Arrayit Corporation (ARYC)

[Alyssa]

Billboard material: Published 21 January 2020 - Influenza virus protein microarrays.The IVPMs were generated and probed similarly to protocols described before (13). Briefly, arrays of recombinant HA were spotted on Nexterion E epoxysilane-coated glass slides (Schott, Mainz, Germany). Eight HAs were included in each array and spotted in triplicate, and 24 arrays were spotted on each slide. Each HA spot had a volume of 30 nl and was spotted at a concentration of 100?µg/ml in phosphate-buffered saline (PBS). After spotting, slides were incubated for 2 h at >95% relative humidity at room temperature and then allowed to dry. Slides were inserted into 96-well microarray gaskets (Arrayit, Sunnyvale, CA, USA) and blocked with 3% milk in PBS containing 0.1% Tween 20 (PBST) for 90 min. After the blocking solution was removed, sera were added at a starting concentration of 1:100 in 1% milk-PBST at a volume of 100?µl/array, and two 10-fold dilutions were performed across each slide. Sera were incubated on the slides for 1 h, and then the slides were washed three times with 220?µl/array PBST before the addition of 50?µl secondary antibody solution, composed of Cy3-labeled anti-human IgA secondary antibody and Cy5-labeled anti-human IgG secondary antibody diluted 1:400 and 1:1,500, respectively, in 1% milk-PBST. After 1 h, the secondary antibody solution was removed, and the arrays were washed three times with 220?µl/array PBST, removed from the 96-well microarray gaskets, rinsed with deionized water, and dried with an air compressor. Dried microarray slides were analyzed with a Vidia microarray scanner (InDevR, Boulder, CO, USA) at an exposure time of 1,000?ms. The AUC was calculated from median fluorescence as the total peak area above a fluorescence of 0.04. AUC values were adjusted based on the reactivity of a standard protein spotted in each array type, A/Perth/16/2009, using reactivity in array 1 as the standard. The AUCs of each array type were multiplied by the mean reactivity of A/Perth/16/2009 in array 1 divided by the mean reactivity of A/Perth/16/2009 in that array type.

IVPMs with monoclonal antibodies (MAbs) were performed as described above but with starting dilutions of 30?µg/ml MAb serially diluted in 1% milk-PBST 1:5 eight times and incubated with 100 µl/array Cy5-labeled anti-IgG secondary antibody diluted 1:3,000 in 1% milk-PBST.

https://mbio.asm.org/content/11/1/e03243-19