SUPPORTING INFORMATION
Feasibility Studies: To demonstrate the feasibility of the MED technique, we first determined if
the epitope bound antibody can be effectively eluted from its epitope. As shown in Fig. S1A,
HeLa cells were stained with antibodies against NFB or STAT3. The antibodies were detected
by immunofluorescence either with or without an elution step between the primary and
secondary antibody incubation steps. The HeLa cells with an elution step after primary antibody
incubation (“Eluted” in Fig. S1A) show a marked decrease in immunofluorescence signal
compared to the control without an elution step. Since the reactivity of the primary antibody
towards secondary antibody survives after acid-based elution (Fig. S1B), the loss of
immunofluorescence is mainly due to the decrease of bound antibodies of NFB and STAT3 via
elution. Fig. S1B delineates the reactivity of renatured “partially-denatured” antibodies (Treated)
towards their cognate epitopes in comparison to that of undenatured antibodies (Control). As
shown in Fig. S1B, all 8 antibodies retain >70% signal of the “Control” after the denaturing and
renaturing process. To demonstrate the reliability of the MED technique, we examined the
correlation between the MED technique and Western blotting. HeLa cells were treated for
varying amounts of time with either 20 ng/ml TNF(R&D Systems) or 100 ng/ml Interferon 2a
(IFN) (Biovision) to induce phosphorylation of IB or STAT3, respectively. The amount of p-IB
and p-STAT3 in each condition was detected either by Western blotting or the MED method (Fig.
S1C & S1D). In Fig. S1C, both the MED method and Western blotting detected a dramatic
increase of p-IB at 5 min TNF induction interval, further increase of p-IB at 10 min interval
and significant decrease of p-IB at 15 min interval. Likewise, the MED method detected the
same induction pattern of p-STAT3 by IFNas detected by Western blotting (Fig. S1D). These
results indicate the MED technique can be used to detect target epitopes of cellular proteins.
As shown in Table S3, most of MED assays’ intra-assay CV and inter-assay CV are within 10%.
For intra-assay CV, the exceptions are NFkB of Assay #3 (12.97%), STAT3 of Assay #2
(24.05%) and Assay #3 (26.81%). We believe the 12.97% intra-assay CV of NFKB is a
reflection of variation of the Luminex assay which produced 15.28% inter-assay variation for
control 1. The relatively higher CV of STAT3 may be due to the low binding affinity of STAT3
antibody that may not have reached equilibrium at the time of elution. For inter-assay CV, the
exception is p-NFkB which is mainly caused by the 50% drop in their signal of Assay #3 in
comparison to Assay #1 and Assay #2. We believe this drop in the signal is most likely due to
degradation of the phosphorylated epitope p-NFkB on the 24-well culture plate 3 days postfixation. To minimize biological variation, all HeLa cells on three 24-well plates were plated at
the same time and fixed the next day at the same time, as well. One plate was used for the
MED assay for Assay #1 and another plate was used the next day for Assay #2 and the third
plate was used on the third day for Assay #3. Even when refrigerated, 3 days may prove to be
too long for keeping all p-NFkB epitopes intact.
Antibodies & Proteins: Rabbit monoclonal antibodies specific for NFB, IB, IB(pS32) (pIB, STAT3, STAT3(pY705) (p-STAT3), p38, S6 Ribosomal Protein(pS235/236) (p-S6) were
provided by Epitomics. Rabbit monoclonal antibodies specific for NFB(pS536) (p-NFB) and
p38(pT180/pY182) (p-p38) were from Cell Signaling Technology. Other antibodies and their
sources were: TfR antibody from Abbiotec; EGFR antibody from Lifespan Biosciences; ErbB2
antibody from Santa Cruz Biotechnology; PDGFR antibody from R&D Systems; CF488A goat
anti-rabbit antibody for immunofluorescence from Biotium, Inc. Recombinant human chimera
proteins (receptor extracellular domain and Fc portion of IgG) for EGFR, ErbB2, PDGFR and
TfR were from R&D Systems.
Peptide & Protein Coupling: Immunogen peptides for NFB, p-NFB, IB, p-IB, STAT3, pSTAT3, p38, p-p38 and p-S6 were provided by Epitomics. Each peptide carried a cysteine at
one terminus for coupling to the Luminex beads via a maleimide linker. The coupling was
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performed by a modified two-step method as described by Kurzeder et al. (18). Briefly, 5X106
carboxylated polystyrene beads (Luminex Corp.) were coated with 50 g of BSA (Sigma-Aldrich)
in a volume of 500 µl according to the manufacturer’s protocol. Excess BSA was removed by
centrifuging followed by washing 3 times with 750 µl of PBS + 5 mM EDTA with vortexing and
sonicating for 30 s each between each wash. The beads were re-suspended in 400 µL of
freshly prepared 0.88 mg/ml sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (Thermo Fisher)
in PBS + 5% DMSO. The beads were incubated at room temperature in the dark on a rotator for
1 h. The beads were washed 3 times with 750 µl PBS+5 mM EDTA as before and were resuspended in 250 µl of PBS + 5 mM EDTA. To each bead set, 20 µl of freshly prepared 10
mg/ml peptide dissolved in DMSO was added. The beads were incubated at room temperature
in the dark on a rotator for 2 h. Excess peptide was removed by centrifugation as before and
washed 3 times with 750 µl PBS, 0.1% BSA, 0.02% Tween-20, 0.05% azide. Finally the
microspheres were re-suspended in 375 µl of PBS with 0.1% BSA, 0.02% Tween-20, 0.05%
azide and stored in the dark at 2-8oC.
Coupling recombinant chimera receptor proteins to Luminex beads was performed according to
manufacturer’s instruction. The protein to bead ratio is 12.5 g chimera protein to 5X106 beads.
MED Assay of Cellular Proteins in Fixed Cells: HeLa cells were plated at a density of 1x105
cells per well in a 24-well plate. After 24 h, the cells were serum-starved for 16-20 h and then
treated with or without the indicated agent. The cells were then fixed in 9% formalin in PBS for
15 min at room temperature followed by two rinses with PBS + 0.1% Tween 20 (PBST). The
cells were then incubated with a blocking solution of 10% calf serum in PBS + 0.1% Triton X100
for 1 h at room temperature. After blocking, the cells were rinsed two more times with PBST
prior to the addition of 200 l per well of antibody cocktail with selected primary antibodies made
in 10% calf serum in PBS. Primary antibody incubations were performed for 3 h at room
temperature. The cells were then washed three times for 5 min each with PBST. Bound
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antibodies were eluted using 200 l Elution Buffer II (LEAP Biosciences) per well for 15 min at
room temperature. The eluted antibodies were then neutralized and stabilized with 58 l of
Renaturing Solution II (LEAP Biosciences) prior to storage at -20°C or use directly in the
Luminex antibody assay for antibody detection and quantification.
MED Assay of Biomarkers in FFPE Tissue Sections: FFPE tissue sections were first rehydrated
using the same procedure as for immunohistochemistry (IHC) staining. Briefly, the FFPE tissue
section slides were baked at 60oC for 45 min followed by treatment of xylene for 5 min twice,
100% alcohol for 5 min twice, 95% ethanol for 3 min, 70% alcohol for 3 min and distilled water
for 3 min. The slides were then immersed in 94oC-98oC antigen retrieval solution (Dako) for 8
min before cooling down at room temperature for 20 min. The slides were subsequently washed
twice with distilled water and air dried. Once each rehydrated tissue section was dry, a
hydrophobic barrier pen (Vector Laboratories) was used to mark the edge of tissue section to
form hydrophobic barrier. One hundred twenty microliters of blocking solution (10% calf serum
in PBS) was applied onto each tissue section for 30 min. The blocking solution was then
removed by aspiration and 120l antibody cocktail was applied for 1 h incubation at room
temperature. The antibody cocktail contained 10% calf serum and 9 antibodies using vendor
recommended dilution for IHC staining. At the end of the incubation, each tissue section was
washed 4 times with PBST and then incubated with 120 l Elution Buffer II (LEAP Biosciences)
for 15 min for antibody elution. After the elution solution was recovered from each tissue
section, additional Elution Buffer II was added for rinsing the tissue section and adjusting the
final volume to 180 l. The eluted antibodies were then neutralized and stabilized with 52 l of
Renaturing Solution II (LEAP Biosciences) prior to storage at -20°C or use directly in the
Luminex antibody assay for antibody detection and quantification.
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Luminex Antibody Assay: The eluted renatured antibodies were detected and quantified
simultaneously by an xMAP assay with Luminex beads each conjugated with an immunogen
peptide or chimera protein bearing the epitope for each antibody. Briefly, 50 l of each antibody
containing sample was incubated with a corresponding mixture of beads for 2 h at room
temperature in a 96-well filter plate (Pall Corp.) on a shaker (600-800 rpm) protected from light.
After washing 3 times with 200 l Wash Buffer (PBS + 0.05% Tween 20 + 0.05% Kathon), 50 l
of Assay Buffer (PBS + 1% BSA + 0.05% Kathon) followed by 50 l of 1:200 PE conjugated
donkey anti-rabbit IgG (Abcam) or/and 1:125 PE conjugated goat anti-mouse (Invitrogen) in
Assay Buffer was added to each well and incubated for 1 h as before. After three washes, the
beads were resuspended in 100 l Assay Buffer and analyzed on the Luminex 200 (Luminex
Corp.).
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Fig. S1. MED method proof of principle. Elution of epitope bound antibodies (A). HeLa cells were
incubated with antibodies against NFB (left two images) or STAT3 (right two images) for 3 hours prior
to washing away excess antibody. The cells in panel A marked “Eluted” were subsequently treated with
antibody elution buffer before rinsing with PBS. Cells were then stained with a fluorescently-conjugated
secondary antibody. Eluted antibodies can be renatured and detected by their corresponding epitopes
(B). Antibodies against NFB, p-NFB, IB, p-IB, STAT3, p-STAT3, p38, p-p38 were treated with acidbased elution buffer followed by renaturing and then detected for their epitope-specific binding by the
Luminex antibody assay with their epitope-specific beads in parallel to their undenatured antibodies
(Control). The signal from “Control” sample was normalized to 100%. Correlation of MED method with
Western blotting (panel C & D). HeLa cells were treated for indicated amount of time with TNF(C) or
IFN(D) to induce phosphorylation of IB (C) or STAT3 (D) and either lysed for use in Western blotting
(bottom panel) or processed via the MED method (top panel).
Fig. S2. Standard curves of 9 biomarker-specific antibodies are plotted of antibody concentration vs MFI
both in logarithmic scale. The adjustment factor is the ratio of the epitope binding efficiency between
the native antibody and the renatured antibody obtained from Table S2. The concentration of recovered
antibody in Table 1 is derived first from the standard curve and then multiplied by the adjustment factor
considering the antibody used for the standard curve is the native antibody.
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