IL-6 VALIDATIONS. Several suppliers reagents correlate well with R&D Systems HS ELISA, the goldstandard assay normally used in our laboratory.

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1 IL-6 VALIDATIONS Several suppliers reagents correlate well with R&D Systems HS ELISA, the goldstandard assay normally used in our laboratory. Assays are not always harmonized with respect to standardization. Slope(m) values reflect degree of harmonization.

2 Suppliers A: Bio-Rad B: Linco Research C: Upstate D: Biosource E: RnD Systems

3 SUPPLIER A Supplier A Multiplex IL-6 vs RnD HS IL-6 ELISA: Serum Set n=19 18 y =.15x +.13 RnD IL-6 pg/ml R =.6757 Mean replicate CV=6.6% R =.68 Slope(m)= Supplier A IL-6 pg/ml

4 Supplier B Supplier B Multiplex IL-6 vs RnD HS IL-6 ELISA: Serum Set n= 8 7 y =.596x RnD IL-6 pg/ml R =.686 Mean replicate CV=8.9% R =.69 Slope(m)= Supplier B IL-6 pg/ml

5 Supplier C Suppler C Multiplex vs RnD HS IL-6 ELISA: Serum Set n= y =.168x R =.39 RnD IL-6 pg/ml 3 Mean replicate CV=16.9% R =.33 Slope(m)= Supplier C IL-6 pg/ml

6 Supplier D Supplier D Multiplex IL-6 vs RnD HS IL-6 ELISA: Serum Set n= y =.3186x +.85 R =.9619 RnD IL-6 pg/ml Mean replicate CV=1.% R =.96 Slope(m)= Supplier D IL-6 pg/ml

7 Supplier E Suppler E FMAP IL-6 vs RnD HS IL-6 ELISA: Citrate Group I 18 RnD HS IL-6 ELISA pg/ml y = 1.13x R =.999 Mean replicate CV =.6% R =.93 Slope(m)= Supplier E FMAP IL-6 pg/ml

8 Supplier B The following 3 slides show run to run reproducibility for Adipokine Panel B IL-6, with assays performed using different kit lots: Run11 (9--) vs Run13 (9-9-) Run18 (1-9-) vs Run13 (9-9-) Run58 (5--5) vs Run13 (9-9-) The last slide shows the presence of outlier points caused by heterophilic antibody reaction in Supplier B s IL-6 assay. This problem emerged with kit lots we used between March 5 and May 5. If the problem is resolved we will update these validations accordingly.

9 Supplier B Supplier B Adipokine Panel B IL-6 Run11 vs Run13: 8/16/ serum set (n=) 8 7 y =.663x +.7 R =.8619 Reproducibility 6 Run11 IL-6 pg/ml 5 3 R = Run13 IL-6 pg/ml

10 Supplier B Supplier B Adipokine Panel B IL-6 Run18 vs Run13: 8/16/ serum set (n=) Reproducibility y =.758x +. R = Run18 IL-6 pg/ml R = Run13 IL-6 pg/ml

11 Supplier B 8. Supplier B Adipokine Panel B IL-6 Run58 vs Run13: 8/16/ serum set (n=) Reproducibility 7. Run13 IL-6 pg/ml Shows 3 outlier points caused by heterophilic antibody reaction in Supplier B s May 5 kit lot Run58 IL-6 pg/ml

12 Supplier B Supplier B Adipokine Panel B IL-6 Run58 vs Run13: 8/16/ Serum Set minus outliers Reproducibility 8. y =.86x R =.833 Run13 IL-6 pg/ml After removal of 3 outlier points, R =.83, indicating the assay is still working except for these random heterophylic antibody interractions. Run58 IL-6 pg/ml

13 Supplier B The following slides shows a recent (3/6) comparison of IL-6 values for supplier B s Adipokine panel B multiplex IL-6 assay with RnD System s Quantikine HS IL-6 ELISA: For 36 serum samples from post-menopausal women with two outliers removed, R =.55, slope =.8, indicating the assays shows poor correlation and lack harmonization with respect to standardization.

14 Supplier B Supplier B Adipokine Panel B IL-6 vs RnD IL-6 HS ELISA Supplier B Adipokine Panel B IL-6 multiplex assay vs RnD Systems Quantikine HS IL-6 ELISA: Serum samples, postmenopausal women Slope =.33 R =.6 RnD HS IL-6 ELISA pg/ml y =.33x R = Supplier B multiplex IL-6 pg/ml For a recent (3/6) comparison of Supplier B s Adipokine Panel B IL-6 assay vs RnD System s HS IL-6 ELISA using 36 serum samples from post-menopausal women, there is poor agreement between the assays, slope =.8 and R =.6. Note that very high values as seen in the two outliers here, should be considered spurious.

15 Supplier B Supplier B Adipokine Panel B IL-6 vs RnD IL-6 HS ELISA: Outliers removed Supplier B Adipokine Panel B IL-6 vs RnD Systems IL-6 HS ELISA: serum samples, post-menopausal women Slope =.8 R =.55 1 y =.8x R =.57 RnD IL-6 ELISA pg/ml Supplier B multiplex IL-6 pg/ml For a recent (3/6) comparison of Supplier B s Adipokine Panel B IL-6 assay vs RnD System s HS IL-6 ELISA using 36 serum samples from postmenopausal women, with two outliers removed, R =.55, showing that there is poor agreement between the assays and that there is a lack of harmonization with respect to standardization (slope =.8).

16 Supplier E The following slides show run to run reproducibility for FMAP IL-6, with assays performed by different lab technicians.

17 Supplier E 3.5 Supplier E FMAP IL-6/Tech 1(Run6) vs Tech (Run6): Serum Set (n=) y = 1.6x R =.99 Reproducibility 3 Run6 IL-6 pg/ml R =.93 Note that both runs used multiplexed IL-6 but were performed by different technicians Run6 IL-6 pg/ml

18 Supplier E 3.5 Supplier E FMAP IL-6: Multiplex/Tech1 vs Singleplex/Tech: Serum set (n=) y =.898x R =.856 Reproducibility Run66 Singleplexed IL-6 pg/ml Run6 Multiplexed IL-6 pg/ml R =.85 Note that not only do these data reflect multiplexed vs singleplexed IL-6 assays, but assays were performed by different technicians also.