Instructions for use

h-NSE ELISATM E-4700R

NSE ELISA
1. INTENDED USE
Immunoenzymatic colorimetric method for determination of hNSE concentration in human serum.
NSE ELISA kit is intended for laboratory use only.
2. PRINCIPLEThe NSE ELISA test is based on simultaneous binding of human Neuron Specific Enolase by two monoclonal
antibodies, one immobilized on microwell plates and the other conjugates with horseradish peroxidase (HPR).
After incubation the bound/free separation is performed by a simple solid-phase washing, then the TMBSubstrate solution (TMB) is added. After an appropriate time has elapsed for maximum colour development,
the enzyme reaction is stopped and the absorbancies are determinated.
The colour intensity is proportional to the hNSE concentration in the sample.
hNSE concentration in the sample is calculated based on a Standard curve.
3. REAGENTS, MATERIALS AND INSTRUMENTATION
3.1 Reagents and materials supplied in the kit
Standards and Controls - lyophilized*
Cat. no.

SymbolStandard

concentration **Volume/Vial

TM E-4701

Standard A

0 ng/ml

2 x 0.75 ml

TM E-4702

Standard B

4 ng/ml

2 x 0.75 ml

TM E-4703

Standard C

20 ng/ml

2 x0.75 ml

TM E-4704

Standard D

50 ng/ml

2 x 0.75 ml

TM E-4705

Standard E

100 ng/ml

2 x 0.75 ml

TM E-4751

Control 1

2 x 0.75 ml

TM E-4752

Control 2

The right concentration arestated on the standard viallabel and on the QC-Report

2 x 0.75 ml* please read carefully paragraph 6.1**approximately concentration: the right concentration for the curve compute are lot-specific andare stated on the standard vial labels and on the QC-Report.
Incubation Buffer

TM E-4713
Content:

Phosphate buffer (50 mM), pH 7.4; BSA (1 g/l)

Volume:1 x 50 ml
Conjugate

TM E-4740
Content:

Monoclonal anti hNSE antibody conjugated with horseradish peroxidase (HRP)

Volume:1 x 1 ml
Microplate

TM E-4731
Content:

1 breakable microplate, Monoclonal anti hNSE antibody adsorbed on the microplate
TMB Substrate

MS E-0055
Content:

H2O2-TMB 0.26g/l (avoid any skin contact)

Volume:1 x 15 ml
Stop Solution

MS E-0080
Content:

Sulphuric acid 0.15 mol/l, (avoid any skin contact)

Volume:1 x 15 ml

Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.

Version 13.0-r

Effective: 2019-07-03

2/7

Wash Solution - 50x concentrated

SA E-0030
Content:

NaCl (45 g/l); Tween 20 (55 g/l)

Volume:1 x 20 ml

3.2 Necessary reagents not supplied
Distilled water.
3.3 Auxiliary materials and instrumentation
Automatic dispenser.
Microplate reader (450 nm, 620 - 630 nm)
Note
The Standards and Controls contain hNSE in a proteic stabilizing matrix solution.
Store all reagents between 2 °C - 8 °C in the dark.
Open the bag of reagent 4 (Coated Microplate) only when it is at room temperature and close it immediately
after use; once opened, the plate is stable up to expiry date.
4. WARNINGS
This kit is intended for research use only.
Use appropriate personal protective equipment while working with the reagents provided.
Follow Good Laboratory Practice (GLP) for handling blood products.
Some reagents contain small amounts of Proclin 300 as preservative. Avoid the contact with skin or mucosa.
The TMB Substrate contains an irritant, which may be harmful if inhaled, ingested or absorbed through theskin. To prevent injury, avoid inhalation, ingestion or contact with skin and eyes.
The Stop Solution consists of a diluted sulphuric acid solution. Sulphuric acid is poisonous and corrosive andcan be toxic if ingested. To prevent chemical burns, avoid contact with skin and eyes.
Avoid the exposure of reagent TMB/H2O2 to directed sunlight, metals or oxidants. Do not freeze the solution.
This method allows the determination of hNSE inside the range of Standard A – Standard E.
Standards values are lot-specific.

5. PRECAUTIONS


Please adhere strictly to the sequence of pipetting steps provided in this protocol. The performance datarepresented here were obtained using specific reagents listed in this Instruction for Use.
All reagents should be stored refrigerated at 2 °C - 8 °C in their original container. Any exceptions are clearlyindicated. The reagents are stable until the expiry date when stored and handled as indicated.
Allow all kit components and specimens to reach room temperature (22 °C - 28 °C) and mix well prior to use.
Do not interchange kit components from different lots. The expiry date printed on box and vials labels must
be observed. Do not use any kit component beyond their expiry date.
If you use automated equipment, the user has the responsibility to make sure that the kit has been
appropriately tested.
The incomplete or inaccurate liquid removal from the wells could influence the assay precision and/or increase
the background. To improve the performance of the kit on automatic systems is recommended to increase thenumber of washes.
It is important that the time of reaction in each well is held constant for reproducible results. Pipetting ofsamples should not extend beyond ten minutes to avoid assay drift. If more than 10 minutes are needed,follow the same order of dispensation. If more than one plate is used, it is recommended to repeat the dose
response curve in each plate
Addition of the TMB Substrate solution initiates a kinetic reaction, which is terminated by the addition of theStop Solution. Therefore, the TMB Substrate and the Stop Solution should be added in the same sequence toeliminate any time deviation during the reaction.
Observe the guidelines for performing quality control in medical laboratories by assaying controls and/orpooled sera.
Maximum precision is required for reconstitution and dispensation of reagents.
Samples microbiologically contaminated, highly lipaemic or haemolysed should not be used in the assay.
Plate readers measure vertically. Do not touch the bottom of the wells.

Version 13.0-r

Effective: 2019-07-03

3/7

6. PROCEDURE
6.1 Preparation of Standards and Controls
Reconstitute each vial of Standard and Control with 0.75 ml of deionized H2O before use.
Important note: Reconstituted Standards and Controls are very sensitive to temperature, so you
should proceed as follows:
1. Reconstitute each vial of Standard and Control with 0.75 ml of deionized water
2. Leave on a rolling mixer for about 5 minutes
3. Take the necessary aliquot for the assay and immediately aliquot and freeze at -20 °C unused Standards
and Controls.
Reconstituted Standards and Controls are stable 1 month at -20 °C; avoid repeated freezing and thawing.
The Standards have approximately the following concentrations:ng/mlStandard A
0Standard B
4Standard C
20Standard D
50Standard E
100

The right Standard concentrations for the curve compute are lot specific and are stated on theStandard vial labels and on the QC-Report.
6.2 Diluted Conjugate
Prepare immediately before use.
Add 20 μl of Conjugate (reagent 4) to 1 ml of Incubation Buffer (reagent 3), the quantity to prepare is directly
proportional to the number of test.
Mix gently leaving in a rotating shaker for at least 5 minutes.
6.3 Preparation of Wash Solution
Dilute contents of wash buffer concentrate (50X) to 1000 ml with distilled or deionised water in a suitablestorage container.
For smaller volumes respect the dilution ratio of 1:50.
The diluted buffer is stable at 2 °C - 8 °C for at least 30 days.
6.4 Preparation of the Sample
The hNSE determination can be carried out in human serum.
The serum would have to be separated from the blood within 60 minutes in order to avoid the increment of
the hNSE from the blood cells release.
Do not use hemolyzed samples.
Avoid use of plasma since meaningful amounts of hNSE could be yielded from platelets.
Samples can be stored at 2 °C - 8 °C for 1 day; for long periods store at -20 °C.
Avoid repeated freeze-thaw cycles. Do not allow the samples at room temperature for long period.
6.5 Procedure
Allow all reagents to reach room temperature (22 °C - 28 °C) for at least 30 minutes.
At the end of the assay, store immediately the reagents at 2 °C - 8 °C avoid long exposure to room
temperature (see paragraph 6.1 for Standards and Controls).
Unused coated microwell strips should be released securely in the foil pouch containing desiccant and stored
at 2 °C - 8 °C.
To avoid potential microbial and/or chemical contamination, unused reagents should never be transferred into
the original vials.
As it is necessary to perform the determination in duplicate in order to improve accuracy of the test results,
prepare two wells for each point of the standard curve (Standard A – Standard E), two for each Control, twofor each sample, one for Blank.

Version 13.0-r

Effective: 2019-07-03

4/7

Reagent

Standard

Standard A – Standard E

Sample/ Controls

25 µl

Sample/ Controls
Diluted ConjugateBlank

25 µl
100 µl100 µlIncubate at room temperature (22 °C - 28 °C) for 1 hour.
Remove the contents from each well and wash the wells 3 times with 300 µl of diluted Wash Solution.
Important note: during each washing step, gently shake the plate for 5 seconds and remove excess solution
by tapping the inverted plate on an absorbent paper towel.
Automatic washer: if you use automated equipment, wash the wells at least 5 times.
TMB Substrate

100 µl100 µl100 µlIncubate at room temperature (22 °C - 28 °C) for 15 minutes in the dark.
Stop Solution

100 µl100 µl100 µlShake the microplate gently. Read the absorbance (E) at 450 nm against a reference wavelength of620 - 630 nm or against Blank within 5 minutes.
7. QUALITY CONTROL
Each laboratory should assay controls at normal, high and low levels range of hNSE for monitoring assay
performance. These controls should be treated as unknowns and values determined in every test procedureperformed. Quality control charts should be maintained to follow the performance of the supplied reagents.
Pertinent statistical methods should be employed to ascertain trends. The individual laboratory should set
acceptable assay performance limits. Other parameters that should be monitored include the 80, 50 and 20%
intercepts of the standard curve for run-to-run reproducibility. In addition, maximum absorbance should beconsistent with past experience. Significant deviation from established performance can indicate unnoticed
change in experimental conditions or degradation of kit reagents. Fresh reagents should be used to determine
the reason for the variations.
8. RESULTS
8.1 Mean AbsorbanceCalculate the mean of the absorbancies (Em) corresponding to the single points of the standard curve(Standard A – Standard E) and of each sample.
8.2 Standard curvePlot the values of absorbance (Em) of the Standards (Standard A – Standard E) against concentration. Draw
the best-fit curve through the plotted points. (e.g.: Cubic Spline, Sigmoid Logistic or Four Parameter Logistic).
8.3 Calculation of Results
Interpolate the values of the samples on the standard curve to obtain the corresponding values of theconcentrations expressed in ng/ml.

Version 13.0-r

Effective: 2019-07-03

5/7

9. REFERENCE VALUES
The serum values are comprised in the following intervals:

Normal range
Pathological valuehNSE
0 - 12 ng/ml> 12 ng/ml

Please pay attention to the fact that the determination of a range of expected values for a “normal” population
in a given method is dependent on many factors, such as specificity and sensitivity of the method used andtype of population under investigation.
Therefore, each laboratory should consider the range given by the manufacturer as a general indication andproduce their own range of expected values based on the indigenous population where the laboratory works.
10. PERFORMANCE AND CHARACTERISTICS
10.1 Specificity
The antibody is directed specifically against the human neuron specific enolase.
Cross reactivity values have been calculated on a weight/weight basis.
NSE Fitzgerald(Cat.No.30AN10 Lot. A99052602)
NNE Biogenesis(Cat. 6880-1004 Lot. 991105A)

100 %<0.22 %

10.2 Sensitivity
The lowest detectable concentration of hNSE that can be distinguished from the Standard A is 0.19 ng/ml at
the 95 % confidence limit.
10.3 Precision
10.3.1 Intra-assay
Within run variation was determined by replicate measurements (16x) of two different control sera in oneassay.
The within assay variability is ≤ 4.4%.
10.3.2 Inter-assay
Between run variation was determined by replicate measurements (10x) of two different control sera indifferent lots. The between assay variability is ≤ 11.2%.
10.4 Correlation
The NSE ELISA (x) kit was compared to another commercially
28 serum samples were analysed according in both test systems.
The linear regression curve is:(y) = 1.34 × (x) – 0.66
r2 = 0.971

availablehNSE

assay

(y).

10.5 Hook EffectThis NSE ELISA kit shows no Hook Effect up to 5000 ng/ml of hNSE.
11. WASTE MANAGEMENT
Reagents must be disposed of in accordance with local regulations.

Version 13.0-r

Effective: 2019-07-03

6/7

12. LITERATURE
1. Sorensen K, Brodbeck U, Paus E, Norgaard-Pedersen B. An enzyme antigen immunoassay for thedeterminationofneuron-specificenolaseinserum
samples.
Clin Chim Acta. 1988 Jul 29;175(3):337-43.
2. Drivsholm L, Osterlind K, Cooper EH, Purves DA. Neuron-specific enolase (NSE) in serum. Comparison ofmonoclonalversuspolyclonal
assay
basedon392
bloodsamples.
Int J Biol Markers. 1995 Jan-Mar;10(1):1-4.
3. Karnak D, Beder S, Kayacan O, Ibis
Am J Clin Oncol. 2005 Dec;28(6):586-90.

E,OflazG.

Neuron-specificenolaseandlungcancer.

4. Berger RP, Dulani T, Adelson PD, Leventhal JM, Richichi R, Kochanek PM. Identification of inflictedtraumatic brain injury in well-appearing infants using serum and cerebrospinal markers: a possible
screeningtool.
Pediatrics. 2006 Feb;117(2):325-32.
5. Ghayumi SM, Mehrabi S, Doroudchi M, Ghaderi A Diagnostic value of tumor markers for differentiating
malignantandbenign
pleuraleffusionsofIranianpatients.
Pathol Oncol Res. 2005;11(4):236-41. Epub 2005 Dec 31.
6. Sawauchi S, Taya K, Murakami S, Ishi T, Ohtsuka T, Kato N, Kaku S, Tanaka T, Morooka S, Yuhki K,Urashima M, Abe T. Serum S-100B protein and neuron-specific enolase after traumatic brain injury.
No Shinkei Geka. 2005 Nov;33(11):1073-80
7. Ramont L, Thoannes H, Volondat A, Chastang F, Millet MC, Maquart FX. Effects of hemolysis and storage
condition on neuron-specific enolase (NSE) in cerebrospinal fluid and serum: implications in clinicalpractice.
Clin Chem Lab Med. 2005;43(11):1215-7.

Symbols:

Version 13.0-r

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberEffective: 2019-07-03

Contains sufficient for<n> tests

For research use
only!
7/7