Amplification of IgG VH and VL (Fab) from single human plasma cells and B cells

Abstract

Amplification of human immunoglobulin has many potential applications such as analysis of clonality, isolation of immunogenic antigens and antigen-specific immunotherapy. Here we describe a method for amplification of human immunoglobulin heavy and light chains from single B lymphocytes or plasma cells. Cells are isolated by FACS, and Ig is amplified by semi-nested RT-PCR. The method is versatile, sensitive and reliable: it provides appropriately paired heavy and light chains, requiring as little as 2 days to produce amplified Fab DNA from human tissues.

Figure 1

Single cell RT–PCR.

Figure 2

Agarose gel electrophoresis of single cell RT–PCR of IgG. Shown are IgG Fab amplified from single cell. Blocks separated by lines represent multiple PCR amplifications of IgG Fab from single cDNA pools. The products of multiple reactions are shown for each cell in order from left to right: VL1B, VL3B, VL4B, VK1B, VK9B, VH5B, VH10B, VH22B. (A) Plasma cells from tonsil. Plasma cells PC12, PC15 and PC16 are λ positive, while PC13 and 14 are κ positive. (B) Plasma cells from medullary carcinoma. PC54 is λ positive, while PC51 is κ positive. (C) B cells from tonsil. B cells BC11 and BC19 are λ positive. BC1 and BC13 are κ positive. BC7 is κ positive, but there was no heavy chain amplification for this cell, indicating non-γ1 for the IgG + B cell heavy chain.

Figure 3

Alignment of sequences from a single plasma cell. Sequences shown are a segment from separate PCR amplifications of a single plasma cell cDNA pool, utilizing different individual IgVH gene family primers (VH10B, VH5B, VH22B, VH14B, VH12B). All VH primer PCR sequences (including regions not shown) were identical and are shown aligned with the parental germline VH gene IGHV1-18*01 for this segment. Two somatic mutations are evident in this segment versus germline (G>C and C>G).