Development of mature BDNF‐specific sandwich ELISA

Abstract

Mature brain-derived neurotrophic factor (mBDNF) plays a vital role in the nervous system, whereas proBDNF elicits neurodegeneration and neuronal apoptosis. Although current enzymelinked immunosorbent assay (ELISA) has been widely used to measure BDNF levels, it cannot differentiate mBDNF from proBDNF. As the function of proBDNF differs from mBDNF, it is necessary to establish an ELISA assay specific for the detection of mBDNF. Therefore, we aimed to establish a new mBDNF-specific sandwich ELISA. In this study, we have screened and found a combination of antibodies for a sandwich ELISA. A monoclonal antibody and sheep anti-BDNF were chosen as capture and detection antibody for sandwich ELISA respectively. The new ELISA showed no cross-reactivity to human recombinant NT-3, NT-4, nerve growth factor and negligible cross-reactivity (0.99-4.99%) for proBDNF compared to commercial ELISA kits (33.18-91.09%). The application of the new mBDNF ELISA was shown through the measurement of mBDNF levels in different brain regions of rats and in the brain of b-site amyloid precursor protein cleaving enzyme 1 (BACE1) À/À and WT mice and compared to western blot.Overall, this new ELISA will be useful for the measurement of mBDNF levels with high specificity. Keywords: ELISA and cross-reactivity, mature BDNF (mBDNF), proBDNF. it has been shown that blood BDNF levels increase in major depressive disorder (MDD) patients treated with pharmacological treatment (Yoshimura et al. 2007;Huang et al. 2008), suggesting that BDNF levels in blood could be a biomarker in MDD and an anti-depressant (Yoshimura et al. 2007;Lin 2009;Hashimoto 2010). In contrast to mBDNF, which is reduced in MDD, proBDNF levels in the blood and lymphocytes have shown to be increased . In addition, a few recent studies have proved that BDNF levels in the brain and blood are firmly connected (Karege et al. 2002;Sartorius et al. 2009;Klein et al. 2011).Received October 18, 2014; revised manuscript received March 23, 2015; accepted March 24, 2015. Address correspondence and reprint requests to Prof. Xin-Fu Zhou, School of Pharmacy and Health Sciences, Sansom Institute, University of South Australia, Adelaide, SA 5000, Australia. E-mail: xin-fu. zhou@unisa.edu.auAbbreviations used: BSA, bovine serum albumin; HRP, horseradish peroxidase; mBDNF, mature brain-derived neurotrophic factor; MDD, major depressive disorder; NGF, nerve growth factor; PBS, phosphatebuffered saline.

BDNF Responses in Healthy Older Persons to 35 Minutes of Physical Exercise, Cognitive Training, and Mindfulness: Associations with Working Memory Function

“…The manufacturer of the ELISA kit we use claims 13% cross-reactivity for recombinant human pro-BDNF. The Elisa kit we used has a very low cross-reactivity for proBDNF compared to the other available ELISA kits (see Polacchini et al [55] and Lim et al [56]),…”

The regulatory role of ProBDNF in monocyte function: Implications in Stanford type‐A aortic dissection disease

“…Mature BDNF ELISA and proBDNF ELISA was performed according to Dr. Zhou's published paper before. 23…”

ProBDNF Signaling Regulates Depression-Like Behaviors in Rodents under Chronic Stress

“…injected with one single dose of sheep anti-proBDNF (Fan et al, 2008;Lim et al, 2015) antibody (10 μg,1 μg/μl) on day 22 with a Hamilton syringe, and then behaviorally assessed on days 23, 24, and 25 ( Figure 3a); (2) i.p. injected with one dose of anti-proBDNF antibody (10 μl, 0.08 μg/μl per g of body weight) on day 22, followed by one half the dose on day 26, and behaviorally assessed on days 27, 28, and 29 ( Figure 3a); and (3) i.m.…”

“…However, AAV-proBDNF increased the depression-like behavior as indicated by a significant increase in the percentage of immobility in the forced swimming test (two-way ANOVA, injection factor: F (1,10) = 0.38, p = 0.544; proBDNF factor: F (1,10) = 5.58, p = 0.028; injection × proBDNF: F (1,10) = 2.62, p = 0.121; Figure 6e). To check the efficacy of AAV-proBDNF treatment to increase proBDNF levels in the brain, we performed an ELISA assay as previously described (Lim et al, 2015) and found that proBDNF levels were significantly increased in the neocortex (Student's t-test, t (10) = − 13.54, po0.001; Figure 6f) and cerebellum (Student's t-test, t (10) = − 2.81, p = 0.019; Figure 6f) but not in hippocampus (Student's t-test, t (10) = − 1.69, p = 0.121; Figure 6f). Histological examination showed that green fluorescence was detected in the brains of rats that received AAV-EGFP but not AAV-proBDNF, and a significant upregulation of proBDNF expression was detected in the brains of rats that received AAV-proBDNF but not AAV-EGFP (Figure 6g).…”

“…Western blot, RT-PCR, ELISA, and immunostaining were performed as previously described (Fan et al, 2008;Lim et al, 2015;Yang et al, 2014), and detailed procedures are described in Supplementary Material.…”