Human IgG antibody profiles differentiate between symptomatic
patients with and without colorectal cancer
Manuscript: GUT_2009_178574
Supplementary Methods
Supplementary Method 1
Serum screening
To identify IgG antibodies in serum of patients, the hEx1 protein arrays were prepared
as described.15 16 Briefly, dry protein arrays were soaked in ethanol for 5 min, washed
in dH2O and incubated in TBSTT buffer (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 0.1%
(v/v) Tween 20, 0.5% (v/v) TritonX-100). E.coli colonies were removed from the
membranes by wiping with tissue paper soaked with TBSTT. The arrays were washed
2 x 10 min in TBST (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 0.1% (v/v) Tween 20), 2
x 5 min in TBS (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl) and 2 x 10 min in TBST. The
arrays were then incubated for 3 hours with blocking solution containing 3% (w/v)
non-fat, dry milk powder (Marvel) in TBST. Subsequently, serum was diluted 1:100
in 20 ml TBST containing 2% (w/v) bovine serum albumin (BSA) and incubated with
the protein arrays for 16 hours under continuous slow agitation. The arrays were then
washed 3 x 30 min in TBST and incubated with the primary antibody (mouse antihuman IgG, GG-7, Sigma, 1:5000 in TBST, 2% (w/v) BSA) for 2 hours. Followed by
washing with TBST for 3 x 30 min the arrays were incubated with an alkaline
phosphatase (AP) -conjugated secondary antibody (goat anti-mouse IgG-AP, A1418,
Sigma, 1:5000 in TBST, 2% (w/v) BSA). After the final washing in TBST for 2 x 30
min and in TBS for 1 x 20 min, for 10 min in TBS, the arrays were incubated for 10
min in AP buffer (1 mM MgCl2, 0.1 M Tris, pH 9.5), followed by 5 min incubation in
25 mM Attophos (JBL Scientific, San Luis Obisco, CA) in AP buffer. Images were
captured with a Fuji LAS 3000 imager.
Supplementary Method 2
Normalisation of mRNA expression levels
The comparative CT method was used to quantitate gene expression. The CT
(threshold cycle) is the cycle number at which the fluorescence generated following
probe cleavage passes a fixed threshold above the baseline. In this method of
quantitation, the difference in CT (ΔCT) between the target gene and the endogenous
standard (β-actin) is used to determine gene expression with the formula 2ΔCT. 2ΔCT is
derived from Xn=X0 (1+Ex)n, the equation which describes the exponential phase of
amplification by PCR, where X0 is the initial number of target molecules, Ex the
efficiency of amplification, and n the number of cycles. At 100% efficiency, the
equation becomes X0(2)n. Reaction efficiencies for the target gene and the
endogenous standard for this method of quantitation should be similar and near 100%.
A normalized value of expression for the target gene with reference to β-actin is then
obtained by the calculation 1/2ΔCT. All mRNA expression values are ratios relative to
β-actin and expressed as x 10-3.