Figure 4. Physical characterization of the EDC/NHS TMV-HA conjugate. (A) Increased EDC/NHS conjugation time from 30 to 60 min caused additional high molecular weight bands to appear in the conjugated reactions (*), in addition to more accumulation in the well (arrow; T-H reactions). (B) Laser Optical scattering microscopy was used to determine the size of the vaccine particle in suspension. Average rotational size was measured for both TMV and TMV-HA, in triplicate. (C) 40 000× Electron microscopy imaging of TMV or the TMV-HA conjugate. Particle average diameter increased by 34% from 18.0 nM to 24.1 nM after HA conjugation. The diameter was derived as the average of 30 electron micrograph images, using the bar = 100 nM as a size standard. (D) TMV and HA were assessed independently at 100 µg, and the TMV-HA conjugate was assessed at 20 µg for the ability to agglutinate turkey RBCs. Data are shown as the inverse of the minimum titer required to stimulate agglutination.