Rapid analysis of rearranged kappa light chain genes of circulating polysaccharide-specific B lymphocytes by means of immunomagnetic beads and the polymerase chain reaction

L Hougs, T Barington, HO Madsen, L P Ryder, A Svejgaard

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Analysis of rearranged immunoglobulin genes used by B lymphocytes of known specificity is an important tool for the study of diversity and selection of B lymphocytes. Usually hybridoma cell lines are used for such analyses, but they are difficult to obtain from humans and may not be representative of the B lymphocytes activated in vivo. Here, we present a method for rapid analysis of the rearranged kappa light chain genes used by human circulating antigen-specific B lymphocytes. After vaccination with Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugated with protein, the HibCP-specific B lymphocytes were isolated by antigen-coated immunomagnetic beads. After the purification, at least 98% of the immunoglobulin-secreting recovered cells were HibCP specific. The RNA was isolated and amplified by cDNA synthesis using a kappa constant region primer followed by polymerase chain reaction (PCR) using in addition a degenerate kappa light chain signal peptide region primer. The PCR product was cloned into the M13mp18 phage. The cloning efficiency was 100-600 clones/ml of blood. Of the 86 clones sequenced, 90% represented rearranged kappa light chain genes from different antibody-secreting cells. Examples of rearranged kappa genes used by HibCP-specific antibody-secreting cells from 4 adult vaccinees are given, representing the 3 largest of the 4 kappa variable region families. This method is a new tool for the investigation of vaccine-induced antibody responses with special reference to immunoglobulin gene usage and variability.
Original languageEnglish
JournalExperimental and Clinical Immunogenetics
Volume10
Issue number3
Pages (from-to)141-51
Number of pages10
ISSN0254-9670
Publication statusPublished - 1993

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Haemophilus influenzae type b
Polymerase Chain Reaction
Clone Cells
Hybridomas
Organism Cloning
Complementary DNA
RNA
Cell Line
Proteins

Keywords

  • Base Sequence
  • Blotting, Southern
  • Epitopes
  • Gene Rearrangement, B-Lymphocyte, Light Chain
  • Humans
  • Immunoglobulin Variable Region
  • Immunoglobulin kappa-Chains
  • Immunomagnetic Separation
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Sequence Analysis, DNA

Cite this

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title = "Rapid analysis of rearranged kappa light chain genes of circulating polysaccharide-specific B lymphocytes by means of immunomagnetic beads and the polymerase chain reaction",
abstract = "Analysis of rearranged immunoglobulin genes used by B lymphocytes of known specificity is an important tool for the study of diversity and selection of B lymphocytes. Usually hybridoma cell lines are used for such analyses, but they are difficult to obtain from humans and may not be representative of the B lymphocytes activated in vivo. Here, we present a method for rapid analysis of the rearranged kappa light chain genes used by human circulating antigen-specific B lymphocytes. After vaccination with Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugated with protein, the HibCP-specific B lymphocytes were isolated by antigen-coated immunomagnetic beads. After the purification, at least 98{\%} of the immunoglobulin-secreting recovered cells were HibCP specific. The RNA was isolated and amplified by cDNA synthesis using a kappa constant region primer followed by polymerase chain reaction (PCR) using in addition a degenerate kappa light chain signal peptide region primer. The PCR product was cloned into the M13mp18 phage. The cloning efficiency was 100-600 clones/ml of blood. Of the 86 clones sequenced, 90{\%} represented rearranged kappa light chain genes from different antibody-secreting cells. Examples of rearranged kappa genes used by HibCP-specific antibody-secreting cells from 4 adult vaccinees are given, representing the 3 largest of the 4 kappa variable region families. This method is a new tool for the investigation of vaccine-induced antibody responses with special reference to immunoglobulin gene usage and variability.",
keywords = "Base Sequence, Blotting, Southern, Epitopes, Gene Rearrangement, B-Lymphocyte, Light Chain, Humans, Immunoglobulin Variable Region, Immunoglobulin kappa-Chains, Immunomagnetic Separation, Molecular Sequence Data, Polymerase Chain Reaction, Sequence Analysis, DNA",
author = "L Hougs and T Barington and HO Madsen and Ryder, {L P} and A Svejgaard",
year = "1993",
language = "English",
volume = "10",
pages = "141--51",
journal = "Experimental and Clinical Immunogenetics",
issn = "0254-9670",
publisher = "S. Karger AG",
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}

Rapid analysis of rearranged kappa light chain genes of circulating polysaccharide-specific B lymphocytes by means of immunomagnetic beads and the polymerase chain reaction. / Hougs, L; Barington, T; Madsen, HO; Ryder, L P; Svejgaard, A.

In: Experimental and Clinical Immunogenetics, Vol. 10, No. 3, 1993, p. 141-51.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Rapid analysis of rearranged kappa light chain genes of circulating polysaccharide-specific B lymphocytes by means of immunomagnetic beads and the polymerase chain reaction

AU - Hougs, L

AU - Barington, T

AU - Madsen, HO

AU - Ryder, L P

AU - Svejgaard, A

PY - 1993

Y1 - 1993

N2 - Analysis of rearranged immunoglobulin genes used by B lymphocytes of known specificity is an important tool for the study of diversity and selection of B lymphocytes. Usually hybridoma cell lines are used for such analyses, but they are difficult to obtain from humans and may not be representative of the B lymphocytes activated in vivo. Here, we present a method for rapid analysis of the rearranged kappa light chain genes used by human circulating antigen-specific B lymphocytes. After vaccination with Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugated with protein, the HibCP-specific B lymphocytes were isolated by antigen-coated immunomagnetic beads. After the purification, at least 98% of the immunoglobulin-secreting recovered cells were HibCP specific. The RNA was isolated and amplified by cDNA synthesis using a kappa constant region primer followed by polymerase chain reaction (PCR) using in addition a degenerate kappa light chain signal peptide region primer. The PCR product was cloned into the M13mp18 phage. The cloning efficiency was 100-600 clones/ml of blood. Of the 86 clones sequenced, 90% represented rearranged kappa light chain genes from different antibody-secreting cells. Examples of rearranged kappa genes used by HibCP-specific antibody-secreting cells from 4 adult vaccinees are given, representing the 3 largest of the 4 kappa variable region families. This method is a new tool for the investigation of vaccine-induced antibody responses with special reference to immunoglobulin gene usage and variability.

AB - Analysis of rearranged immunoglobulin genes used by B lymphocytes of known specificity is an important tool for the study of diversity and selection of B lymphocytes. Usually hybridoma cell lines are used for such analyses, but they are difficult to obtain from humans and may not be representative of the B lymphocytes activated in vivo. Here, we present a method for rapid analysis of the rearranged kappa light chain genes used by human circulating antigen-specific B lymphocytes. After vaccination with Haemophilus influenzae type b capsular polysaccharide (HibCP) conjugated with protein, the HibCP-specific B lymphocytes were isolated by antigen-coated immunomagnetic beads. After the purification, at least 98% of the immunoglobulin-secreting recovered cells were HibCP specific. The RNA was isolated and amplified by cDNA synthesis using a kappa constant region primer followed by polymerase chain reaction (PCR) using in addition a degenerate kappa light chain signal peptide region primer. The PCR product was cloned into the M13mp18 phage. The cloning efficiency was 100-600 clones/ml of blood. Of the 86 clones sequenced, 90% represented rearranged kappa light chain genes from different antibody-secreting cells. Examples of rearranged kappa genes used by HibCP-specific antibody-secreting cells from 4 adult vaccinees are given, representing the 3 largest of the 4 kappa variable region families. This method is a new tool for the investigation of vaccine-induced antibody responses with special reference to immunoglobulin gene usage and variability.

KW - Base Sequence

KW - Blotting, Southern

KW - Epitopes

KW - Gene Rearrangement, B-Lymphocyte, Light Chain

KW - Humans

KW - Immunoglobulin Variable Region

KW - Immunoglobulin kappa-Chains

KW - Immunomagnetic Separation

KW - Molecular Sequence Data

KW - Polymerase Chain Reaction

KW - Sequence Analysis, DNA

M3 - Journal article

VL - 10

SP - 141

EP - 151

JO - Experimental and Clinical Immunogenetics

JF - Experimental and Clinical Immunogenetics

SN - 0254-9670

IS - 3

ER -