DRG BDNF Elisa

PRINCIPLE OF THE TEST 
The BDNF enzyme-linked immunosorbent assay (ELISA) Kit is a sandwich ELISA that allows the preferential quantification of mature BDNF in less than 3 hours. This kit consists of a pre-coated mouse monoclonal anti-BDNF capture antibody, a biotinylated anti-BDNF detection antibody and horseradish peroxidase (HRP)-conjugated streptavidin. The addition of a substrate (3,3’,5,5’- tetramethylbenzidine, TMB) yields a colored reaction product which is directly proportional to the concentration of BDNF present in samples and protein standards. This BDNF ELISA kit employs a recombinant human BDNF standard approved by the World Health Organization (WHO, www.nibsc.org). This kit is suitable to measure mature BDNF in human serum and suitably treated plasma samples only. The antibodies used in this ELISA kit bind epitopes within the mature domain of the protein and therefore recognize the mature as well as the proform of BDNF. However, cross-reactivity to the full-length proBDNF protein is low.

SUMMARY AND EXPLANATION 
Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family of growth factors that play an important role in a variety of physiological functions, for instance mediating neuronal survival and apoptosis, maintaining synaptic plasticity and regulating synaptic transmission. Altered BDNF levels in the central nervous system and blood are implicated in a variety of neurodegenerative diseases such as amyotrophic lateral sclerosis, neuropathic pain and Alzheimer’s disease.

SPECIMEN COLLECTION AND HANDLING
Serum 
Allow the serum to clot in a serum separator tube (about 30 minutes to 4 hours) at room temperature. Centrifuge at approximately 1,000 x g for 15 minutes. 
Analyze the serum immediately or aliquot and store frozen at -20 °C to -80 °C. 
Dilute serum samples with Assay Diluent A to measure BDNF concentrations. 
For human serum samples, a minimum dilution of 1⁄50 – 1⁄100 is required. 
Plasma 
Collect plasma using citrate as anticoagulant, other coagulants have not been qualified for use. 
Centrifuge for 15 min at 2 °C - 8 °C at 1,500 x g within 30 minutes of collection. 
For eliminating the platelet effect we suggest further centrifugation for 10 min at 2 °C - 8 °C at 10,000 x g. 
Analyze immediately or aliquot and store samples at -20 °C to -80 °C. 
Dilute plasma samples at least 1⁄20 with Assay Diluent A to measure BDNF concentrations.

Calculation of Results 
1. Average the readings for each BDNF standard concentration, blank and sample. 
2. Plot a standard curve with the BDNF standard concentration on the x-axis and the OD at 450 nm on the y-axis. 
3. If values for the BDNF standards are adjusted for background absorbance, then subtract the blank value from the OD450 of the samples as well. 
4. Use appropriate software to reduce the data and generate a four-parameter logistic curve-fit. Do not use linear regression. 
5. Perform a regression analysis to calculate the concentration of BDNF in the samples. Multiply the result by the sample dilution factor.