Neuroserpin ELISA Assay

Additional Information

Assay Background

Neuroserpin is an inhibitory serpin that is expressed predominantly in central nervous system. Although the physiological target of neuroserpin is still unclear, cumulative evidence suggest that it plays an important role in controlling proteolytic degradation of extracellular matrix (ECM) during synaptogenesis and the subsequent development of neuronal plasticity. In the adult brain, neuroserpin is secreted from the growth cones of neurons in areas where synaptic changes are associated with learning and memory, i.e. cerebral cortex, hippocampus, and amygdala. The neuroprotective role of neuroserpin has been demonstrated in transgenic mice lacking neuroserpin expression. The deficiency of neuroserpin in these mice was associated with motor neuron disease characterized by axonal degradation. In humans, defects in neuroserpin, caused by point mutations in the neuroserpin gene, underlie a hereditary disorder called the familial encephalopathy with neuroserpin inclusion bodies (FENIB). Recombinant human neuroserpin is a 44.6 kDa non-glycosylated protein containing 394 amino-acid residues.

Assay Procedure

1. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal it.
2. Add 100 μl of standards, samples and zero controls (standard diluent buffer) into wells. Incubate for 1.5 h at 36 °C.
3. Aspirate each well and wash, repeating the process four times for a total five washes. Wash by filling each well with 1× Wash Buffer (350 μl) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating, decanting or blotting against clean paper towels.
4. Add 100 μl 1X Antibody conjugate into each well. Cover wells and incubate for 1 hour at 36°C.
5. Aspirate each well and wash as step 3.
6. Add 100 μl of 1X HRP-Streptavidin solution to each well. Cover wells and incubate for 30 minutes at 36°C.
7. Aspirate each well and wash as step 3.
8. Add 100 μl of TMB Reagent to each well. Incubate for 15 minutes at 36°C in dark.
9. Add 100 μl of Stop Solution to each well. The color of the solution should change from blue to yellow.
10. Read the OD with a microplate reader at 450nm immediately.

Typical Standard Curve