A Multiplex Serological Assay for the Detection of Antibody Responses to Arboviruses

In our lab, we measure immune responses to infectious diseases in order to better understand how they are transmitted in populations. Recent developments in our field are multiplex assays, which allow us to measure many different antibodies in a single reaction using minimum volume. To begin, obtain eight 96 deep well plates.

Prepare each plate by adding tip combs, monobasic sodium phosphate, Triton solution, sulfo-NHS, EDC, and antigen solution. To set up the coupling cycle on the automated processor, switch on the machine, set up the protocol parameters using the software. Load the plates into the allocated slots on the machine, then select the protocol and start the cycle after completion, unload the plates and switch off the machine as per manufacturer's instructions.

Visually check that plate eight contains the beads visible as a brown pellet. Transfer the couple beads from plate eight into individual 1.5 milliliter tubes for storage. Store the coupled beads at four degrees Celsius until use.

To count the beads, thoroughly vortex the coupled beads. Transfer 10 microliters to a cell counting chamber, then follow the manufacturer's instructions to read the bead count per milliliter. Check aggregation by visualizing the image on the cell counter.

Set up the plate layout. Dilute samples, and standard in PBT buffer in a separate non-binding plate. Thoroughly vortex the coupled beads mix for 30 seconds, then sonicate for at least 60 seconds.

Now, transfer the adequate bead antigen volumes to a centrifuge tube and adjust the volume with PBT buffer. Thoroughly mix the premix. Distribute 50 microliters of the premix to the imaging microplate, then pipette 50 microliters of the diluted samples to the beads in the imaging microplate and mix on a plate shaker.

Place the plate on a magnetic rack for 60 seconds at room temperature. Hold the plate tightly on the rack and discard the supernatant. Prepare a diluted solution of the secondary antibody with an appropriate amount of PBT buffer.

Add 50 microliters of the antibody to the washed beads in the imaging plate. Incubate the plate on a shaker for 15 minutes at room temperature and perform three more washes with PBT. After inserting the plate into the fluorescence reader, set up the protocol and save it.

Linearity of antibody response was confirmed for selected arbovirus antigens tested at optimal concentrations on the ORPAL standard pool. Parallel linearity of antibody response was observed when comparing the manual and three automated coupling methods. Median fluorescence intensity values obtained from 19 different plates for each antigen and control fell within the range of the mean.

A strong correlation was observed between the median fluorescence intensity values obtained from multiplex and singleplex assays using the ORPAL standard pool. In the Senegal cohort, a clear bimodal distribution was observed in the antibody response to Chick VVLP and subtle bimodal tails were also seen for DENV1 NS1 and RVFV NP.We have shown that our serological tests can simultaneously measure antibodies to many arboviruses. Our validated assay to measure multiple responses to different arboviruses will help in our efforts to understand the transmission of these viruses across Africa.

Our protocol provides huge time savings and reproducibility in the preparation of reagents for multiplex assays.