Phosphorylated NF-H ELISA Assay


The Phosphorylated NF-H ELISA Assay is intended for the quantification of Phosphorylated Neurofilament NF-H (pNF-H) in plasma, serum, CSF and tissue extracts. The Eagle Biosciences Phosphorylated Neurofilament NF-H ELISA Assay Kit is for research use only and should not be used for diagnostic procedures.

SKU: ARG81164 Categories: , ,

Phosphorylated NF-H ELISA Assay

The Phosphorylated NF-H ELISA Assay is For Research Use Only

Size: 1×96 wells
Dynamic Range: 0.156-10 ng/ml
Incubation Time: 3 hours
Sample Type: Serum, Plasma, CSF, and Tissue Extracts
Sample Size: 50 µL
Alternative Names: pNF-H ELISA, Human Phosphorylated Neurofilament H

Assay Background

Neurofilaments are type IV intermediate filament heteropolymers composed of light, medium, and heavy chains. Neurofilaments comprise the axoskeleton and functionally maintain neuronal caliber. They may also play a role in intracellular transport to axons and dendrites. This gene encodes the heavy neurofilament protein. This protein is commonly used as a biomarker of neuronal damage and susceptibility to amyotrophic lateral sclerosis (ALS) has been associated with mutations in this gene. [provided by RefSeq, Oct 2008] The pNF-H protein has been detected in large amounts following experimental spinal cord and brain injury in rats (18). Levels of greater than 100ng/mL of pNF-H were detectable in blood samples following serious spinal cord injury and lower but still easily detectable levels were seen in blood of animals given experimental brain injury.

More recent studies have revealed considerable amounts of this protein in the blood of transgenic mice carrying mutations of human copper/zinc superoxide dismutase-1 which are associated with amyotrophic lateral sclerosis. These mice develop an axonal degeneration pathology similar to that seen in humans with ALS, and blood pNF-H levels can be used to monitor progression of the disease. Interestingly, pNF-H was detectable before the onset of obvious disease symptoms.

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Additional Information

Assay Principle

This assay employs the sandwich enzyme immunoassay technique for the detection of pNF-H protein in plasma, serum, CSF and tissue extracts samples. pNF-H protein in samples and standards will bind to the capture mouse monoclonal antibody coated on the microtiter plate. After appropriate washing steps, HRP-conjugated anti-pNF-H monoclonal antibody binds to the captured protein. After washing away any unbound antibody-enzyme reagent, a substrate solution (TMB) is added to the wells and color develops in proportion to the amount of pNF-H protein bound in the initial step. The color development is stopped by the addition of acid and the intensity of the color is measured at a wavelength of 450nm ±2nm. The concentration of pNF-H protein in the sample is then determined by comparing the O.D of samples to the standard curve.

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 50 µl per well of standards and diluted samples in duplicates into appropriate wells. Incubate the plate at RT for 2 hours on a microplate shaker at 250-300 rpm (alternately, cover the plate with a plate sealer and incubate the plate at 4°C for overnight on a microplate shaker at 250-300 rpm)
  3. Aspirate each well and wash, repeating the process 2 times for a total 3 washes. Wash by filling each well with 1XTBST (300 µl) using a squirt bottle, manifold dispenser, or autowasher. Gently shake the plate for few seconds before removal of liquid, and complete removal of liquid at each is essential to good performance. After the last wash, remove any remaining buffer by aspirating, decanting or blotting against clean paper towels.
  4. Turn the plate 180°, wash as according to step 3.
  5. Add 100 µl of working 1X HRP-conjugated pNF-H Antibody (1:4000 diluted) into each well. Incubate the plate for 2 hour at RT on a microplate shaker (250-300 rpm).
  6. Wash as according to step 3 and step 4. (Before washing step, bring TMB substrate to room temperature.)
  7. Add 100 µl of TMB substrate to each well. Incubate and shake plate on a shaker with gentle shaking for 5-20 minutes at RT in dark (average time might be 7-10 min). Substrate will change from colorless to different strengths of blue.
  8. Add 50 µl of 2N H2SO4 to each well. The color of the solution should change from blue to yellow. Mix thoroughly by gently shaking/tapping the plate. Take care to avoid creating bubbles which will create a strong interfering absorbance signal.
  9. Read the OD with a microplate reader at 450 nm immediately.

Package Inserts

Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.

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