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Specific WNV-reactive equine serum samples were distributed in valley regions (prevalence 1.3%), savannah grasslands (2.4%), the western region of Zulia (0.4%) and the Central Lake Basin (0.3%).
Serum samples were distributed randomly in 96-well microtiter plates and the assay operators were blinded to case/control identity of all samples.
Blood was collected in a 10 cc serum separator vacutainer tube and centrifuged 30 min later at 3000 r.p.m. for 10 min. Serum samples were distributed into 0.5 ml aliquots and stored at −70°C until the experiment.
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There were fewer positive samples in the non-cancer group and these samples were distributed relatively evenly across all samples.
The samples were then centrifuged at 1900 g for 15 min at 20 ± 5 °C and the serum obtained from each sample was distributed to two fresh polypropylene test tubes in two aliquots of 1 ml each, one for rhNGF determination and the other for anti-NGF antibody detection by ELISA methods following the manufacturer's instructions.
All serum samples were run in duplicate and randomly distributed on the plates.
Serum samples were allocated to 96-well plates so that age, gender, study site (UK, Japan) and disease group (control, gastric cancer) were evenly distributed.
Serum samples were collected from 2003 through 2007.
Liver and serum samples were collected and stored at −80 °C.
Serum samples were albumin-depleted prior to electrophoresis.
Serum samples were heat inactivated for 60 min at 56 °C and serially diluted in serum-free DMEM.
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