M e a n. F l u o r e s c e n c e I n t e n s i t y P r o t e i n ( p g / m L )

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2 P r o t e i n ( p g / m L ) P r o t e i n ( p g / m L ) Figure. Representative curves for one analyte from a multiplex panel showing the extended ranges of 0.6 to 0,000 pg/ml for mouse IL-3 (left) and.5 to 30,000 pg/ml for human TNF alpha (right).

3 f lu o r e s e n c e i n t e n s i t y f lu o r e s e n c e i n t e n s i t y 40% 20% 00% 80% 60% 40% 20% 0% 40% 20% 00% 80% 60% 40% 20% 0% Figure 2. Linearity of dilution in native samples (A) and spike-recovery (B) studies for stnf RI. Mean recovery and standard error are plotted. A: Each sample was tested at a :4 dilution and at six further dilutions for serum and plasma and three for other sample types. A two-fold dilution series was used. Analyte concentrations were interpolated from the standard curve. Percentage recovery is relative to the :4 dilution. B: Three different concentrations of protein standard were spiked into three different sample dilutions. Analyte concentration was interpolated from the standard curve. Percentage recovery was calculated after subtraction of the no-spike control value P l a s m a ( H e p a r i n ) P l a s m a ( E D T A ) P l a s m a ( c i t r a t e ) 0 C u l t u r e s u p e r n a t a n t S e r u m S t a n d a r d c u r v e p g / m L B A L 0 0 S y n o v i a l f l u i d C S F M i l k ( d e f a t t e d ) 0 S a l i v a S t a n d a r d c u r v e p g / m L p g / m l S e r u m P l a s m a ( c i t r a t e ) P l a s m a ( H e p a r i n ) P l a s m a ( E D T A ) C u l t u r e s u p e r n a t a n t S t a n d a r d c u r v e Figure 3. Confirmation of parallelism using the same data as figure 2 for native linearity of dilution (A and B) and spike-recovery (C). A: For each sample, analyte concentration in the :4 dilution was calculated by interpolation against the standard curve. The concentration of each subsequent dilution was then calculated and plotted, based on the dilution factor, from the concentration of the :4 dilution. B: The spiked concentration after non-spike control subtraction is plotted. The range of concentrations tested by spikerecovery was limited by the level of native protein.

4 Figure 4. Example of combinatorial testing using unique detector antibody / protein standard pools. Over the 88 analytes and 3.8x0 22 possible custom panels tested, only one combination (Eotaxin and IP-0) was identified as incompatible. The two tallest bars represent two pools tested against the IP-0 capture particle; both contain Eotaxin assay components, but have no other analyte in common.

5 Protein (pg/ml) I P - 0 s i n g l e p l e x C D 3 s i n g l e p l e x I P - 0 m u l t i p l e x C D 3 m u l t i p l e x M o u s e I P - 0 ( p g / m L ) M o u s e C D 3 ( p g / m L ) P F 4 s i n g l e p l e x L e p t i n s i n g l e p l e x P F 4 m u l t i p l e x L e p t i n m u l t i p l e x M o u s e P F 4 ( p g / m L ) M o u s e L e p t i n ( p g / m L ) Figure 5. Representative examples of standard curve alignment testing in singleplex and in multiplex. Leptin and PF-4 were tested in an 8 analyte multiplex panel; CD3 and IP-0 in a 5 and 0 analyte panel respectively. Testing of mouse serum samples in the same experiments resulted in an average CV of 3% between singleplex and multiplex assays RA SLE AD TNFα IL-2 IL-4 IL-5 IL-6 IL-0 GM-CSF Figure 6. Seven cytokines were quantified in normal serum and serum from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and atopic dermatitis (AD). Each dot represents an individual patient. Data was consistent with relevant publications 3-5.

6 SimpleStep ELISA (pg/ml) CV% Figure 7. Inter-assay CV% values. To determine intra-assay CVs, a single biological sample is tested at three sample dilutions with four replicates of each dilution. This experiment is performed three times on different days to determine inter-assay CVs. 4,000 R² = ,000 2,000, ,000,500 2,000 2,500 3,000 3,500 4,000 FirePlex immunoassay (pg/ml) Figure 8. SimpleStep ELISA kits and a FirePlex immunoassay was used to determine human BCA, IL-7A, GM-CSF, G-CSF, TARC, IL-0 and RANTES concentrations in supernatant from a PBMC cell culture which had been stimulated with.5% PHA-M for 24 hours.

7 Firefly assay (pg/ml) pg/ml Firefly immunoassay Luminex assay Figure 9. Eleven human cytokines (IFN-gamma, IL-4, IL- beta, MCP, TNF-alpha, IL-0, IL-5, IL-7A, IL-2, IL-6, IL-2p70) in stimulated PBMC cell culture supernatants were analyzed with both Luminex 30,000 25,000 R² = ,000 5,000 0,000 5, ,000 0,000 5,000 20,000 25,000 30,000 35,000 40,000 Bead-based (pg/ml) assays (Millipore catalog# HCytoMAG-60K, tested by Boston University Analytical Instrumentation Core) and a FirePlex immunoassay panel.

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