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

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.

Figure 1

A flow chart summarizing the protocol. Under optimal conditions, an experienced laboratory can complete the entire procedure from vaccination to antibody in as little as 28 d.

Figure 2

Representative flow data summarizing the gating strategy. First, the live cell gate is set, including blasting cells, then CD19^high/CD20^{low to neg}/CD3^neg and CD27^high/CD38^high. Finally, appropriate IgG IgM, and IgD gates are set to obtain the precise population of interest, improving the immunoglobulin constant region-specific priming efficiency.

Figure 3

Characterization of the antibodies produced. Purified antibodies run on a 10% PAGE gel under reducing conditions (a). The heavy and kappa chain bands differ slightly in MW from antibody to antibody but typically fall between 50 and 60 kDa for the heavy chain and between 20–25 kDa for the kappa chain. ELISA curves for recombinant antibodies from day −7 IgG antibody-secreting cells (ASCs) from donors immunized with Fluvirin (b) or Pneumovax23 (c). In total, 73% (Fluvirin) and 67% (Pneumovax) of the antibodies bound either native antigen. Numbers in the legend indicate the well number of the antibody.