Abstract
We describe an adaptation of conventional ELISA methods to an ELISA-Array format using non-contact Piezo printing of up to 30 spots of purified recombinant viral fusion proteins and vaccine on 96 well high-protein binding plates. Antigens were printed in 1 nanoliter volumes of protein stabilizing buffer using as little as 0.25 nanograms of protein, 2000-fold less than conventional ELISA. The performance of the ELISA-Array was demonstrated by serially diluting n = 9 human post-flu vaccination plasma samples starting at a 1/1000 dilution and measuring binding to the array of Influenza antigens. Plasma polyclonal antibody levels were detected using a cocktail of biotinylated anti-human kappa and lambda light chain antibodies, followed by a Streptavidin-horseradish peroxidase conjugate and the dose-dependent signal was developed with a precipitable TMB substrate. Intra- and inter-assay precision of absorbance units among the eight donor samples showed mean CVs of 4.8% and 10.8%, respectively. The plasma could be differentiated by donor and antigen with titer sensitivities ranging from 1 × 103 to 4 × 106, IC50 values from 1 × 104 to 9 × 106, and monoclonal antibody sensitivities in the ng/mL range. Equivalent sensitivities of ELISA versus ELISA-Array, compared using plasma and an H1N1 HA trimer, were achieved on the ELISA-Array printed at 0.25 ng per 200um spot and 1000 ng per ELISA 96-well. Vacuum-sealed array plates were shown to be stable when stored for at least 2 days at ambient temperature and up to 1 month at 4–8 °C. By the use of any set of printed antigens and analyte matrices the methods of this multiplexed ELISA-Array format can be broadly applied in translational research.
Highlights
The activity of humoral antibodies provide the best correlation to long-term immune memory and protection (Antia et al 2018)
Many operating conditions for printing followed the standard recommendations of the manufacturer of the sciFLEXARRAYER S12 instrument, several specific parameters were optimized for this ELISA-Array application
The probes of the printed arrays bound to the Fc region of IgG within the human plasma, are detected with horseradish peroxidase (HRP)
Summary
The activity of humoral antibodies provide the best correlation to long-term immune memory and protection (Antia et al 2018). Some plasmablasts will home to the bone marrow where they terminally differentiate into long-lived plasma cells stably secreting antibodies that circulate in serum for many years (Abbas, Lichtman, and Pillai 2014; Yoshida et al 2010). Serum can be used to measure the binding kinetics, magnitude, specificity and cross-reactivity of the secreted antibodies in response to infection or vaccination. Humoral responses are typically quantified by titer in naïve, acute, convalescent and recovery sera in the context of natural infection or pre- and post- vaccination and correlated to in vitro activity assays and clinical signs of immune protection Humoral responses are typically quantified by titer in naïve, acute, convalescent and recovery sera in the context of natural infection or pre- and post- vaccination and correlated to in vitro activity assays and clinical signs of immune protection (e.g. Antia et al, 2018; Lowell et al, 2017; Madore et al, 2010)
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