Survivin autoantibodies are not elevated in lung cancer when assayed controlling for specificity and smoking status Ingrid Broodman1, Martijn M. VanDuijn2, Christoph Stingl2, Lennard J.M. Dekker2, Anastasios E. Germenis3, Harry J. de Koning4, Rob J. van Klaveren5, Joachim G. Aerts5, Jan Lindemans1, Theo M. Luider2

Authors' Affiliations1 Department of Clinical Chemistry, Erasmus University Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands

2 Department of Neurology, Erasmus University Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands

3 Department of Immunology & Histocompatibility, School of Medicine, University of Thessaly, 41110 Larissa, Greece

4 Unit of Public Health, Erasmus University Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands

5 Department of Pulmonology, Erasmus University Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands

Supplementary Figures S1-S6

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Supplementary Figure 1. Standard curve of rabbit monoclonal antibody to recombinant survivin

using DAC-ELISA (A) and sandwich ELISA (B).

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Supplementary Figure 2. Antibody response to recombinant survivin was measured in seven NSCLC

sera previously reported to be positive for survivin antibodies (16) and in seven healthy non-smoking

control sera. Optical densities (ODs) were measured at a 1:100 dilution in DAC-ELISA that contained

the antigen, survivin (green), and that without antigen (grey). Specific binding (red) was obtained by

subtracting background ODs (grey) obtained in the absence of survivin. No significant difference was

found between ODs with antigen of the NSCLC patients (square) (median 0.209, IQR 0.154-0.427) and

those of the healthy non-smoking controls (rhombus) (median 0.241, IQR 0.209-0.325) (Mann-

Whitney U-test (MW), P = 0.620). In addition, no significant differences were found in specific binding

(MW, P = 0.535).

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Supplementary Figure 3. Standard curve of rabbit anti-survivin spiked in non-smoking control sera

using sandwich ELISA. Optical densities (ODs) of serial dilutions of rabbit anti-survivin in six healthy

non-smoking control sera (D1-D6) were identical to those spiked in diluent (St). No sample matrix

interference was found in the sandwich ELISA.

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Supplementary Figure 4. Standard curve of rabbit anti-survivin spiked in NSCLC patient sera using

sandwich ELISA. Optical densities (ODs) of serial dilutions of rabbit anti-survivin in ten NSCLC patient

sera (C1-C10) were identical to those spiked in diluent (St). No sample matrix interference was found

in the sandwich ELISA.

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Supplementary Figure 5. Standard curve of human autoantibodies to HuD using sandwich ELISA.

Sandwich ELISA with HuD antigen and serial dilutions from 1:10 to 1:640 (0.16-10% serum) of an anti-

HuD-positive serum from a patient with paraneoplastic neurological syndrome (PNS).

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Supplementary Figure 6. Results of sandwich ELISA were confirmed by western blot analysis.

Examples of sample negatives measured by sandwich ELISA to recombinant survivin with CaM-tag

(33 kDa; 50 ng/lane) analyzed by western blotting. Western blots (A and B) of blank control (5% BSA-

TBST, Bl, light blue); positive control (Rabbit monoclonal to survivin, C1-C4; 1:5,000, 1:25,000,

1:125,000, 1:625,000, purple); nine NSCLC patients previously found positive by DAC-ELISA (16) (P1-

P9, orange); five cases analyzed before diagnosis of NSCLC (b, dark blue box) and after diagnosis (a,

red box); two smoking-habit controls (c, green box); and two healthy non-smoking controls (d, yellow

box). Western blot analysis on recombinant survivin was negative for survivin antibodies in patient

sera.

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