A method for reproducible measurements of serum BDNF: comparison of the performance of six commercial assays

Abstract

Brain-Derived Neurotrophic Factor (BDNF) has attracted increasing interest as potential biomarker to support the diagnosis or monitor the efficacy of therapies in brain disorders. Circulating BDNF can be measured in serum, plasma or whole blood. However, the use of BDNF as biomarker is limited by the poor reproducibility of results, likely due to the variety of methods used for sample collection and BDNF analysis. To overcome these limitations, using sera from 40 healthy adults, we compared the performance of five ELISA kits (Aviscera-Bioscience, Biosensis, Millipore-ChemiKine(TM), Promega-Emax(®), R&D-System-Quantikine(®)) and one multiplexing assay (Millipore-Milliplex(®)). All kits showed 100% sample recovery and comparable range. However, they exhibited very different inter-assay variations from 5% to 20%. Inter-assay variations were higher than those declared by the manufacturers with only one exception which also had the best overall performance. Dot-blot analysis revealed that two kits selectively recognize mature BDNF, while the others reacted with both pro-BDNF and mature BDNF. In conclusion, we identified two assays to obtain reliable measurements of human serum BDNF, suitable for future clinical applications.

Figure 1. BDNF serum levels measured with the indicated ELISA assays.

Box plot of serum BDNF concentrations (ng/ml) from healthy volunteers (n = 38–40, see Table 2) represented as mean of two independent measures. The upper line of the box marks the 75th percentile, the middle line is the median value and the lower line specifies the 25th percentile. Whiskers above and below the box indicate the 90th and 10th percentiles, respectively. Dots indicate the outlier values within each group.

Figure 2. Inter-assay variation of the BDNF ELISA kits.

Scatter plot showing the BDNF values distribution measured by the same operator on two different days using two plates of the same lot for each brand (Day 1 & Day 2). Each dot represents a BDNF value from one subject and the dashed lines link together two assessments of the same subject. The reproducibility was checked performing one-way ANOVA for repeated measures and the P values are specified.

Figure 3. Inter-assay variation of the Aviscera-Bioscience, Biosensis and Millipore-ChemikineTM kits over a third replica.

Scatter plots show the BDNF values distribution measured by the same operator on three different days for each brand. Distributions for Day 1 and Day 2 are the same as Fig. 2, here reported for comparison; distributions for Day 3 were obtained 1 year after Day 2, using a plate of a different lot (serum samples were stored at −80 °C). The reproducibility was checked performing one-way ANOVA for repeated measures and post-hoc Bonferroni correction applied when a significant comparison was found. P values and cumulative CVs expressed in percentage are given.

Figure 4. Line-blot for qualitative analysis of anti-BDNF antibodies specificity.

(A) The antibodies from each ELISA kit were tested for specificity against pro-BDNF or mature BDNF. The BDNF standards blotted were commercial pro-BDNF (Alomone; 10 pg/lane), mature BDNF (1 and 2 from Alomone and Sigma, respectively; both 1000 pg/lane) and the standard BDNF protein included in each kit (Aviscera-Bioscience and Biosensis: 10 pg/lane; Millipore-ChemiKine^TM, Millipore-Milliplex^®- and R&D System-Quantikine^®: 100 pg/lane; Promega-Emax^®: 1000 pg/lane). BSA (1000 pg/lane) was used as a negative control. The mouse monoclonal anti-BDNF antibody, (1:1000; Sigma) was tested as a control. (B) Central region of the same blot shown in A, from an overexposed film to better visualize the reactivity against pro-BDNF. (C) Reactivity of antibodies from Biosensis, Promega-Emax^® pAb and Sigma on a dot blot in which the same quantity of pro-BNDF and mature BDNF were spotted (100 pg each). Each antibody from the ELISA kits was used at the dilution suggested by the manufacturer’s instructions. mAb: Promega-Emax^® monoclonal capture antibody for plate coating. pAb: Promega-Emax^® polyclonal detection antibody.

Figure 5. Graphic summary of BDNF ELISA kit performances.

Polar plot showing the kit performances. The black solid line indicates the assessed performances, the dashed line shows those declared by the manufacturers, while bold grey line indicates the best values (100%). Data are from a triplicate experiment for Aviscera-Bioscience, Biosensis and Millipore-ChemiKine^TM while from a duplicate for the other kits. See text for a detailed description.