Measuring BDNF levels in mammalian brain tissues using sandwich ELISA with mAb #1 and mAb #9

1. Materials Monoclonal antibodies: mAb BDNF-#1 and mAb BDNF-#9 are available at Developmental Studies Hybridoma Bank (DSHB), University of Iowa (http://dshb.biology.uiowa.edu/) Plates: F96 MicroWell Plates, MaxiSorp White Polystyrene (Nunc, Cat. no. 436110) Coating buffer: 50 mM NaHCO3, 50 mM Na2CO3, pH 9.7 Incubation buffer: 0.1 M Phosphate buffer (0.1 M KH2PO4 + 0.1 M Na2HPO4), pH 7.6 Extraction buffer: 50 mM Sodium acetate, 1 M NaCl, 0.1% Triton X100, add Acetic acid until pH 4.0 is reached Before use add one Complete or Complete Mini protease inhibitors cocktail tablet (Roche, Cat. no. 11697498001 or 11836153001), to be used as recommended by the manufacturer. Blocking buffer: 4% BSA in PBS Washing buffer: 0.1% Tween-20 in PBS Substrate: BM Chemiluminescence ELISA Substrate (POD) (Roche, Cat. no. 11582950001) Purified recombinant BDNF Microplate washer: ELx50 (BioTek) Luminometer: Wallac Victor2 1420 Multilabel Counter (Perkin Elmer) 2. Experimental procedure: BDNF extraction: Dissect brain structures, weigh tissue fragments, then freeze them rapidly in liquid nitrogen. For long term storage transfer the frozen tissue samples to -80 C.

Re-suspend brain tissues in approx. 10 vol/weight extraction buffer (for example, 100 L extraction buffer for 10 mg tissue) and sonicate them to homogeneity on ice with a probe sonicator (e.g. Misonix Ultrasonic Liquid Processors). Sonicate in short bursts (5-7 sec) to avoid excessive sample heating. Keep homogenates on ice for 30 min, then repeat sonication as well as incubation on ice. Centrifuge homogenates for 30 min at 100000xg and 4 C, then transfer clear supernatants into clean tubes and discard pellets. Measure total protein concentration (e.g BCA Protein Assay Kit, Pierce Cat. no. 23227). These supernatants may be stored at -80 C and must be centrifuged again for 30 min at 20000xg and 4 C immediately after thawing and before being loaded into the wells of an ELISA plate. ELISA plate preparation: Coat wells of an ELISA plate with mAb #1 diluted in coating buffer (2 g/mL, 200 L/well). Incubate overnight at room temperature without shaking. Close plate with lid to prevent evaporation. Remove coating solution and wash plate three times with washing buffer using the microplate washer. Add blocking buffer to all wells and incubate the plate for 2h at 30 C. Remove blocking buffer and wash plate three times. Add 150 L incubation buffer to each well, then add either samples or standards (50 L in extraction buffer) to the appropriate wells. Incubate at room temperature for three hours on a rotating platform (300 rpm). Remove samples and standards, wash plate five times, then add the secondary antibody for detection: mAb #9 directly conjugated to horseradish peroxidase (Peroxidase labeling kit (POD), Roche, Cat. no. 11829696001), diluted in incubation buffer containing 4% BSA (200 L/well). Optimal dilution for the secondary antibody usually ranges from 1:4000 to 1:10000 and should be determined experimentally for each new batch of POD-conjugated mAb #9. Incubate for 3 hours at room temperature on the rotating platform. Remove secondary antibody and wash plate five times, then add 100 L/well chemiluminescent substrate (to be used according to the manufacturers instructions). Measure luminescence with an appropriate luminometer, then use a standard curve to determine the BDNF amounts in the samples. Results can then be indicated as ng BDNF/mg total soluble protein or g wet weight if tissue sample is large enough. At least duplicate determinations are recommended for both standards and samples.

BDNF standard curve: Successive 1:2 dilutions of purified recombinant BDNF in extraction buffer are used for the standard curve. A typical concentration range is between 25 pg/mL and 25 ng/mL (i.e. from 1.25 pg/well to 1.25 ng/well).

BDNF recovery: To evaluate recovery, known amounts of purified recombinant BDNF were added to some of the brain tissue homogenates. By means of the sandwich ELISA procedure described above, 97-100% of the added BDNF could be recovered and detected in these samples.