Rapid analysis of rearranged kappa light chain genes of circulating polysaccharide-specific B lymphocytes by means of immunomagnetic beads and the polymerase chain reaction.
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.
Hougs
L
L
Department of Clinical Immunology, Rigshospitalet, National University Hospital, Copenhagen, Denmark.
Barington
T
T
Madsen
H O
HO
Ryder
L P
LP
Svejgaard
A
A
D001483:Base Sequence; D015139:Blotting, Southern; D000939:Epitopes / Q000235:genetics; D015328:Gene Rearrangement, B-Lymphocyte, Light Chain / Q000235:genetics*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D018189:Immunomagnetic Separation; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D000939:Epitopes; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains