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Supplementary materials
These supplementary materials describe the development of the IC6 competitive
ELISA assay.
Methods
Antigen generation
The antigen peptide was generated from the consensus collagen type VI alpha 1
sequence (NP_001839). It was directed against the N-terminal globular domain
and selected for minimization of homology to other human proteins and
optimization of immunogenicity. The resulting sequence was
ADWGQSRDAEEAISQ. Several sequences from different regions of the protein
was used, but only this passed quality control steps for ELISA assay validation.
Monoclonal antibody development
4–6 weeks-old Balb/C mice were immunized subcutaneously with about 200 l
emulsified antigen and 50 g of the IC6 epitope sequence (ADWGQSRDAEEAISQGGC-KLH). Consecutive immunizations were performed at 2-week intervals until
stable sera titer levels were reached in Freund’s incomplete adjuvant. Blood
samples were collected from the 2nd immunization. At each blood sampling, the
serum titer was determined and the mouse with highest antiserum titer was
selected for fusion. After the 4th immunization, this mouse was rested for 1
month and then boosted intravenously with 50 g CO6-MMP in 100 l 0.9%
sodium chloride solution three days before isolation of the spleen for cell fusion.
Fusion and antibody screening
The fusion procedure performed as described by Gefter et al 9. Briefly, mouse
spleen cells were fused with SP2/0 myeloma fusion partner cells. The hybridoma
cells were cloned using a semi-solid medium method and transferred into 96well microtiter plates for further growth and incubated in a CO2-incubater.
Standard limited dilution was used to promote monoclonal growth. Supernatants
were screened using an indirect ELISA with streptavidin-coated microtitre plates
and Biotin-ADWGQSRDAEEAISQ as a capture peptide.
Characterization of clones
Native reactivity and peptide binding of the monoclonal antibodies was
evaluated by displacement of native samples (human/rat/mouse serum, plasma
and urine) in a preliminary ELISA using biotinylated peptide coater on a
streptavidin-coated microtitre plate and the supernatant from the growing
monoclonal hybridoma. Specificity of the clones to a free peptide
(ADWGQSRDAEEAISQ) was tested. Isotyping of the monoclonal antibodies was
performed using the Clonotyping System-HRP kit, cat. no. 5300-05 (Southern
Biotech, Birmingham, AL, USA). The selected clones were purified using protein
G columns according to manufacturer’s instructions (GE Healthcare Life Science,
Little Chalfont, Buckinghamshire, UK). Selected monoclonal antibodies were
labeled with horseradish peroxidase (HRP) using the Lightning link HRP labeling
kit according to the instructions of the manufacturer (Innovabioscience,
Babraham, Cambridge, UK).
ELISA assay generation/optimization
The initial steps of assay generation concerned optimizing buffer type, coater
concentration, antibody concentration and incubation conditions. When these
conditions were set, testing of dilution recovery in our desired matrices were
determined as well as establishing sigmoid standard curve conditions. When
these were established, technical performance (intra/inter assay variability,
stress test, lower detection limit etc. were determined.
ELISA characterization
Sensitivity and specificity of the developed IC6 ELISA was evaluated measuring
nonsense peptides as coaters or as samples in the regular assay. A selection of
peptides from other Collagen Types were used for this purpose.
Standard curves
Standard curves were generated by serial dilution of the calibrator peptide in
each assay, pipetted on every plate and automatically fitted using a 4-parameter
fit (y=(A-D)/(1+(x/C^B)+D)) model in Softmax Pro version 5. These fits were
used to calculate biomarker levels through numerical curve regression.
Technical evaluation
From 2-fold dilutions of pooled serum and plasma samples, linearity was
calculated as a percentage of recovery of the 100% sample. The lower detection
limit (LDL) was calculated from 21 determinations of the lowest standard (the
zero standard) and calculated as the mean +3x standard deviation. The interand intra-assay variation was determined by 10 independent runs of 8 QC
samples, with each run consisting of two replicas of double determinations of the
samples. Finally, for each assay, a master calibrator prepared from synthetic
peptides accurately quantified by amino acid analysis was used for calibration
purposes. The analyte stability was determined for six serum samples (three rat
and three human) for 10 freeze and thaw cycles.
Results
Clone characterization
The antibody with the best native reactivity, affinity and stability in the assay
was chosen from the antibody-producing clones generated after the fusion
between spleen cells and myeloma cells. The clone selected was determined to
be the IgG1 subtype. The clone was reactive to human serum and plasma and rat
serum (Figure 1B).
IC6 ELISA protocol
The IC6 competitive ELISA procedure was as follows: A 96-well streptavidincoated ELISA plate from Roche, cat.11940279, was coated with the biotinylated
peptide Biotin-ADWGQSRDAEEIAISQ dissolved in coater buffer (25mM PBS-BTB,
pH 7.4) at 6 ng/ml in 100l, incubated for 30 min at 20°C in the dark and
subsequently washed in washing buffer (20 mM Tris, 50 mM NaCl, pH 7.2).
Thereafter 20 µl of peptide calibrator or sample were added to appropriate
wells, followed by 100 µl of HRP-conjugated monoclonal antibody NB300
dissolved in incubation buffer (25 mM PBS-BTB, pH 7.4) at 190 ng/ml and the
plate was incubated for 20 hours at 4°C and washed. Finally, 100 µl
tetramethylbenzinidine (TMB) (Kem-En-Tec cat.: 438OH) was added, the plate
was incubated for 15 min at 20°C in the dark and in order to stop the reaction,
100 µl of stopping solution (1% H2SO4) was added and the plate was analyzed in
the ELISA reader at 450 nm with 650 nm as the reference (Molecular Devices,
SpectraMax M, CA, USA). A calibration curve was plotted using a 4-parametric
mathematical fit model.
Technical evaluation
The typical standard curve is presented in Figure 1A, showing a 4-parametric fit
for the assay. The lower limit of detection (LDL) for the assay was 0.032 ng/mL.
Dilution recovery was within 100 +/-20% for dilution ranges 1:2-1:8 for rat
samples and through the full range (down to 1:16) for human samples (Supp.
Table 1 and Supp. Fig 1). The inter- and intra-assay variation was a mean 11.75
and 11.05% respectively (Supp. Table 2). The analyte stability was acceptable for
4 consecutive freeze/thaw cycles within 100+/- 20%.
Legends
Supplementary Figure 1 – Dilution recovery
Graph depicting signal of standard curve peptide and 2-fold dilution series of
human and rat samples. A strong inhibition from samples is shown as well as
dilution recovery.
Supplementary table 1 - Assay dilution recovery
Table showing dilution recovery of different matrices. Dilution recovery is
defined as the signal multiplied by the dilution degree, relative to the signal in
the reference (undiluted) sample. Maintaining a dilution recovery is a
prerequisite for being able to measure analytes across a wide range of
concentrations.
Supplementary table 2 – Intra- and inter-assay variability
Intra- and inter-assay variability measured using 10 repetitive measurements of
the same human control samples on the same plates or in individual plates,
respectively.
Supplementary table 1 - Assay dilution recovery
Dilution factor
Neat
2x
4x
8x
16x
Rat serum
(n=4)
Human
Serum (n=4)
EDTA-plasma
(n=2)
dRE%
dRE%
dRE%
100
77
90
103
178
100
90
89
103
119
100
81
80
82
92
Supplementary table 2 – Intra- and inter-assay variability
Sample
Conc
Intra-assay
(ng/ml)
variability
(%)
HS1
0.76
15.34
HS2
1.83
14.30
HS3
1.94
10.71
HS4
2.48
9.56
HS5
2.40
11.20
HS6
2.64
10.23
HS7
3.09
8.79
HS8
4.97
8.30
Mean
2.51
11.05
Citrateplasma
(n=2)
sRE%
Heparinplasma
(n=2)
100
84
81
87
95
Inter-assay
variability
(%)
14.80
10.15
8.68
8.86
11.18
12.33
13.11
14.92
11.75
100
88
78
86
99