The affinity of antibodies for their cognate antigens is a critical aspect of humoral immunity. The immune system has gone to great lengths to evolve a mechanism that enables real time increases in antibody affinity during the course of an immune response. This occurs in germinal centers (GC), which form in spleen and lymph nodes following immunization. GC B cell competition for limiting amount of antigen drives the selection of B cells expressing higher affinity Abs. Remarkably little is known about affinity maturation of B cells in immune responses to all but a handful of small model antigens. It has proven challenging to measure the avidity of specific Abs in polyclonal sera to more complex antigens, including viruses. In this chapter we present a simple, flow cytometry based, method that determines the average avidity of GC B cells for the influenza A virus hemagglutinin glycoprotein, the target antigen of traditional influenza vaccines. Flow cytometry using fluorescent hemagglutinin and B cell marker specific Abs enables high throughput qualitative and quantitative detection of individual B cells. By using a graded amount of antigen and gating on GC B cells we define the AC50 the amount of antigen required to stain 50% of hemagglutinin specific B cells. This number is in remarkable agreement with the avidity of the B cell population. This method can be generally employed to include antibody avidity measurements basic and clinical studies of immunity to viruses and other medically relevant immunogens.
Keywords: Affinity; Avidity; B cell; Flow cytometry; Germinal center; Hemagglutinin; Immunoglobulin; Influenza.