tau total elisa · 2015. 3. 3. · quantification using the standard curve, containing pbs, bsa,...

Tau total ELISA

Enzyme Immunoassay for the quantitative determination of human TAU in human CSF for use as an aid in the diagnosis of Alzheimer’s Disease.

RE59631

12 x 8

For illustrative purposes only.

To perform the assay the instructions for use provided with the kit have to be used.

Distributed by:

I B L I N T E R N A T I O N A L G M B HFlughafenstrasse 52a Phone: +49 (0)40-53 28 91-0 [email protected] Hamburg, Germany Fax: +49 (0)40-53 28 91-11 www.IBL-International.com

Instructions for Use

hTAU total ELISA (REF 0108000101) ENGLISH

Version 2015-12 1 / 11

CONTENT

1. INTENDED USE 2

2. SUMMARY AND EXPLANATION 2

3. TEST PRINCIPLE 2

4. WARNINGS AND PRECAUTIONS 3

5. STORAGE AND STABILITY 3

6. SPECIMEN COLLECTION AND STORAGE 3

7. MATERIALS SUPPLIED 4

8. MATERIALS REQUIRED BUT NOT SUPPLIED 5

9. PROCEDURE NOTES 5

10. PRE-TEST SETUP INSTRUCTIONS 6

10.1. Preparation of lyophilized or concentrated components 6

10.2. Predilution of samples 6

11. TEST PROCEDURE 7

12. QUALITY CONTROL 7

13. CALCULATION OF RESULTS 7

13.1. Determining the standard curve 7

13.2. Typical Calibration Curve 8

14. EXPECTED VALUES 8

15. LIMITATIONS OF THE PROCEDURE 8

16. PERFORMANCE 8

17. PRODUCT LITERATURE REFERENCES 9

18. NOTE 10

19. SYMBOLS 11

hTAU total ELISA (REF 0108000101)

Version 2015-12

1. INTENDED USE

Enzyme Immunoassay for the quantitative determination of T

of Alzheimer’s disease.

2. SUMMARY AND EXPLANATION

In 2010, the number of dementia patients worldwide was estimated at 36 million. Assuming an ongoing lack of

sufficient preventive and curative treatments, this is expected to double every 20 years. Alzheimer’s

for roughly 60-70% of all dementia cases. Both prevalence and incidence increase with age. Prevalence is around 1%

in those aged 65-69, and more than 30% in those aged 90 or older.

The first case of Alzheimer’s disease was defined and reported in 1907 by German scientist Dr. Alois Alzheimer. He

described two hallmarks of the Disease -

The development of the Disease is characterized by three stages, as defined by the US National Institute on Aging

workgroups.

- a preclinical stage of Alzheimer’s

- the mild cognitive impairment (MCI) stage due to AD

- the dementia stage due to AD

Amyloidosis occurs as early as the preclinical stage. The first cognitive deficits can manifest themselves in MCI stage,

while in the dementia stage patients are unable to do any work or daily chores.

The concentration of Tau in CSF is therefore recogniz

Moreover, a number of independent studies showed the ratio of TAU

diagnostic marker for Alzheimer’s disease.

3. TEST PRINCIPLE

This kit uses a monoclonal antibody specific for the amino acid region 160

area of the microtiter plate. Tau protein from sample

detected by another specific Tau protein binding monoclonal antibody.

detected via a conjugated horseradish peroxidase using the chromogenic substrate Tetramethylbenzidine (TMB). The

concentration of TAU is proportional to the obtained optical den

ELISA

- 1. Incubation step -

The capture antibody on the

microtiter plate binds Tau from

CSF or standards and controls

After washing,

Tau

quantitative determination of Tau protein in human CSF for use as aid i

SUMMARY AND EXPLANATION


eventive and curative treatments, this is expected to double every 20 years. Alzheimer’s

70% of all dementia cases. Both prevalence and incidence increase with age. Prevalence is around 1%

30% in those aged 90 or older.

isease was defined and reported in 1907 by German scientist Dr. Alois Alzheimer. He

the plaques and tangles in the brains of Alzheimer’s patients.


preclinical stage of Alzheimer’s disease,

mpairment (MCI) stage due to AD and

D


while in the dementia stage patients are unable to do any work or daily chores.

in CSF is therefore recognized as a useful biomarker in diagnosing Alzheimer’s

Moreover, a number of independent studies showed the ratio of TAU-protein to amyloid

isease.

al antibody specific for the amino acid region 160-180 of Tau441

protein from samples, standards and controls binds to this antibody and

protein binding monoclonal antibody. The binding of the monoclonal antibody is


is proportional to the obtained optical density.

ELISA

-2. Incubation step - - TMB/H

After washing, POD recognizes

au bound conjugated antibody

on the microtiter plate.

TMB acts as a hydrogen donor

for the reduction of H

water by the POD.

ENGLISH

2 / 11

rotein in human CSF for use as aid in the diagnosis


eventive and curative treatments, this is expected to double every 20 years. Alzheimer’s disease accounts

70% of all dementia cases. Both prevalence and incidence increase with age. Prevalence is around 1%

isease was defined and reported in 1907 by German scientist Dr. Alois Alzheimer. He

the plaques and tangles in the brains of Alzheimer’s patients.



ed as a useful biomarker in diagnosing Alzheimer’s disease.

protein to amyloid-beta (1-42) to be a superior

441, immobilized on the surface

, standards and controls binds to this antibody and Tau is

The binding of the monoclonal antibody is


ELISA

TMB/H2O2 Coloring -

TMB acts as a hydrogen donor

reduction of H2O2 to

water by the POD.


Version 2015-12 3 / 11

4. WARNINGS AND PRECAUTIONS

1. For in-vitro diagnostic use only. For professional use only.

2. Before starting the assay, read the instructions completely and carefully. Use the valid version of the package

insert provided with the kit. Make sure that everything is understood.

3. Should the kit package be severely damaged, please contact IBL or your supplier in writing, no later than one

week after receiving the kit. Do not use damaged components in test runs. Instead, keep them safe for complaint-

related issues.

4. Obey lot number and expiry date. Do not mix reagents of different lots. Do not use expired reagents.

5. Follow good laboratory practice and safety guidelines. Wear lab coats, disposable latex gloves and protective

glasses where necessary.

6. Reagents of this kit containing hazardous material may cause eye and skin irritations. See MATERIALS SUPPLIED

and labels for details. Material Safety Data Sheets for this product are available directly from IBL on request.

7. Chemicals and reagents prepared or used must be treated as hazardous waste according to national biohazard and safety guidelines or regulations.

8. Professionals should guide the cleaning staff regarding potential hazards and handling.

9. All reagents of this kit containing human serum or plasma have been tested and were found negative for anti-HIV

I/II, HBsAg and anti-HCV. However, a presence of these or other infectious agents cannot be excluded absolutely. For this reason reagents should be treated as potential biohazards in use and for disposal.

10. Avoid contact with Stop solution. It may cause irritations and burns.

5. STORAGE AND STABILITY

The kit is shipped at ambient temperature and should be stored at 2-10 °C. Keep away from heat or direct sunlight.

The storage and stability of specimens and prepared reagents is stated in the corresponding chapters.

The microtiter strips are stable up to the expiry date of the kit in the broken, but tightly closed bag when stored at

2-10 °C.

6. SPECIMEN COLLECTION AND STORAGE

The Alzheimer’s Biomarker Standardization Initiative provides the following recommendations for the pre-analytical

and analytical aspects for AD biomarker testing in CSF ([1]).

Specimen Collection

Lumbar puncture may be performed at the vertebral body L3-L5 with the patient either sitting or lying down. Use a small

diameter (0.7 mm and 22 G), preferably atraumatic needle. A small-gauge needle will make a smaller hole in the dura, aiding

healing, and an atraumatic needle will reduce the chance of blood contamination in the CSF.

Each laboratory should use one kind of polypropylene tubes only. Glass or polystyrene tubes should in no circumstances be

used. Tubes of the smallest volume should be used, and these should be filled to at least 50% of their volume. It is important to

have carefully recorded and validated details concerning each stored sample so that any investigator when using these samples

has a precise history of the sample. Centrifugation is only required for visually hemorrhagic samples. Centrifuge as soon as

possible – within 2 hours of LP (on site or at nearest laboratory). Centrifugation speed has no effect; however it is recommend

applying 2000 g for 10 minutes at room temperature.

Specimen shipment

Samples may be sent by regular post as long as the duration of transport is less than 5 days.

Specimen Storage

It is recommended to freeze samples and store at -80°C for long time storage. It is recommended to limit the number of freeze

/thaw cycles to a maximum of 1-2.


Version 2015-12 4 / 11

Each laboratory should use the same polypropylene tubes. Glass or polystyrene tubes should in no circumstance be used

for the collection, storage or dilution of CSF sample.

All dilutions of CSF must be made using polypropylene tube or direct in Microtiterplate D1 – MTP.

For the dilution of CSF it is important to pipette Sample Diluent first into a polypropylene tube and add the CSF directly

into the Sample Diluent.

Samples with an erythrocyte count >500/µL should not be used without centrifugation.

Storage: -80 °C Keep away from heat or direct sunlight.

Stability: < 2 years

7. MATERIALS SUPPLIED

Quantity

D1 - MTP

Microtiter plate, 12 x 8-well strip coated with a

monoclonal antibody (mouse) to Tau, blocked and

stabilized. Ready to use.

96 Wells

D2 – 40X WB

Wash Buffer, 40X concentrates containing PBS, BSA,

Tween 20 and proclin 300. To Dilute 1:40 with distilled

water.

1 bottle natural, 50 mL

D3.1 – STD

D3.2 – STD

D3.3 – STD

D3.4 – STD

D3.5 – STD

D3.6 – STD

Lyophilized Tau standards (LYO) 1000, 500, 250, 100, 50

and 25 pg for inclusion in 1 ml D6 for preparing a standard

curve for quantification of TAU in unknown cerebrospinal

fluid samples.

6 x 3 Tubes with cap

- red 1000 pg

- natural 500 pg

- violet 250 pg

- yellow 100 pg

- green 50 pg - blue 25 pg

D4 – BL

Negative control (blank) OD <0.2 (450 nm), containing PBS, BSA, Tween 20 and proclin 300. Ready to use.

1 tube, white cap, 1 mL

D5 – HRP-CONJ

Monoclonal antibody (mouse) specific to Tau, conjugated

with horseradish peroxidase, 15X concentrate, containing

PBS, BSA, Tween 20 and proclin 300.

1 bottle brown, black cap,

1 mL

D6 – ASSAYBUF

Dilution buffer, containing PBS, BSA, Tween 20 and proclin

300. Ready to use.

1 bottle natur, 50 mL

D7 – CONTROL 1

Lyophilized quality control (LYO) with a defined high Tau

concentration corresponding batch specific certificate for

quantification using the standard curve, containing PBS,

BSA, Tween 20 and proclin 300.

3 tubes, brown cap

D8 – CONTROL 2

Lyophilized quality control (LYO) with a defined low Tau

concentration corresponding batch specific certificate for

quantification using the standard curve, containing PBS,

BSA, Tween 20 and proclin 300.

3 tubes, orange cap

D9 – TMB

TMB Substrate Solution. Ready to use. 1 bottle brown, 20 mL

D10 – STOP Stop Solution (1 M sulphuric acid). Ready to use. 1 bottle natural, 25 mL

REF


Version 2015-12 5 / 11

8. MATERIALS REQUIRED BUT NOT SUPPLIED

1. Micropipettes (e.g. Multipette or similar devices, < 3 % CV). Volume: 0-20 µL; 10-100 µL; 100-1000 µL

2. Plate shaker, horizontal shaking preferred, range 100-300 rpm (e.g. Rotamax 120)

3. Vortex mixer 4. Wash bottle, automated or semi-automated microtiter plate washing system

5. Bidistilled or deionised water

6. Paper towels, pipette tips and timer

7. Microtiter plate reader capable of reading absorbance at 450 nm (reference wavelength 600-650 nm)

8. Tubes for sample dilution (disposable polypropylene tubes)

9. 8-Channel Micropipettor with reagent reservoirs

9. PROCEDURE NOTES

1. Any improper handling of samples or modification of the test procedure may influence the results. The indicated

pipetting volumes, incubation times, temperatures and pretreatment steps must be followed strictly and in line

with the instructions. Use calibrated pipettes and devices only.

2. Once the test has been initiated, all steps should be completed without interruption. Make sure that required

reagents, materials and devices are prepared ready at the appropriate time. Allow all reagents and specimens to

reach room temperature (18-25 °C) and gently swirl each vial of liquid reagent and sample before use. Mix

reagents without foaming.

3. Avoid contamination of reagents, pipettes and wells/tubes. Use new disposable plastic pipette tips for each

component and specimen. Do not interchange caps. Always cap vials that are not used. Do not reuse wells/tubes

or reagents. Unused wells should be returned immediately to the resealed pouch including the desiccant.

4. It is advised to determine in duplicate in order to be able to identify potential pipetting errors.

5. Use a pipetting scheme to verify an appropriate plate layout.

6. Incubation time affects results. All wells should be handled in the same order and time sequences. It is

recommended to use an 8-channel Micropipettor for pipetting solutions in all wells.

7. Microtiter plate washing is important. Improperly washed wells will give erroneous results. It is recommended to

use a multichannel pipette or an automatic microtiter plate washing system. Do not allow the wells to dry

between incubations. Do not scratch coated wells during rinsing and aspiration. Rinse and fill all reagents with

care. While rinsing, check that all wells are filled precisely with Wash Buffer, and that there are no residues in the

wells.

8. Ensure the CSF samples are visually okay (e.g. samples with an erythrocyte count >500/µL should not be used

without centrifugation).


Version 2015-12 6 / 11

10. PRE-TEST SETUP INSTRUCTIONS

The contents of the kit for 96 determinations can be divided into three separate runs.

The volumes stated below are for one run with all strips (96 determinations).

10.1. Preparation of lyophilized or concentrated components

Volume/

Unit

with

(mL) Relation Remarks Storage Stability

Lyo

D3.1 – STD

D3.2 – STD

D3.3 – STD

D3.4 – STD

D3.5 – STD

D3.6 – STD

1.0

D6 – ASSAYBUF

Vortex short, 2-5 s.

RT

(18-25 °C)

Prepare freshly

and use only

once.

1.0 mL

D5 – HRP-CONJ

14

D6 – ASSAYBUF

1:15

Prepare freshly and use

only once.

Mix without foaming.

RT

(18-25 °C)

Prepare freshly

and use only

once.

50 mL D2 – 40X WB 1950 water

(twice distilled) 1:40 Mix vigorously. 2-10°C 4 weeks

Lyo

D7 – CONTROL 1

D8 – CONTROL 2

1.0 D6 - ASSAYBUF

vortex short, 2-5 s. RT

(18-25 °C)

Prepare freshly

and use only

once.

10.2. Predilution of samples

Patient sample Dilution with Relation Remarks

CSF generally D6 – ASSAYBUF

1:4

e.g.: 50 µL + 150 µL

All dilutions of CSF must be made in polypropylene

tubes. This also applies to automated processes.

For the dilution of CSF, it is important to first provide

the Sample Diluent in a PP tube and pipette the

cerebrospinal fluid directly into the Sample Diluent.

The dilution of the sample can also take place in the

D1 – MTP .

For this provide 75 µl D6 – ASSAYBUF and pipetting

25 µl Liquor and mixed it 2-3 times with the pipette.

REF


Version 2015-12 7 / 11

11. TEST PROCEDURE

1. Pipette 100 µL of each Standard, Control and diluted patient sample into the respective wells of microtiter

plate. Cover plate with lid or foil.

2. Incubate microtiter plate for 120 min at RT (18-25 °C) horizontal shaking, 100-300 rpm.

3. Wash plate 5x with 300 µL of diluted wash buffer using an automatic ELISA plate washer.

Alternatively, when performed manually discard incubation solution. Wash plate 5x with 300 µL of diluted

Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel.

4. Pipette 100 µL of diluted Enzyme Conjugate in each well. Cover plate with lid.

5. Incubate microtiter plate for 90 min at RT (18-25 °C).

6. Wash plate 5x with 300 µL/well of diluted wash buffer using an automatic ELISA plate washer.

Alternatively, when performed manually discard incubation solution. Wash plate 5x with 300 µL/well of

diluted Wash Buffer. Remove excess solution each step. At the end tapping the inverted plate on a paper

towel.

7. Pipette 100 µL of TMB Substrate Solution into each well.

8. Incubate microtiter plate for 30 min at RT (18-25 °C) in the dark.

9. Stop the substrate reaction by adding 150 µL of TMB Stop Solution into each well. Briefly mix contents by

gently shaking the plate.

10. Measure optical density with a photometer at 450 nm within 15 min after pipetting the Stop Solution.

12. QUALITY CONTROL

The test results are only valid if the test has been performed according to the instructions. Moreover the user must adhere strictly to the rules of GLP (Good Laboratory Practice) or comparable standards/laws. User and/or laboratory

must have a validated system for obtaining diagnosis according to GLP. All kit controls must be within the acceptable

ranges as stated on the labels and the QC certificate. If the criteria are not met, the run is not valid and should be

repeated. Each laboratory should use known samples as further controls. Participating in appropriate quality

assessment trials is recommended.

In case of any deviation the following technical issues should be proven: Expiration dates of (prepared) reagents,

storage conditions, pipettes, devices, incubation conditions and washing methods.

13. CALCULATION OF RESULTS

13.1. Determining the standard curve

For test validation OD of D4 (blank) has to be < 0.2 and controls D7 and D8 should be inside range of batch specific

certificate.

Use logarithmic values (LN) of standard OD’s and standard concentrations for plotting them onto x-axis (OD) and y-

axis (concentration) of a linear diagram or for using them in linear regression analysis to estimate concentration of

each sample. Logarithmic values of measured sample OD’s have to be used for this regression analysis followed by

exponentiation to calculate concentration in pg/ml or ng/ml. Alternative obtained ODs of the standards (y-axis, linear)

are plotted against their concentration (x-axis, logarithmic) either on semi-logarithmic graph paper or using an

automated method. A good fit is provided with cubic spline, 4 Parameter Logistics or Logit-Log.

To calculate the standard curve, apply each signal of the standards (one obvious outlier of duplicates might be

omitted and the more plausible single value might be used).

The concentration of the samples can be read from the standard curve. The initial dilution has to be taken into

consideration when reading the results from the graph.

Results of samples of higher pre-dilution have to be multiplied by the dilution factor. Samples with concentrations

above the highest standard can be diluted as described in PRE-TEST SETUP INSTRUCTIONS and re-assayed.


Version 2015-12

13.2. Typical Calibration Curve

(Example. Do not use for calculation!)

Standard Tau-Protein

(pg/mL) OD

3.1 1000 3

3.2 500 1,

3.3 250 0,

3.4 100 0,

3.5 50 0,

3.6 25 0,

14. EXPECTED VALUES

Many age-related reference ranges are given for the tau protein.

The German Society for CSF diagnostics and Clinical Neurochemistry e. V. in the German Society of Neurology

recommends the following reference ranges

<50 Years 51-70 Years

<300 pg/mL <450 pg/m

15. LIMITATIONS OF THE PROCEDURE

Specimen collection and storage have a significant effect on the test results. See SPECIMEN COLLECTION AND

STORAGE for details.

For cross-reactivities, see PERFORMANCE.

16. PERFORMANCE

Analytical Sensitivity

(Limit of Detection) 25

Cut-off

Clinical sensitivity

Clinical specificity

Precision Range

Intra-Assay 25

Inter-Assay 25

Inter-Lot 25

Linearity Range

10

Recovery Range

100

Method Comparison

ODmean OD/ODmax

(%)

3,073 100

1,664 54,1

0,747 24,3

0,333 10,8

0,245 7,8

0,149 4,8

related reference ranges are given for the tau protein.


the following reference ranges

Years >70 Years

<450 pg/mL <500 pg/mL

LIMITATIONS OF THE PROCEDURE


reactivities, see PERFORMANCE.

25 pg/mL Mean signal negative

hTAU total

(0108000101)

399 pg/mL

86%

100%

Range (pg/mL) Range (%)

25 – 1000 0.6 – 6.9

25 – 1000 2.4 – 6.6

25 - 1000 4.9 – 7.9

Range (pg/mL) Range

100 - 600 1:16

Range (pg/mL) Range (%)

100 - 600 80 - 100

hTAU total = 0.989 x INNOTEST hTAU Ag + 55.24

ENGLISH

8 / 11



e control (blank) + 3SD

Each laboratory must

establish its own cut-

off values

n=72

n=41

CVMean (%)

3.5

4.3

5.9

variance (%)

5.9

RecoveryMean (%)

88

55.24 r² = 0.90

n = 329


Version 2015-12 9 / 11

17. PRODUCT LITERATURE REFERENCES

1. Blennow, K., Dubois, B., Fagan, A. M., Lewczuk, P., de Leon, M. J., Hampel, H. Clinical utility of cerebrospinal

fluid biomarkers in the diagnosis of early Alzheimer's disease Alzheimers Dement. (2014) 2, 5552-5260. 2. Lewczuk, P., Kornhuber, J.; Neurochemical dementia diagnostics in Alzheimer's disease: where are we now

and where are we going?; Expert Rev Proteomics; 2011 Aug; 8(4); 447-58

3. Vanderstichele H, Bibl M, Engelborghs S, Le Bastard N, Lewczuk P, Molinuevo JL et al. Standardization of

preanalytical aspects of cerebrospinal fluid biomarker testing for Alzheimer's disease diagnosis: a consensus

paper from the Alzheimer's Biomarkers Standardization Initiative. Alzheimers Dement 2012;8(1):65–73.

4. Alzheimer A, Stelzmann RA, Schnitzlein HN, Murtagh FR. An English translation of Alzheimer’s 1907 paper,

„Über eine eigenartige Erkrankung der Hirnrinde“. Clin Anat. 1995;8:429-31.

5. Thies W, Bleiler L. 2013 Alzheimer’s disease facts and figures. Alzheimers Dement. 2013;9(2):208-45.

6. McKhann GM, Knopman DS, Chertkow H, Hyman BT, Jack CR et al. The diagnosis of dementia due to

Alzheimer’s disease: recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement 2011;7(3):263-9.

7. Albert MS, DeKosky ST, Dickson D, Dubois B et al. The diagnosis of mild cognitive impairment due to

Alzheimer’s disease: recommendations from the National Institute on Aging-Alzheimer’s Association

workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement 2011;7(3):270-9.

8. Sperling RA, Aisen PS, Beckett LA, Bennett DA et al. Toward defining the preclinical stages of Alzheimer’s

disease: recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on

diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement 2011; 7(3):280-92.

9. Deutsche Gesellschaft für Liquordiagnostik und Klinische Neurochemie e. V. in der Deutschen Gesellschaft für

Neurologie: Ausgewählte Methoden der Liquordiagnostik und Klinischen Neurochemie. 2004. 2. Auflage.


Version 2015-12 10 / 11

18. NOTE


Version 2015-12

19. SYMBOLS

Cat.-No.

No. of tests.

Store at:

Read instructions before use!

Use by:

Lot-No.

In Vitro Diagnostic Medical Device.

Manufacturer

Keep away from heat or direct sun

Caution!

.

Read instructions before use!

In Vitro Diagnostic Medical Device.

Manufacturer

Keep away from heat or direct sun light.

ENGLISH

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tau total elisa · 2015. 3. 3. · quantification using the standard curve, containing pbs, bsa,...

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