Efficient method for construction comprehensive murine Fab antibody libraries displayed on phage

Ørum, Henrik; Andersen, Peter S.; øster, Anne; Johansen, Lene K.; Riise, Erik; Bjornvad, M.E.; Svendsen, I.; Engberg, Jan

doi:10.1093/nar/21.19.4491

Cited by 108 publications

(44 citation statements)

References 25 publications

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“…RNA was isolated from the samples by acid phenol extraction according to the procedure of Chirgwin et al (7). mRNA was converted to cDNA as described by Ørum et al (29).…”

Section: Methodsmentioning

confidence: 99%

Human Recombinant Antibodies againstPlasmodium falciparumMerozoite Surface Protein 3 Cloned from Peripheral Blood Leukocytes of Individuals with Immunity to Malaria Demonstrate Antiparasitic Properties

et al. 2006

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Immunoglobulins from individuals with immunity to malaria have a strong antiparasitic effect when transferred to Plasmodium falciparum malaria infected patients. One prominent target of antiparasitic antibodies is the merozoite surface antigen 3 (MSP-3). We have investigated the antibody response against MSP-3 residues 194 to 257 (MSP-3 194-257 ) on the molecular level. mRNA from peripheral blood leukocytes from clinically immune individuals was used as a source of Fab (fragment antibody) genes. A Fab-phage display library was made, and three distinct antibodies designated RAM1, RAM2, and RAM3 were isolated by panning. Immunoglobulin G1 (IgG1) and IgG3 full-length antibodies have been produced in CHO cells. Reactivity with the native parasite protein was demonstrated by immunofluorescence microscopy, flow cytometry, and immunoblotting. Furthermore, the antiparasitic effect of RAM1 has been tested in vitro in an antibody-dependent cellular inhibition (ADCI) assay. Both the IgG1 and the IgG3 versions of the antibody show an inhibitory effect on parasite growth.Clinical immunity to Plasmodium falciparum malaria is gradually acquired over a dozen years of intense exposure to the parasite (12). Acquired immunity to malaria has been termed premunition and is characterized as being nonsterile and incomplete (43). The exact mechanism responsible for premunition is not known with certainty. However, a number of clinical studies carried out in the early sixties (9, 16, 23)-and subsequently confirmed and extended in the nineties (2, 33)-showed an unambiguous antiparasitic effect of antibodies transferred from adults with immunity to malaria to malariainfected infants. Clinical effects observed in one of these studies correlated with the effect measured in the in vitro assay termed antibody-dependent cellular inhibition (ADCI) (2, 4). In the ADCI assay, immune antibody cooperates with monocytes in an in vitro malaria culture, and the antiparasitic effect is demonstrated by parasite growth inhibition. It has been shown that the antibody-merozoite complex by a contact-dependent mechanism stimulates the monocyte to secrete substances toxic to the asexual blood stages. The specific substances responsible for the subsequent, non-contact-dependent parasite growth inhibition include tumor necrosis factor alpha together with other molecules that are yet to be identified (4). The ADCI assay has been used for identification and characterization of the merozoite surface protein 3 (MSP-3) (27).An invariable structural feature of all reported MSP-3 sequences is the presence of three regions each of which contains three, four, or five conserved heptad repeat units. Previously published structural analyses suggest that the heptad repeat regions have an amphipathic alpha-helical secondary structure. A coiled-coil bundle conformation including these regions is a theoretical possibility supported by experimental data (24). The C-terminal part of MSP-3 contains a leucine zipper-like domain possibly implicated in dimerization and the formation...

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“…RNA was isolated from the samples by acid phenol extraction according to the procedure of Chirgwin et al (7). mRNA was converted to cDNA as described by Ørum et al (29).…”

Section: Methodsmentioning

confidence: 99%

Human Recombinant Antibodies againstPlasmodium falciparumMerozoite Surface Protein 3 Cloned from Peripheral Blood Leukocytes of Individuals with Immunity to Malaria Demonstrate Antiparasitic Properties

et al. 2006

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“…Therefore, we set out to apply the proteasesensitive helper phage in cell surface selection. Furthermore, the presence of a Myc tag between the displayed antibody fragment and protein III in the fusion coat protein as well as the protease-sensitive linker in helper phage-encoded protein III enables elution of phage bound via antibodies and concomitant functional elimination of helper phage-encoded protein III (17,36).…”

Section: Selection Of Antibodies Directed Against Extracellularmentioning

confidence: 99%

Identification of Keratinocyte-specific Markers Using Phage Display and Mass Spectrometry

Jensen

Ravn

et al. 2003

Molecular & Cellular Proteomics

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Specific molecular markers for various normal and pathogenic cell states and cell types provide knowledge of basic biological systems and have a direct application in targeted therapy. We describe a proteomic method based on the combination of new and improved phage display antibody technologies and mass spectrometry that allows identification of cell type-specific protein markers. The most important features of the method are (i) reduction of experimental noise originating from background binding of phage particles and (ii) isolation of affinity binders after a single round of selection, which assures a high diversity of binders. The method demonstrates, for the first time, the ability to detect, identify, and analyze both secreted and membrane-associated extracellular proteins as well as a variety of different cellular structures including proteins and carbohydrates. The optimized phage display method was applied to analysis of human skin keratinocytes resulting in the isolation of a panel of antibodies. Fourteen of these antibodies were further characterized, half of which predominantly recognized keratinocytes in a screen of a range of different cell types. Three cognate keratinocyte antigens were subsequently identified by mass spectrometry as laminin-5, plectin, and fibronectin. The combination of phage display technology with mass spectrometry methods for protein identification is a general and promising approach for proteomic analysis of cell surface complexity.

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“…Following ligation, the recombinant DNA was precipitated, washed and dissolved in 15 µl of distilled water. 1 µl of recombinant DNA was transformed by electroporation [18] into 40 µl of E. coli XL1-Blue every time. After transformation, 1 ml SOC medium was added and the culture was shaken for 1 h at 37 ºC, and then 10 ml of SB containing 20 µg/ml ampicillin and 10 µg/ml tetracycline was cultivated for an additional hour at 37 ºC on a shaker.…”

Section: Library Constructionmentioning

confidence: 99%

Generation and selection of immunized Fab phage display library against human B cell lymphoma

et al. 2007

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npgThe approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production of monoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody (HAMA) response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the κ light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94×10 7. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains (V H ) and variable light domains (V L ) were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.

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Efficient method for construction comprehensive murine Fab antibody libraries displayed on phage

Cited by 108 publications

References 25 publications

Human Recombinant Antibodies againstPlasmodium falciparumMerozoite Surface Protein 3 Cloned from Peripheral Blood Leukocytes of Individuals with Immunity to Malaria Demonstrate Antiparasitic Properties

Human Recombinant Antibodies againstPlasmodium falciparumMerozoite Surface Protein 3 Cloned from Peripheral Blood Leukocytes of Individuals with Immunity to Malaria Demonstrate Antiparasitic Properties

Identification of Keratinocyte-specific Markers Using Phage Display and Mass Spectrometry

Generation and selection of immunized Fab phage display library against human B cell lymphoma

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