Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

M Dziegiel; L K Nielsen; P S Andersen; A Blancher; E Dickmeiss; J Engberg

doi:10.1016/0022-1759(95)00013-Z

Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

M Dziegiel, L K Nielsen, P S Andersen, A Blancher, E Dickmeiss, J Engberg

Research output: Contribution to journal › Journal article › Research › peer-review

Abstract

A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a κ-Fd heterodimer. The successful use of intact cells for selection of specific Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the V _H 1.9III and V _κ Humκv325 germline genes, respectively.

Original language	English
Journal	Journal of Immunological Methods
Volume	182
Issue number	1
Pages (from-to)	7-19
Number of pages	13
ISSN	0022-1759
DOIs	https://doi.org/10.1016/0022-1759(95)00013-Z
Publication status	Published - 11 May 1995
Externally published	Yes

Keywords

Amino Acid Sequence
Bacteriophages
Base Sequence
Cloning, Molecular
DNA, Complementary
Genetic Vectors
Humans
Isoantibodies
Molecular Sequence Data
Polymerase Chain Reaction
Recombinant Fusion Proteins
Rh-Hr Blood-Group System
Journal Article
Research Support, Non-U.S. Gov't

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10.1016/0022-1759(95)00013-Z

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title = "Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D",

abstract = "A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a κ-Fd heterodimer. The successful use of intact cells for selection of specific Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the V H 1.9III and V κ Humκv325 germline genes, respectively.",

keywords = "Amino Acid Sequence, Bacteriophages, Base Sequence, Cloning, Molecular, DNA, Complementary, Genetic Vectors, Humans, Isoantibodies, Molecular Sequence Data, Polymerase Chain Reaction, Recombinant Fusion Proteins, Rh-Hr Blood-Group System, Journal Article, Research Support, Non-U.S. Gov't",

author = "M Dziegiel and Nielsen, {L K} and Andersen, {P S} and A Blancher and E Dickmeiss and J Engberg",

year = "1995",

month = may,

day = "11",

doi = "10.1016/0022-1759(95)00013-Z",

language = "English",

volume = "182",

pages = "7--19",

journal = "Journal of Immunological Methods",

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T1 - Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

AU - Dziegiel, M

AU - Nielsen, L K

AU - Andersen, P S

AU - Blancher, A

AU - Dickmeiss, E

AU - Engberg, J

PY - 1995/5/11

Y1 - 1995/5/11

N2 - A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a κ-Fd heterodimer. The successful use of intact cells for selection of specific Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the V H 1.9III and V κ Humκv325 germline genes, respectively.

AB - A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a κ-Fd heterodimer. The successful use of intact cells for selection of specific Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the V H 1.9III and V κ Humκv325 germline genes, respectively.

KW - Amino Acid Sequence

KW - Bacteriophages

KW - Base Sequence

KW - Cloning, Molecular

KW - DNA, Complementary

KW - Genetic Vectors

KW - Humans

KW - Isoantibodies

KW - Molecular Sequence Data

KW - Polymerase Chain Reaction

KW - Recombinant Fusion Proteins

KW - Rh-Hr Blood-Group System

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

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U2 - 10.1016/0022-1759(95)00013-Z

DO - 10.1016/0022-1759(95)00013-Z

M3 - Journal article

C2 - 7769246

SN - 0022-1759

VL - 182

SP - 7

EP - 19

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

IS - 1

ER -

Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

Abstract

Keywords

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