VEGF levels were measured by ELISA: 0.4 μg/ml
anti-VEGF165 (R&D Systems, Abingdon, UK) in 0.05
M carbonate buffer, pH 9.6, was adsorbed onto 96-well plates (Dynatech
Laboratories, Sussex, UK) for at least 15 hours at 4°C (100μ
l/well). The plates were washed five times in 0.1 M PBS, pH 7.2,
supplemented with 0.05% Tween 20 (PBS-T) before and after blocking for
2 hours at room temperature (RT) with 5% dried powdered milk (Marvel
in PBS-T). Subsequently 100 μl of triplet rhVEGF standards (R&D
Systems) diluted in wash buffer [ranging from 10 pg/ml to 250 ng/ml
and blank (assay buffer)] or plasma was added to each well. After
incubation for 2 hours at RT and five washes as before, 100 μl
biotinylated goat anti-human VEGF (500 μg/ml in assay buffer; R&D
Systems) was then added to each well, and plates were left for a
further 2 hours at RT. After washing, 100 μl of Extravidin-peroxidase
(Sigma-Aldrich, Poole, Dorset) at 1:1000 dilution was added, and plates
incubated at RT for 45 minutes. The plates were washed five times, and
substrate [o-phenylenediamine dihydrochloride in 0.05 M
citrate buffer, pH 5, with hydrogen peroxide (Sigma-Aldrich)] was
added to allow color development. The reaction was stopped using 3 M
HCl (Sigma-Aldrich), and the absorbance was read immediately in an
ELISA reader at 492 nm. The assay has a minimum sensitivity of 15
pg/ml, with an intra-assay coefficient of variation of 4.9%
(n = 18) and an interassay coefficient of variation of
9.1% (n = 20) at 1.6 ng/ml.
Level of sFlt-1 able to bind immobilized VEGF were measured in a
modified ELISA: 0.4 μg/ml rabbit polyclonal anti-VEGF (R&D Systems)
in 0.05 M carbonate buffer, pH 9.6, was adsorbed onto 96-well plates
(Dynatech Laboratories) for at least 15 hours at 4°C (100 μl/well).
The plates were washed and blocked as previously described, saturated
with 100 μl/well of 250 ng/ml rhVEGF (R&D Systems), and left at RT
for 2 hours. Plates were washed, and 100 μl of triplet rhFlt-1/Fc
chimera (R&D Systems) standards diluted in wash buffer [ranging from
100 pg/ml to 500 ng/ml and blank (assay buffer)] or plasma was added
to each well. Plates were incubated for an additional 2 hours at RT and
then washed as before. One hundred microliters of biotinylated goat
anti-human Flt-1 (500 μg/ml in assay buffer; R&D Systems) was added
to each well, and plates were left for another 2 hours at RT. The assay
was completed with extravidin-peroxidase, substrate, and hydrochloric
acid and read at 492 nm as described above. This assay has a lower
limit of sensitivity of 50 pg/ml, an intra-assay coefficient of
variation of 3.7% (
n = 12), and an interassay
coefficient of variation of 8.8% (
n = 18) at 10 ng/ml.
Levels of vWf were determined using an established ELISA
13 with reagents from Dako (Ely, UK). HbA1c was measured in diabetic
patients using a standard high-pressure liquid chromatography method in
our routine clinical chemistry laboratory, which is based on ion
exchange chromatography principles.
14 Erythrocyte
hemolysates were passed through a MonoS ion exchange column (Pharmacia,
Uppsala, Sweden), where the charge is altered by passing an increasing
gradient of lithium chloride through the column, thus promoting the
elution of the hemoglobin fractions from the column. Fractions of HbA1c
are detected by measuring the absorption of light of the different
fractions at 415 nm using a Thermoquest Spectra system (Thermoquest,
Manchester, UK), and the concentration of each fraction is directional
proportional to the absorbance at 415 nm. The proportion of HbA1c is
calculated as the ratio of the area of the HbA1c peak to the sum of the
areas of the HbA1c and HbA peaks.