Detection of brain-derived neurotrophic factor (BDNF) in rat blood and brain preparations using ELISA: pitfalls and solutions.
Quantification of endogenous brain-derived neurotrophic factor (BDNF) can be performed with an enzyme-linked immunosorbent assay (ELISA). Although BDNF has been determined in blood and brain preparations in numerous studies with ELISA kits, the methodological issues regarding measurements of BDNF concentrations in the blood and particularly in the brain have only been superficially investigated. We aimed at validating and optimizing the BDNF ELISA kit with respect to measurements in rat blood and brain samples. We found that the pre-analytical conditions were critical for plasma samples, but not serum or whole blood samples. The intra- and inter-assay variation and the accuracy and yield of the BDNF ELISA kit in rat serum and brain tissue were conducted with the optimal dilutions of frontal cortex and hippocampus extract. The optimal dilutions of frontal cortex and hippocampus extracts were determined to be 20 and 120 times, and we established that the intra-assay coefficient of variation (CV%) was 8 in hippocampus and 2 in frontal cortex and serum. The inter-assay variation was also low with a CV% of 11 or less in hippocampus, frontal cortex, and serum. Finally, we found that the accuracy and yield of the BDNF measurements were high in serum and low in hippocampus and frontal cortex. We conclude that the BDNF ELISA kit determines serum BDNF accurately and with high reproducibility. Furthermore it can be used for measurement of BDNF in rat brain preparations when particular precautions are taken and in particular with care regarding the dilution of the brain tissue samples.
Elfving
Betina
B
Centre for Psychiatric Research, Aarhus University Hospital, Skovagervej 2, DK-8240 Risskov, Denmark.
Plougmann
Pia Høgh
PH
Wegener
Gregers
G
D000042:Absorption; D000818:Animals; D001921:Brain / Q000378:metabolism*; D019208:Brain-Derived Neurotrophic Factor / Q000097:blood* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005625:Frontal Lobe / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism; D008297:Male; D010949:Plasma / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D015203:Reproducibility of Results; D044967:Serum / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D019208:Brain-Derived Neurotrophic Factor