARS) Coronavirus Human Monoclonal Antibody Fab Fragment by Using a Combinatorial Immunoglobulin Gene Library Derived from Patients Who Recovered from SARS

“…Both serum samples were positive for SARS-CoV by an enzyme-linked immunosorbent assay (ELISA), with titers greater than 1:2,000. Construction of an immunoglobulin gene library from peripheral lymphocytes was performed as described previously (10). Briefly, total RNA was purified from lymphocytes and was subjected to reverse transcription-PCR.…”

Production of High-Affinity Human Monoclonal Antibody Fab Fragments to the 19-Kilodalton C-Terminal Merozoite Surface Protein 1 ofPlasmodium falciparum

“…Lymphocytes were separated from the blood by Ficoll-Paque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. Construction of an immunoglobulin gene library from the lymphocytes was performed as previously described (39). Briefly, total RNA was purified from lymphocytes and subjected to reverse transcription-PCR.…”

“…Several methods have been developed to produce human monoclonal antibodies (1,2,11,44). We have reported that the bacterial expression system is useful for the preparation of human Fab fragments specific to pathogens (6,17,(39)(40)(41)(42). In the present study, we use a combinatorial immunoglobulin gene library derived from lymphocytes of patients with falcip-arum malaria to produce human monoclonal antibody Fab fragments that specifically react to P. falciparum MSP-1 19 .…”

Feasibility of polyelectrolyte-driven Fab fragment separation

“…Treatment of a monoclonal antibody (mAb) with papain is one way to produce antigen-binding fragments or fragment antigen binding (Fab), also resulting in production of a Fc fragment [1-2]. As Fab fragments exhibit low immunogenicity while obtaining similar binding properties compared to an entire mAb they find wide applications in diagnostics and research and can also be used as carriers for cytotoxic compounds due to their rapid blood clearance [3][4][5][6][7][8][9][10][11]. Fab and Fc can be separated using protein A affinity chromatography, however, this separation technique is insufficient for Fabs derived from mAbs belonging to the V H 3 subfamily, due to a binding affinity of protein A to this type of Fab [12,13].…”