Quantikine human bdnf immunoassay

The serum and EDTA-plasma samples of group one were analyzed at five different time points (Figure 1). The sample was taken out of the −80 °C freezer and 120 min were allowed for thawing at room temperature (21 °C) before analysis (“Baseline”; T1). After analysis, the same aliquot was stored for two hours at room temperature and then analyzed again (“2 hrs storage at room temperature”; T2) to test the short term stability of BDNF at room temperature. Subsequently, this aliquot was deep frozen, thawed again the next day, and analyzed to test the stability of BDNF after a repeated freeze/thaw cycle (“After 2nd freeze/thaw cycle”; T3). Then, this aliquot was again deep frozen and stored at −80 °C for two months before the next analysis (“2 months storage at −80 °C”; T4). After that, this aliquot was again deep frozen and stored at −80 °C for another four months before the last analysis (“6 months storage at −80 °C”; T5). BDNF was measured using the Quantikine human BDNF Immunoassay from R&D Systems, Inc., Minneapolis, MN, USA following manufacturer’s instructions [20 ].

For BDNF analysis, 4.5 mL of venous blood from the antecubital vein was collected with a clotted blood tube at three time points. The first blood sample was taken at the beginning of the experimental session. The second blood sample was collected immediately after the learning phase and the third one was taken immediately after the intervention (exercising or relaxing).
All samples clotted within 30 minutes at a temperature of 21°C. After the clotting period, samples were centrifuged for 10 minutes with 4800 rounds per minute using the Heraeus Labofuge 200 (Thermo Fisher Scientific, Germany). Serum was immediately pipetted into separate SafeSeal microtubes (Sarstedt, Nürnberg, Germany). Samples were stored at −23°C for a mean of 13.5 days and then transferred to a −80°C freezer until the analysis started. BDNF levels in serum were measured using the Quantikine® Human BDNF Immunoassay from R&D Systems (Wiesbaden, Germany) with intra- and interassay coefficients of variation in the range between 8.8 and 11.4 for values between 7.24 and 41.6 ng/mL. The minimum detectable BDNF dose was less than 20 pg/mL, according to the manufacturer's information. BDNF analysis was performed by the Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics in Leipzig.

In each of the two blood samples, 4.5 ml of venous blood from the antecubital vein were collected with a clotted blood tube. Collection and analysis of blood samples was performed according to the following uniform protocol: all samples clotted within 30 minutes at a temperature of 21C. After the clotting period, samples were centrifuged for 10 minutes with 4800 rounds per minute using the Heraeus Labofuge 200 (Thermo Fisher Scientific, Germany). Immediately afterwards, serum was pipetted into separate SafeSeal micro tubes (Sarstedt, Nürnberg, Germany). Samples were stored at -30°C for one night and then transferred to a -80°C freezer for another three weeks. Then, BDNF levels in serum were measured using the Quantikine® Human BDNF Immunoassay from R&D Systems (Wiesbaden, Germany) with intra- and inter-assay precision of 6.2% and 8.1%, respectively.
The minimum detectable BDNF dose was less than 20 pg/mL, according to the manufacturer’s information. All samples were tested twice for reliability.