|Whole virion ELISA results from Tulika Singh||mkoenig5||2017-07-24|
Tulika conducted a whole virion ELISA to assess the binding of rhesus macaque plasma to Zika virus during infection and secondary challenge. Her summary of the experiment and results is included below and the results are included in the attached PDF
I did a whole virion ELISA in order to assess the binding of rhesus macaque plasma at sequential timepoints to Zika virus. We hypothesize that prior to exposure to Zika virus, plasma is not specific to Zika virus and thus will not bind the virion; but as the humoral immune response develops after challenge with Zika virus, we expect that antibodies specific to Zika will expand and display stronger binding to Zika virus.To do test this, I ran the following ELISA: I coated flat-bottomed 96 well plates with 4G2 anti-flavivirus mAb, which allows us to immobilize Zika virion on the plate. Then, I incubated this with serial dilutions of plasma, starting at 1:12.5 and diluting 4-fold 11 times. The plasma binding to Zika virions was detected with a goat anti-human mAb conjugated to horseradish peroxidase. This enzyme catalyzes a substrate (commercially known as TrueBlue/SureBlue) and generates a colorimetric luminescence at different intensities, which can be measured. The plasma serial dilution generates a curve – where lower dilutions have more plasma antibodies and bind more with the virus, and higher dilutions will have less plasma and thus display weaker binding (curves shown on slides 3–5). From each curve we can calculate an ED50: the dilution at which the plasma displays half as much binding as its maximum binding.To evaluate the plasma binding profile along the timeline of ZIKV primary and secondary challenge, I tested plasma at multiple time-points from the baseline of Day 0 (day of Zika challenge) through the re-challenge on Day 70 (shown on slide 2). Based on the data, specificity and binding to Zika virus increases dramatically, to near-maximal threshold, between days 5 and 15 post primary infection. High binding to Zika is maintained for the next 60 days, indicative of sterilizing immunity. Upon re-challenge, binding to Zika virus does not increase significantly in the plasma, indicating that the pre-existing humoral response was protective. This is consistent with the lack of viremia upon re-challenge, as well as lack of plasmablast expansion upon re-challenge.Tulika Singh Duke Molecular Genetics and Microbiology Graduate Student Permar Lab Duke University School of Medicine Durham, N.C.