Measurements of brain-derived neurotrophic factor: Methodological aspects and demographical data

doi:10.1016/j.brainresbull.2007.03.009

Brain Research Bulletin

Volume 73, Issues 1–3, 15 June 2007, Pages 143-149

https://doi.org/10.1016/j.brainresbull.2007.03.009 Get rights and content

Abstract

Although numerous studies have dealt with changes in blood brain-derived neurotrophic factor (BDNF), methodological issues about BDNF measurements have only been incompletely resolved. We validated BDNF ELISA with respect to accuracy, reproducibility and the effect of storage and repeated freezing cycles on BDNF concentrations. Additionally, the effect of demographic characteristics in healthy subjects on BDNF was verified. Whole blood and serum was collected from 206 healthy subjects and a subgroup was genotyped for BDNF Val66Met polymorphism. The effect of age, gender, BDNF genotype and thrombocyte count on whole blood BDNF was assessed. The BDNF ELISA measurement was accurate, 91.6 ± 3.0%, and showed high reproducibility, whereas inter-assay and intra-subject variations were modest, 8.4 ± 5.2% and 17.5 ± 14.1%, respectively. Storage of whole blood samples at 4 °C significantly decreased BDNF concentration, while repeated freezing cycles and storage at −20 °C was without any effect. Storage at −20 °C of serum, but not whole blood, was associated with a significant decrease in BDNF concentration. Women had significantly higher whole blood BDNF concentrations than men (18.6 ± 1.3 ng/ml versus 16.5 ± 1.4 ng/ml), and showed a right-skewed BDNF concentration distribution. No association between whole blood BDNF concentrations and thrombocyte count, age, or BDNF genotype was found. In conclusion, the BDNF ELISA assay determines whole blood BDNF accurately and with high reproducibility. Female gender is associated with higher whole blood BDNF concentrations whereas age, thrombocyte count and BDNF Val66Met polymorphism were un-associated.

Introduction

Brain-derived neurotrophic factor (BDNF) was characterised for the first time more than a decade ago [48] and it belongs to the neurotrophin family that also includes nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). The neurotrophin BDNF is crucially involved in brain development, neurogenesis, neural circuit formation, and plasticity [40]. It has an activity dependent release and is transported across the blood–brain barrier [33]. In the blood, BDNF is stored in platelets and released upon agonist stimulation by thrombin, Ca²⁺, collagen or shear stress [11] and the BDNF concentration can be measured with a commercially available assay [37]. Other peripheral sources of BDNF include the endothelial cells, smooth muscle cells, and eosinophils [8], [30], [38].

The peripheral effects of BDNF are only sparsely known; but it is believed to be involved in regeneration of neurons during nerve injury [31] and affect the immune system [13], [39], [41]. Animal studies indicate that brain and blood BDNF concentrations undergo similar changes during maturation and aging, suggesting that blood BDNF levels may reflect the BDNF levels in brain [18]. Indeed, rodent studies show a tight correlation between brain and blood BDNF values [18].

Numerous clinical studies have identified changes in serum or blood BDNF concentrations in patients with neuropsychiatric disorders such as depression [43], schizophrenia [44], Alzheimer's disease [21], multiple sclerosis [2], and anorexia [27]. Furthermore, in Alzheimer's disease protein and mRNA levels of BDNF are changed in the same direction, [12], [21], [26], and the same applies to exposure to stress [15], [26] and antidepressants [7], [26]. Methodological issues regarding measurements of BDNF concentration have, however, only been superficially encountered. Measurements of BDNF is highly valuable since it may be a marker of disease development in Alzheimer's disease [35] and it correlates with disease severity in major depression [43]. The aim of this study is, firstly, to validate a commonly used ELISA based assay. The reproducibility and accuracy of blood BDNF measurement as well as the influence of various storage conditions was assessed. Secondly, the relation between serum and whole blood BDNF concentrations was investigated to assess if studies on serum or whole blood are directly comparable. Thirdly, whole blood BDNF concentrations were investigated in healthy controls and related to gender, age, and the BDNF Val66Met functional polymorphism.

Section snippets

Subjects

Two-hundred and six healthy subjects, 122 women and 84 men, with a mean age of 44.3 ± 13.3 (range 20–70) years were recruited, mainly from newspaper advertisements. None of the subjects had a history of neuropsychiatric disorders, current alcohol abuse, and they were all drug free. Written informed consent was obtained according to the declaration of Helsinki II, and the study was approved by the Ethics Committee of Copenhagen and Frederiksberg [(KF)02-058/99, (KF)12-091/00, (KF)12-113/00,

Accuracy and yield of the BDNF assay

The average yield for samples spiked with three different concentrations of BDNF (100, 200, or 400 pg/ml), was high: 91.6 ± 3.0% (n = 6).

Internal standards and repeated measurements

When the individual standard curves from three kits were applied, the two internal standard samples BDNF concentrations varied by 34.0 ± 20.9%. This was due to a large variation in the absorbance values of the standard that was applied in each kit and used to yield the standard curve for each plate. For comparison, the manufacturer claims an inter-assay variation of

Discussion

This is the first study to systematically validate the BDNF ELISA assay with respect to accuracy, yield, inter-assay, and intra-subject variability. In addition, the stability of BDNF in samples under various storage conditions and the relation between serum and whole blood BDNF was assessed. We found that the BDNF ELISA assay is accurate and highly reproducible [3], [29]. Based on our data we recommend that duplicate determinations are carried out. Further, to ensure stability of the assay for

Acknowledgements

Terry Jernigan is acknowledged for good advice. The technical assistance supplied by Inge Møller, and the donation of serum samples from Bodil Jakobsen at Clinical Immunology Department, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark, is gratefully acknowledged. This work was supported by research grants from the Lundbeck Foundation, the Danish Medical Research Council, Novo Nordisk Foundation, sawmill owner Jeppe Juhl and Ovita Juhl Memorial Foundation, Augustinus

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Technical commentMeasurements of brain-derived neurotrophic factor: Methodological aspects and demographical data

Abstract

Introduction

Section snippets

Subjects

Accuracy and yield of the BDNF assay

Internal standards and repeated measurements

Discussion

Acknowledgements

Biol. Psychiatry

J. Neuroimmunol.

Neuroscience

Cell

Brain. Res.

Biol. Psychiatry

Psychiatry Res.

Neurosci. Lett.

J. Psychiatr. Res.

Exp. Neurol.

Neurobiol. Aging

Biol. Psychiatry

Clin. Microbiol. Infect.

Schizophr. Res.

Neuropharmacology

J. Neuroimmunol.

Trends Neurosci.

J. Neuroimmunol.

Psychoneuroendocrinology

Biol. Psychiatry

Psychiatry Res.

Brain Res. Dev. Brain Res.

Biochem. Biophys. Res. Commun.

The comparative accuracy of 8 commercial rapid immunochromatographic assays for the diagnosis of acute dengue virus infection

Clin. Infect. Dis.

Genetic variant BDNF (Val66Met) polymorphism alters anxiety-related behavior

Science

Technical comment
Measurements of brain-derived neurotrophic factor: Methodological aspects and demographical data