The study used four PBA imitators and eight antibody preparations — two Ab clones for each target. RNA-virus detection monoplex was against internal nucleoprotein (NP). As the target imitator, influenza A virus (IAV), subtype H5N2 [NCBI, KX879578-85] (Smirnov et al., 2000), kindly provided by Dr. M. Shmarov, was used. For capture and detection, mouse monoclonal antibodies to the nucleocapsid protein of the avian influenza virus clones NP3 and NPS, respectively, obtained by Dr. A. Yu. Kozlov and kindly provided by Prof. T. V. Grebennikova, were used. The clone of NPS was conjugated to biotin isothiocyanate using EZ-Link^TM Sulfo-NHS-Biotin kit (Thermo Fisher, #21326) according to the manufacturer’s protocol.

DNA-containing virus detection monoplex was designed against surface protein (hexon) antigen from Adenovirus type 6 (AdV6). Strain Tonsil 99 (Bialexa, Russia) preparation was used as the model antigen. Mouse monoclonal antibodies to AdV6 hexon (clone SY-25), kindly provided by Prof. T. V. Grebennikova, were used as the capture antibody. Clone 1AD mouse monoclonal antibodies to AdV6 hexon (Bialexa, Russia) conjugated with biotin was used as a detection antibody.

As a non-pathogenic bacteria imitator, Salmonella enterica serovar typhimurium MvP728 (STm), prepared from a strain of wild-type S. typhimurium NCTC12023 kindly provided by Dr. V. G. Lunin, was used. Virulence was suppressed in these strain by a double deletion dHtrA/dPurD. Clone ST1 mABs (Bialexa, Russia or 1E6, Abcam) against LPS S. typhimurium was used as capture antibody. The same biotinylated aliquot was used as detection antibody.

Cholera toxin B-subunit (CTB) (Sigma, #C9903) was used as a toxin imitator. The preparation CTB mABs (clone CT8) and CTB mABs (clone CT9) preparations conjugated with biotin (Bialexa, Russia) were used as the primary and detecting antibodies, respectively.

Pathogenic biological agent, potentially present in bioaerosol, are typically collected and transferred into a liquid medium before analysis. At the same time, the fine-dispersed dust, which will inevitably penetrate the sample in the process of collecting the desired targets, is a side effect of the analysis. As matrices for the analysis, we compared four media using combination of SASS^® 4000 Aerosol Concentrator attached to SASS^TM 2300 Wetted-Wall Air Sampler (Research International, United States). We used 5 min of aerosol collection in an open forest park on a sunny, windy summer day at a temperature of 20°C – 25°C for collection of the ambient air contents (1–10 microns size dust in accordance with the characteristics of the device) in PBS pH 7.4 (Amresco, #E404), or in deionized water type 1, 18.2 MOm. These two types of media are named here as PBS-DUST and H2O-DUST. Initial media without dust PBS and H2O as a control were used through the study. Five minutes intervals was enough to pass through 15 000 L of aerosol.

Mono- and multi-spiked samples were prepared by adding PBAs to each of the four matrices of a certain concentration and then by double dilution six times (seven concentrations in total). To extract the internal target — nucleoprotein — from the virion of the influenza A virus, 0.1% Igepal CA-630 [Sigma, #I3021] was added to the matrices before the start of the assay.

During the initial studies, a wide range of target concentrations was analyzed. The range of concentrations of each PBA for presentation in the paper was determined as follows: the lowest concentration in the multiplex at which the MFI values of PBA in all four matrices are higher than the LOD. In addition to this concentration, six sequential doubly increased concentrations were included for each PBA. Finally the following PBA concentrations were analyzed: IAV in concentration from 4000 to 63 ng/ml (about 1.5^∗10¹⁰ to 1^∗10⁸ virions/ml); AdV6 in concentration from 2000 to 32 ng/ml (about 8^∗10⁹ to 1^∗10⁸ virions/ml); STm from 10⁷ to 10¹ CFU/ml, then the most informative range of concentrations were chosen ranging from 10⁶ to 1.5^∗10⁴ CFU/ml and CTB from 128 to 2 ng/ml.

Immunoassay was performed using xMAP technology from Luminex. Primary ABs in concentration 10 μg/10⁶ microspheres was conjugated with 4 regions: NP3 (anti-NP IAV) with no.15, SY-25 (anti-hexon AdV6) with no.45, ST1 (anti-LPS STm) with no.72 and CT8 (anti-CTB) with no.78. The microsphere coupling was carried out by carbodiimide chemistry coupling protocol in accordance with the protocol given by Luminex (2016). The solutions necessary for the coupling of microspheres were prepared using the following reagents: NaH₂PO₄⋅H₂O (Helicon, #Am-O823) for activation buffer (0.1 M NaH₂PO₄, pH 6.2); MES hydrate [Sigma, #M2933] for coupling buffer (50 mM MES, pH 5.0), and 1-ethyl-1-3-dimethylaminopropyl-carbodiimide hydrochloride (EDC) (Sigma, #22980) and N-hydroxysulfosuccinimide (s-NHS) (Sigma, #24520). The coupling procedure is described briefly as follows: 1 × 10⁶ beads were activated with 10 μl of 50 mg/mL EDC and 10 μl of 50 mg/mL s-NHS in 80 μl activation buffer for 20 min at 25°C with gentle mixing by vortex at 10 min intervals. After that, the activated beads were washed two times, and resuspended in 500 μL of coupling buffer with the addition of 10 μg/10⁶ microspheres coupling Abs. After incubation for 2 h with mixing (by 20 rpm rotation) at room temperature in the dark and 3 washing steps, the microspheres were resuspended in 1 ml of blocking/storage buffer with minimum overnight storing before analysis. The microspheres remaining after the coupling procedure were counted using an automatic cell counter TC-20 (Bio-RAD, United States).

As a microsphere blocking/storage buffer, PBS-TBN (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% NaN3) for no.15-NP3 and no.78-CT8 was used. PBS-BN (PBS, 1% BSA, 0.05% NaN3) was used for no.45-SY-25. Pierce^TM Protein-Free (PBS) Blocking Buffer (Pierce, #37584) for no.72-ST1 was used. To prepare PBS-TBN and PBS-BN, BSA (Sigma, #B-4287), Tween 20 [Helicon, #Am-O777], and NaN3 [Helicon, #Am-O639] were used.

Immunological analysis was carried out according to the manufacturer’s instructions (Luminex, 2016). Briefly, in a 96-well polistirol flat-bottom plate, [Greiner, #GR-655001] 50 μl PBS-TBN with 2500 microspheres of each region and 50 μl of one of four spiked matrices were added. Shaking-incubation conditions were as follows: 1 h, +37°C and 800 rpm. Further washing included two cycles of adding and mixing on the shaker (30 s 800 rpm) and removal of 100 μl of PBS-TBN in each well. The same washing conditions were applied for all washing steps. Spheres were resuspended in 50 μl of PBS-TBN. Detecting antibodies at a concentration of 8 μg/ml in 50 μl PBS-TBN were used. The mixture was incubated on a shaker for 1 h (37°C and 800 rpm) and then washed. Before the third incubation, 50 μl of PBS-TBN for bead resuspension was added followed by 50 μl of SAPE solution (Thermo, #S866) at a concentration of 8 μg/ml in PBS-TBN. The third incubation was performed for 30 min at +25°C and 800 rpm, followed by the final washing step and resuspension in 100 μl of PBS-TBN. The sample was analyzed in the MagPix analyzer (Luminex, United States). It was considered sufficient to have at least 100 or more microspheres of each region per well.

For the disintegration of two multi-spiked matrices PBS-DUST and H₂O-DUST with all four PBAs (IAV, AdV6, STm and CTB in concentrations of 1000 ng/ml, 500 ng/ml, 2.5^∗10⁵ CFU/ml and 32 ng/ml, respectively) in the device for laboratory ultrasound studies of the Volna-L [630 W, 22 ± 1.65 kHz] series, Model UZTA-0,63/22-OL manufactured by the Center of Ultrasonic Technologies LLC (Biysk, Russia) was used. The procedure was carried out using a cup horn for the indirect sonication mode. According to the manufacturer, the intensity of ultrasonic exposure for this method of use is from 3 to 10 W/cm², which is regulated by the output power controller from 30 to 100%, respectively, with linear dependence.

Three samples of each matrix in a volume of 4 ml each, in triplicates, at the level of 30, 65, and 100% power for 15 min were sonicated. At time intervals of 5 s, 30 s, 1, 2, 5, 10, and 15 min, 70 μl of sample was removed from the sonicator. For comparison, samples for each run similar in composition but not exposed to ultrasound were analyzed.

The results of the study were processed with parametric statistical methods using the statistical program GraphPad Prism 6 (GraphPad Software, Inc., United States). The type of data representation and the methods of statistical processing are described in figure legend. LODs were determined as the lowest concentration tested with a signal greater than the mean background fluorescence plus three times the standard deviation. The fluorescent threshold (FT) line indicator depending on the matrix for each target was slightly different. In view of that heterogeneity, each mean value of the PBA of every matrix in mono- and multiplex was normalized relative to each other by subtracting the corresponding FT value. All measures were assayed in three replicates.

The immunological xMAP multiplex test system development underwent an extensive optimization process. The amount of capture antibodies (2, 5, 10, 20 μg per 1 million microspheres), time for the first two of the three incubations (30–60 min), incubation temperature (+25°C or +37°C), with or without shaking, concentration of detecting antibodies (4, 6, or 8 μg/ml), concentration of SAPE (4, 6, or 8 μg/ml) and the number of washes (2–4 cycles) were optimized for each particular target (Supplementary Figures S1, S2). Various blocking reagents (PBS-TBN, PBS-BN and Pierce^TM Protein-Free (PBS) Blocking Buffer) were used to reduce the non-specific background and improve specificity. SuperBlock^TM (PBS) (Thermo, #37580), Blocker^TM casein in PBS (Thermo, #37582) and gelatin from the skin of cold water fish (Sigma, #G7765) were also tested, but none of these were subsequently used because of the lack of blocking effect (Supplementary Figure S3).

For the influenza virus A NP, an optimization of the lysing reagent was also performed. N-lauroylsarcosine sodium salt (NLS) (Helicon, #Am-O719), ethylenediaminetetraacetic acid (EDTA) (Helicon, #Am-O105B), sodium deoxycholate (Sigma, #D6750) and Igepal CA-630 (Sigma, #I3021) were tested. Earlier, the lysis capacity of NLS was tested for two concentrations of NLS (0.1 and 0.5%) (Leirs et al., 2016). All four detergents in concentration 0.1% were tested in this study (Supplementary Figure S4). The best results were shown for 0.1% Igepal CA-630, both for prior lysis and for lysis combined with Ab-coupled microspheres incubation. There was no significant effect of 0.1% Igepal CA-630 on the results of the other PBA multiplex detection. Therefore, 0.1% Igepal CA-630 was used for the initial incubation buffer conditions.

To evaluate the effect of dust in monoplex vs. multiplex regimens, MFI values in PBS and water in the presence of dust were combined on separate plots. Compared to monoplex conditions, raw MFI LOD was 1.5–3 times higher (Supplementary Figure S6) but after subtraction of FT, this trend was no longer seen (Figure 3).

Multiplexing of the influenza A virus did not change the MFI in PBS with dust and significantly worsened the values in water with dust at a higher concentration range (Figure 3A). In the multiplex conditions for AdVH and STm LPS, the sensitivity limit was similar to the monoplex for all the matrices (Figures 3B,C). In the case of CTB detection, PBS-DUST was also the preferred matrix for both sensitivity and MFI levels.

Therefore, multiplexing significantly improved the values for all targets in PBS with the presence of dust compared to monoplex. It is possible to suggest that a non-specific effect of dust in the multiplex mixture is distributed between the four populations of microspheres thus decreasing the effect of dust to individual bead targets.

To evaluate the analytical properties of aiRDeTeX 1.0, cross-reactivity (Table 1) and within-run precision (Table 2) were tested for all targets. For cross-reactivity of aiRDeTeX 1.0, the MFI values for all targets were assessed in all individual monoplex assays in triplicate (Table 1). Although it would be better to study cross-reactivity over the whole range of concentrations, in present research only highest concentrations of each target had been used, according to some previous studies (Tamborrini et al., 2010; Silbereisen et al., 2015). The value of MFI for every target is significantly higher only in the respective immunoassay. This finding indicates that aiRDeTeX 1.0 specifically detects different targets and does not show cross-reactivity.

To evaluate the precision of aiRDeTeX1.0, the coefficient of variation (CV) was calculated for the whole range of concentrations. CV was calculated as the SD divided by the mean of three repetitions in frame of one plate and multiplied to 100%. The within-run precision of aiRDeTeX1.0 was not higher than 10% (Table 2).

Ultrasound is one of the few sample preparation approaches that could be used before immunological assay in automatic (flow through) bioaerosol surveillance systems as a method for cell lysis and inactivation effect on PBAs (Joyce et al., 2011; Wu et al., 2012). This method seems to be particularly useful for hidden targets (the internal viral NP IAV) or bacterial antigen release (LPS). To assess the effect of ultrasound conditions on multiplex detection, different intensiveness (30, 65, 100%) and times (5″, 30″, 1′, 2′, 5′, 10′, 15′) were tested (Figure 4). The results showed that for three out of four targets, ultrasound treatment decreased the MFI (Figures 4A–D,G,H). Statistical analysis between different points after sonication was carried out by the method of multiple comparisons using the Tukey criterion (Supplementary Figures S7–S10). Only bacterial LPS antigen shows significant MFI improvement upon treatment for more than 2 min (Figures 4E,F). Lower US treatment times decreased the MFI both for PBS and water. According to these data, US could improve bacterial immunodetection performance but intensiveness and treatment times should be carefully assessed for all individual targets. For protein (toxin) or viral detection, the use of US is questionable even for internal targets.