Authors
Birgit Obermeier, Reinhard Mentele, Joachim Malotka, and Klaus Dornmair
Summary
We describe a method for relating oligoclonally expanded B cells at particular body locations to their secreted products, soluble immunoglobulins. The technique makes use of the virtually infinite variability of mature immunoglobulin (Ig) chains, which results from VDJ recombination and somatic hypermutation (SHM). We create patient-specific transcriptome databases by PCR-cloning of Ig transcripts of B cells from the relevant location. In parallel, we analyze the Ig proteome at the location of interest. To this end, we isolate the Ig molecules by affinity chromatography and isoelectric focusing, and analyze them by trypsin digestion and subsequent mass spectrometry. Then the patient-specific Ig proteomes and transcriptomes are compared by searching for the peptides identified by mass spectrometry in the transcriptome databases. We place particular focus on the peptides that carry characteristic amino acids introduced by VDJ recombination and SHM. Specific matches suggest that the particular B cells produce the oligoclonally expanded antibodies. We applied the method to cerebrospinal fluid from multiple sclerosis patients. However, the technique will also be applicable to antibody populations that are expanded in body fluids or tissues of any species.Further details
The protocol was published on Protocol Exchange in 2008. To see the entire protocol, click on the source link.Advertisement
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