Determination of reagent cross-reactivity. When adding a new, candidate protein to an

Transcription

1 This journal is The Royal Society of Chemistry 23 Supplementary Information Supplementary Text Determination of reagent cross-reactivity. When adding a new, candidate protein to an existing multiplexed digital ELISA, two experiments were performed to ensure that the new protein and detection reagents did not cross-react with the existing reagents and result in elevated false positive AEB signals for any of the proteins. In experiment # (see table below for design), a calibration curve ( to ) was generated for the new protein based on its specific capture and detection reagents, with and without: a) the addition of of the existing multiplexed proteins to each sample; and b) a mixture of all the biotinylated detection antibodies for the multiplexed proteins used at the detection antibody labeling step. In experiment #2 (see table below for design), and of each of the existing multiplexed proteins were spiked into a sample, and detected using the multiplexed digital ELISA, with and without: a) of the candidate protein in the sample; and b) the biotinylated detection antibody against the candidate protein at the detection antibody labeling step. If unanticipated increases in false positive signals were observed in either of these experiments then the new protein was not selected to be part of the multiplex. Examples of the data generated in Experiments # and #2 are shown in Supplementary Figure 2. Experiment #: Run compared to Run 2 Capture Analyte Detector Capture Analyte Detector New protein Existing protein # Existing protein #2 Existing protein #N

2 This journal is The Royal Society of Chemistry 23 Experiment #2: Run compared to Run 2 Capture Analyte Detector Capture Analyte Detector New protein Existing protein # Existing protein #2 Existing protein #N 2

3 This journal is The Royal Society of Chemistry 23 Supplementary Figures AEB.. Non-encoded beads AF-488 (low) beads AF-488 (high) beads HF-75 beads.. [PSA] () Supplementary Figure. Plots of AEB against concentration of PSA for digital ELISAs developed with non-encoded beads and beads labeled with fluorescent dyes to enable decoding. 3

4 This journal is The Royal Society of Chemistry 23 A AEB of IL- beads.. IL- reagents only IL- with multiplex reagents and of 3 other cytokines in multiplex. [IL- ] () B [IL- ] () AEB from IL-b beads IL-b reagents only AEB from IL-b beads IL-b reagents plus of 3 cytokines and their detection antibodies.36±.4.8± ± ± ± ± ± ± AEB plex 4 plex + Eotaxin 4 plex 4 plex + Eotaxin 4 plex 4 plex + Eotaxin 4 plex 4 plex + Eotaxin TNF a IL 6 IL b IL a Supplementary Figure 2. Examples of experiments to determine cross-reactivity in multiplexed digital ELISA. A) Example of Experiment # described above. IL- was being added to an existing 3-plex of TNF-, IL-6, and GM-CSF. IL- beads were run in conventional singleplex mode (red crosses), and also with each of TNF-, IL-6, and GM-CSF, and a mixture of the biotinylated detection antibodies for these 3 cytokines added to the assay (blue squares). The 3-fold increase in background signals for IL- beads was expected from the use of four-fold higher concentration of detection 4

5 This journal is The Royal Society of Chemistry 23 antibodies, but no further increase was observed from the presence of of 3 other antigens, so cross-reactivity was acceptable. B) Example of Experiment #2 described above. Eotaxin was being added to an existing 4-plex of TNF-, IL-6, IL-, and IL-. The 4-plex was run with all 4 cytokines at, with and without eotaxin and. g/ml of its biotinylated detection antibody to assess the effect on backgrounds. For each of the proteins, the backgrounds increased between fold upon addition of eotaxin, an increase not anticipated by the 2% increase in detection antibody concentration. We inferred significant cross-reactivity with eotaxin reagents giving rise to false positive signals, so eotaxin was not added to this multiplex assay. 5

6 This journal is The Royal Society of Chemistry 23 AEB of TNF- beads.. A. Only IL-6 spiked in AEB of IL-6 beads... [IL-6] (). [IL-6] () AEB of IL- beads.. AEB of IL- beads.. AEB of TNF- beads.... [IL-6] () [IL-6] () B. Only IL- spiked in AEB of IL-6 beads..... [IL- ] () [IL- ] () AEB of IL- beads.. AEB of IL- beads... [IL- ] (). [IL- ] () AEB of TNF- beads AEB of IL- beads... [IL- ] ()... [IL- ] () C. Only IL- spiked in AEB of IL-6 beads.. [IL- ] () Supplementary Figure 3. Plots of AEB against protein concentration for 4 beads specific to 4 cytokines measured in bovine serum samples spiked with: A) only IL-6; B) only IL- ; and C) only IL-. AEB of IL- beads..... [IL- ] () 6

7 This journal is The Royal Society of Chemistry 23 Supplementary Tables Supplementary Table. Effect of fluorescence of unmodified and encoded beads on the channel used to detect fluorescence (resorufin) from the reaction of single enzymes. Bead type Average fluorescence in resorufin detection channel (574 nm/65 nm ex/em) Unmodified, non-encoded beads 48±4 AF-488 fluorescent beads 39±9 cy5 fluorescent beads (low) 4±2 cy5 fluorescent beads (high) 48± HF-75 fluorescent beads 42±2 Supplementary Table 2. AEB values of 4 bead types in a 4-plex measured in samples spiked with IL-6 before and after software correction of crosstalk. Significant crosstalk was observed at IL-6 in all three non-il-6 bead types, and these false positive signals are greatly reduced by correction without affecting the IL-6 bead data. Beads measured [IL-6] IL-6 beads TNF- beads IL- beads IL- beads Before crosstalk correction After crosstalk correction AEB s.d. CV AEB s.d. CV.2. 8.% % % % % %.. 6.% 5.2% 5.7% 3. 6.% 2.2% 3.% % 6. 6.% 5..% 7.% 3.% 3.7%. 7

8 Supplementary Table 3. AEB as a function of concentration for calibration curves shown in Figure 5 and Supplementary Fig. 3. Experiment TNF- beads IL-6 beads IL- beads IL- beads [cytokine] AEB s.d. CV (%) [cytokine] AEB s.d. CV (%) [cytokine] AEB s.d. CV (%) [cytokine] AEB s.d. CV (%) TNF- only spiked in % 2 3% % % % % IL-6 only spiked in % % 23% 2% % % 7% 2% 3% 2% IL- only spiked in % 3 3% 7% % % % % % % 2% 7% % IL- only spiked in % 22% 2 % % 37% 2 7% % 2 2% % % % 3% All 4 cytokines spiked in % % % % 2% 3% % % 2% 3% % % 7% 2% 8 Electronic Supplementary Material (ESI) for Lab on a Chip This journal is The Royal Society of Chemistry 23

9 This journal is The Royal Society of Chemistry 23 Rissin, D. M.; Kan, C. W.; Campbell, T. G.; Howes, S. C.; Fournier, D. R.; Song, L.; Piech, T.; Patel, P. P.; Chang, L.; Rivnak, A. J.; Ferrell, E. P.; Randall, J. D.; Provuncher, G. K.; Walt, D. R.; Duffy, D. C. Nat. Biotechnol. 2, 28,