SUPPLEMENTARY MATERIALS AND METHODS
Production of Recombinant PSP/reg and ELISA
Reagents to detect PSP/reg (reg1

) were developed as described in previous reports(1,
2). Briefly, PCR-amplified cDNA encoding PSP/reg was cloned into the pCR2.1 vector
(Invitrogen) by using sequence-specific primers (forward primers: 5’GAAAAGACAAGAG
GCCCAGACAGAGTT 3’, 5’ GTATCTCTCGAGAAAAGACAAGAGGCCCAGA 3’
(elongation); reverse primer: 5’ CTAGTTTTTGAACTTGCATAC 3’). After confirmation by sequencing, the cDNA was cleaved and ligated into the vector pPIC9 (Invitrogen) to regenerate the

-factor signal peptide in frame with the sequence encoding the mature protein.
The inclusion of a KEX2 cleavage site between the signal peptide and the mature peptide ensured correct processing during synthesis and secretion. The Pichia pastoris strain KM71 was transformed, and the most productive clone was selected for expansion and production of recombinant protein. The isolated peptide was N-terminally sequenced to verify correct processing.
Isoform-specific ELISAs were designed using the sandwich technique as described previously (1). Guinea pig anti-PSP/reg antibodies (affinity-purified IgG) were diluted in
Tris-buffered saline (TBS) and coated onto Maxisorp plates (FIRMA) at 4 °C overnight. The plates were then blocked with 1% bovine serum albumin (BSA) in TBS for 1 h at room temperature. Samples were pre-diluted in TBS/BSA and loaded in duplicate wells. The standard curve was generated from serial dilutions of recombinant PSP/reg protein. A second antibody, rabbit anti-PSP/reg, was then incubated and detected by phosphatase-conjugated anti-rabbit IgG. To test the correlation between dilution and concentration, we serially diluted human pancreatic juice and human pancreatic tissue. Recovery rates of recombinant PSP/reg diluted into serum from a healthy volunteer were as follows: 71% at a 1:10 dilution, 118% at
1:20, and 95% at a 1:40 dilution. Intra-plate and inter-plate variances were <5% and <10%,

respectively, for concentrations within the range of the standard (0.1

3.5 ng/ml). The limit of detection was <0.1 ng/ml.
SUPPLEMENTARY REFERENCES
1. Schiesser M, Bimmler D, Frick TW, et al. Conformational changes of pancreatitisassociated protein (PAP) activated by trypsin lead to insoluble protein aggregates. Pancreas
2001;22:186-192.
2. Graf R, Schiesser M, Lussi A, et al. Coordinate regulation of secretory stress proteins
(PSP/reg, PAP I, PAP II, and PAP III) in the rat exocrine pancreas during experimental acute pancreatitis. J Surg Res 2002;105:136-144.

SUPPLEMENTARY FIGURE LEGENDS
Figure 1. Recombinant pancreatic stone protein after purification from Pichia pastoris medium. A) SDS-PAGE analysis of recombinant PSP/reg. On the basis of BSA standards and quantitative amino acid analysis, 0.8 and 2
 g protein were loaded. B) Western blot analysis of PSP/reg after the generation of antibodies against the recombinant protein. Recombinant
PSP/reg (P) and a tryptic digest of PSP/reg (P+T) demonstrated that the recombinant protein had a selective cleavage site typical for this protein family, which produces a secretory product found in pure pancreatic juice (PPJ). Arrowhead indicates front of gel.
Figure 2. Kinetics of serum amylase and lipase levels in polytrauma patients.
A) Serum pancreatic amylase levels from polytrauma patients without infection (white bars, n = 18), with local infection (grey bars, n = 32), or with sepsis (dark grey bars, n = 33) were measured by an enzymatic assay from the day of admission until day 21. B) Serum lipase levels from the same polytrauma patients were measured by enzymatic assay.
Figure 3. Kinetics of leukocyte numbers in sera of polytrauma patients.
Figure 4. Flow cytometric analysis of CD11b binding to leukocytes from whole blood. Whole blood was stained with PE-labeled CD11b antibody and analyzed by FACS. Depicted are the forward scatter (FSC)/side scatter (SSC) plot with the specific gates for neutrophils (G1), monocytes (G2), and lymphocytes (G3) and the histogram analysis of these gates with the fluorescence for non-specific binding (black line) and specific binding (grey line).
Figure 5.
Expression of CD62L and CD11b on the PMN surface. Whole blood was incubated with medium or with PSP/reg at various doses of 0, 10, 50, 100, 500 &1000 ng/mL for 1 h, and expression of CD62L (left) and CD11b (right) on PMN was analyzed by FACS after staining

with PE-labeled antibodies. In addition, boiled PSP/reg (500 ng/ml) was tested (grey columns). n= 6, *p-value < 0.05 and **p-value < 0.01 for the comparison of PSP/reg with medium.
SUPPLEMENTARY TABLE
Table 1. Sensitivity, specificity, and positive and negative predictive values for three cut-off points of serum PSP/reg levels in patients with sepsis compared with patients without infectious complications on days 3 and 5.
Day 3
Sensitivity (%)
Specificity (%)
Positive predictive value (%)
Negative predictive value (%)
Day 5
Sensitivity (%)
Specificity (%)
Positive predictive value (%)
Negative predictive value (%)
30 ng/mL
70.4
72.2
65.5
76.5
30 ng/mL
74.1
66.7
62.5
77.4
60 ng/mL
55.5
83.3
71.4
71.4
60 ng/mL
63.0
75.0
65.4
73.0
80 ng/mL
40.7
83.3
64.7
65.2
80 ng/mL
51.9
77.7
63.6
68.3