Intended use
This immunoassay kit allows for the specific measurement of total rat brain-derived neurotrophic factor (BDNF)concentrations in cell culture supernates, serum, and plasma.
Introduction
BDNF is a 13 kDa, 119 amino acid (aa) residue non-glycosylated polypeptide whose primary structure is conserved among all mammalian species examined. Initially synthesized as a 247 aa residue prepropeptide, the BDNF molecule is divided into an 18 aa residue signal sequence, a 110 aa residue prosequence, and a 119 aa residue mature segment. Similar to other neurotrophic factors, there is a possibility that the N-terminus is alternatively spliced, giving rise to a longer pre-prosegment (but identical mature segment) with different functional properties. As a mature molecule, BDNF is 52% identical to NGF at the amino acid level, exists as a noncovalently-linked homodimer in solution, and contains six cysteine residues that are believed to form three intrachain disulfide linkages. BDNF in plasma is detected in the pg/mL range, while BDNF in serum is measured in the ng/mL range, the difference apparently attributable to platelet degranulation and BDNF release during clotting. The conservation of BDNF structure potentially allows a BDNF ELISA to be widely applied across species.
There are at least two receptors for BDNF, the first being the low affinity 75 kDa Nerve Growth Factor Receptor (LNGFR), and the second being the high affinity 145 kDa TrkB (tropomyosin receptor kinase-B). The LNGFR is a 399 aa residue Type-I (extracellular N-terminus) transmembrane glycoprotein that is currently considered to be a member of the TNF receptor superfamily. Although all neurotrophins bind LNGFR with approximately the same affinity (Kd ~ 1 nM), the significance of such binding is uncertain. What seems clear is that the LNGFR alone can engage certain signal transduction pathways. The biological significance of the activation of these pathways is not well understood. For BDNF specifically, LNGFR may serve as a retrograde transport molecule in neurons, promote Schwann cell migration near injury, and/or modulate TrkB activity in those cells that co-express both LNGFR and TrkB. The second receptor for BDNF is the TrkB high affinity receptor (Kd ~ 10 pM), a receptor that also binds NT-3 and NT-4/5. TrkB is a 792 aa residue type I (extracellular N-terminus) transmembrane glycoprotein that exhibits a number of distinct extracellular domains. These include two N-terminal cysteine-rich regions that flank an intervening leucine-rich domain and two membrane proximal C2 Ig-like domains. Comparing rat and human proteins, these regions exhibit over 90% identity in aa sequence. Alternative splice events have been identified for the TrkB gene in the mouse, the rat, and the human. In each case, nonsignalling, cytoplasmically-truncated variants are produced, leading to speculation that alternative splicing may be one method by which cells down-regulate neurotrophin activity. While full TrkB activity is believed to require TrkB homodimerization, evidence suggests that full length TrkB and TrkC receptors may also form functional heterodimers in select cells where both receptors are co-expressed. These include cerebellar granule neurons and neurons of the hippocampal dentate nucleus. Among the cells known to express TrkB are motoneurons of the spinal cord, pyramidal cells of the hippocampus, almost all neurons in the developing brain, and thymocytes, leading to speculation that BDNF plays a role in lymphopoiesis.
The number of functions attributed to BDNF is quite large. During development, BDNF has been
implicated in neuronal differentiation, maturation, survival and synapse formation. One of its most promising roles centers on neuroprotection, possibly protecting forebrain neurons from ischemic attack and motor neurons from axotomy-induced death.
Test principle
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal
antibody specific for BDNF has been pre-coated onto a microplate. Standards and samples are
pipetted into the wells and any BDNF present is bound by the immobilized antibody. An
enzyme-linked monoclonal antibody specific for BDNF is added to the wells. Following a wash
to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells
and color develops in proportion to the amount of BDNF bound in the initial step. The color
development is stopped and the intensity of the color is measured.
