ILP z3170416

From CellBiology

ILP Z3170416 NSE on CSF samples 12Aug08

NSE Kit Info:

DRG NSE EIA-4610

Lot Number: 1081

Expiry: 31/01/2009

Aim

To detect Neuron-Specific Enolase (NSE) levels in CSF of patients with delirium and control group.

Hypothesis

NSE level of CSF of patients with delirium is higher than that of control group. NSE is released from neuronal cells in apoptotic event and can be detected in the CSF of delirium patients.

Methods

Solutions/Material Supplied:

1. Standards/Controls:

-STD0 0.75mL

-STD1 0.75mL

-STD2 0.75mL

-STD3 0.75mL

-STD4 0.75mL

-CTR1 0.75mL

-CTR2 0.75mL

2. Incubation buffer 50mL Phosphate buffer 50mM pH 7.4; BSA 1g/L

3. Enzyme conjugate 0.4mL Anti-monoclonal h-NSE-HRP conjugate

4. Coated microplate X1 Anti-monoclonal-h-NSE adsorbed on microplate

5. TMB Substrate solution 12mL hydrogen peroxide-TMB 0.25 g/L

6. Stop solution 12mL Sulphuric acid 0.15 mol/L

7. Concentrated Wash solution 20mL NaCl 45 g/L; Tween-20 55 g/L


Preparation of reagents:

- Preparation of Wash Solution – 10mL of Concentrated Wash solution diluted to 500mL RO water.

- Preparation of Standards/Controls – 750uL of RO water added into each standard and control tube available.

Standards/Controls S0 S1 S2 S3 S4 Control1 Control2

Concentration (ng/mL) 0 4 20 60 100 3-9 20-60

- Diluted Conjugate – 200uL Enzyme Conjugate is added to 10.0mL of Incubation Buffer


Procedure:

- 25uL of each standards and controls is pipetted into each well. Standards and controls are duplicated.

- 100uL of each CSF sample is added into each well. Samples are duplicated.

- 100uL Diluted Conjugate is added into each well.

- Microtitre plate arrangement - refer to word document

- Plate incubated at room temperature for 1 hour (1235-1335).

- Contents from each well were removed. Wells were washed with 300uL of Diluted Wash Solution. The plate is dried by blotting on a clean wipe. The washing procedure was repeated 3 times by draining the wash completely.

- 100uL Substrate Solution is pipetted into each well.

- Plate incubated at room temperature for 15 minutes in the dark (1445-1500) covered with aluminium foil.

- 100uL Stop Solution added into each well. An observable colour change from blue to yellow occurred to indicate thorough mixing.

- Absorbance at 450nm is read with a plate reader.

Results

- Refer to Word document NSE (CSF) 12Aug Report

Discussion

NSE levels are significantly higher in CSF of the control group compared to delirium group.

References

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