tetano
Editor, Senior Moderator
J Immunol Methods. 2020 May 4:112789. doi: 10.1016/j.jim.2020.112789. [Epub ahead of print]
Adaption of a conventional ELISA to a 96-well ELISA-Array for measuring the antibody responses to influenza virus proteins and vaccines.
Waltari E[SUP]1[/SUP], Carabajal E[SUP]2[/SUP], Sanyal M[SUP]3[/SUP], Friedland N[SUP]3[/SUP], McCutcheon KM[SUP]2[/SUP].
Author information
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 ? 10[SUP]3[/SUP] to 4 ? 10[SUP]6[/SUP], IC[SUB]50[/SUB] values from 1 ? 10[SUP]4[/SUP] to 9 ? 10[SUP]6[/SUP], 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.
Copyright ? 2019. Published by Elsevier B.V.
KEYWORDS:
ELISA-Array; Infectious disease; Protein array; Titer
PMID:32380014DOI:10.1016/j.jim.2020.112789
Adaption of a conventional ELISA to a 96-well ELISA-Array for measuring the antibody responses to influenza virus proteins and vaccines.
Waltari E[SUP]1[/SUP], Carabajal E[SUP]2[/SUP], Sanyal M[SUP]3[/SUP], Friedland N[SUP]3[/SUP], McCutcheon KM[SUP]2[/SUP].
Author information
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 ? 10[SUP]3[/SUP] to 4 ? 10[SUP]6[/SUP], IC[SUB]50[/SUB] values from 1 ? 10[SUP]4[/SUP] to 9 ? 10[SUP]6[/SUP], 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.
Copyright ? 2019. Published by Elsevier B.V.
KEYWORDS:
ELISA-Array; Infectious disease; Protein array; Titer
PMID:32380014DOI:10.1016/j.jim.2020.112789