Viral Disruption for Conjugation
To develop a method for viral conjugation, we began with our whole-virus ELISA protocol, in which the virus is disrupted using SDS. For the first bead conjugation, SRV was disrupted with SDS. We also tried undisrupted whole virus and whole virus disrupted with Triton X-100 detergent. To compare the results obtained with the various disruption methods, the beads were run on the Bio-Plex system.
Optimizing Conjugation by Virus/Plasma Titration
After determining that we could successfully conjugate whole disrupted virus to the beads, the next step was to optimize the amount of virus for bead conjugation, as well as to determine the optimum plasma concentration. The protocol provided with the Bio-Plex amine coupling kit suggested using 512 g of protein for conjugation. We conjugated beads with 2, 5, 8, 10, and 12 g of virus (disrupted with Triton X-100), and then reacted the conjugated beads with both positive and negative plasma at dilutions of 1:50, 1:100, and 1:200.
ELISA assays followed conventional ELISA protocols.
Multiplexing was performed according to the following protocol:
1. Prepare plasma samples.
2. Load beads into wells.
3. Wash beads.
4. Incubate plasma with antigen-conjugated beads.
5. Wash beads.
6. Incubate beads with phycoerythrin detection antibody (Figure 1).
7. Wash beads.
8. Read on Bio-P