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Nat Protoc. 2009;4(3):372-84. doi: 10.1038/nprot.2009.3.

Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen.

Author information

1
Department of Clinical Immunology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.

Abstract

We describe herein a protocol for the production of antigen-specific human monoclonal antibodies (hmAbs). Antibody-secreting cells (ASCs) are isolated from whole blood collected 7 d after vaccination and sorted by flow cytometry into single cell plates. The antibody genes of the ASCs are then amplified by RT-PCR and nested PCR, cloned into expression vectors and transfected into a human cell line. The expressed antibodies can then be purified and assayed for binding and neutralization. This method uses established techniques but is novel in their combination and application. This protocol can be completed with as little as 20 ml of human blood and in as little as 28 d when optimal. Although previous methodologies to produce hmAbs, including B-cell immortalization or phage display, can be used to isolate the rare specific antibody even years after immunization, in comparison, these approaches are inefficient, resulting in few relevant antibodies. Although dependent on having an ongoing immune response, the approach described herein can be used to rapidly generate numerous antigen-specific hmAbs in a short time.

PMID:
19247287
PMCID:
PMC2750034
DOI:
10.1038/nprot.2009.3
[Indexed for MEDLINE]
Free PMC Article

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