CircuLex 14-3-3 Gamma ELISA Kit

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CircuLex

14-3-3 Gamma ELISA Kit

User’s Manual

For Research Use Only, Not for use in diagnostic procedures

ELISA Kit for Measuring 14-3-3 Gamma

CircuLex 14-3-3 Gamma ELISA Kit

Cat# CY-8082

Intended Use................................................ 1

Storage......................................................... 1

Introduction.................................................. 2

Principle of the Assay.................................. 3

Materials Provided....................................... 4

Materials Required but not Provided........... 5

Precautions and Recommendations............. 6

Sample Collection and Storage.....................7

Detailed Protocol......................................... 8-10

Calculations.................................…............ 10

Measurement Range.................................... 10

Troubleshooting.......................................... 10

Reagent Stability......................................... 11

Assay Characteristics.................................. 12-14

Example of Test Results..............................15-17

References................................................... 17-18

License........................................................ 18

Related Products......................................... 18

Intended Use

The CycLex Research Product CircuLex 14-3-3 Gamma ELISA Kit is used for the quantitative measurement of 14-3-3 Gamma (gamma isoform protein) in CSF (Cerebrospinal fluid), cell lysates and other biological samples in units of concentration.

This assay kit is for research use only and not for use in diagnostic or therapeutic procedures.

Storage

• Upon receipt store all components at 4°C .

• Don’t expose reagents to excessive light.

Cat#: CY-8082 1 Version#: 150601

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CircuLex

14-3-3 Gamma ELISA Kit

User’s Manual

For Research Use Only, Not for use in diagnostic procedures

Introduction

14-3-3 proteins are a family of conserved regulatory molecules expressed in all eukaryotic cells. There are seven genes, β, γ, ε, σ, ζ, τ and η, that encode 14-3-3s in most mammals. 14-3-3 proteins have the ability to bind phospho-Ser/Thr motifs, as well as phosphorylation-independent interactions, on a multitude of functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors. The family dynamically regulates the activity of target proteins in various signaling pathways that control diverse physiological and pathological processes. More than 100 signaling proteins have been reported as 14-3-3 proteins’ targets.

Either an abnormal state of 14-3-3 protein expression or dysregulation of 14-3-3/terget protein interactions contributes to the development of many human diseases. Clinical investigations have demonstrated a correlation between up-regulated 14-3-3 protein levels and poor survival of cancer patients. Several studies have also suggested that 14-3-3 isoforms are differentially regulated in cancer and neurological syndromes. Especially, elevated amounts of the gamma isoform (14-3-3 Gamma) are found in the cerebrospinal fluid (CSF) of Creutzfeldt-Jakob disease (CJD) patients and could be as a specific marker of the disease as well as tau protein level.

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CircuLex

14-3-3 Gamma ELISA Kit

User’s Manual

For Research Use Only, Not for use in diagnostic procedures

Principle of the Assay

The CycLex Research Product CircuLex 14-3-3 Gamma ELISA Kit employs the quantitative sandwich enzyme immunoassay technique. An antibody specific for 14-3-3 Gamma has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and the immobilized antibody binds any 14-3-3 Gamma present. After washing away any unbound substances, a detection antibody specific for 14-3-3 Gamma is added to the wells, followed by binding with horseradish peroxidase (HRP) conjugated anti-IgG antibody. Following a wash to remove any unbound HRP conjugate, the remaining conjugate is allowed to react with the substrate H

2

O

2

- tetramethylbenzidine. The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured at 450 nm. The absorbance is proportional to the concentration of 14-3-3 Gamma. A standard curve is constructed by plotting absorbance values versus the units of 14-3-3 Gamma concentration of calibrators, and units of concentration of unknown samples are determined using this standard curve.

Summary of Procedure

Add 100 µL of 14-3-3 Gamma Standard Solutions and diluted samples to wells

Incubate at 25-30°C for 1 hour

Wash the wells

Add 100 µL of 1x 14-3-3 Gamma Detection Antibody

Incubate at 25-30°C for 1 hour

Wash the wells

Add 100 µL of 1x HRP conjugated Anti-IgG Antibody

Incubate at 25-30°C for 1 hour

Wash the wells

Add 100 µL of Substrate Reagent

Add 100 µL of Stop Solution

Measure absorbance at 450 nm

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CircuLex

14-3-3 Gamma ELISA Kit

User’s Manual

For Research Use Only, Not for use in diagnostic procedures

Materials Provided

All samples and standards should be assayed in duplicate. The following components are supplied and are sufficient for the one 96-well microplate kit.

Microplate: One microplate supplied ready to use, with 96 wells (12 strips of 8-wells) in a foil, zip-lock bag with a desiccant pack. Wells are coated with anti-14-3-3 Gamma antibody.

10X Wash Buffer: One bottle containing 100 mL of 10X wash buffer that contains Tween

®

-20

Sample/Standard Dilution Buffer: One bottle containing 50 mL of 1X Sample/Standard Dilution

Buffer; use for the dilutions of sample and 14-3-3 Gamma Standard. Ready to use.

14-3-3 Gamma Standard: One vial containing 64,000 AU of human 14-3-3 Gamma. Lyophilized.

100X 14-3-3 Gamma Detection Antibody: One vial containing 140 uL of anti-14-3-3 Gamma antibody.

Detection Antibody Dilution Buffer: One bottle containing 12 mL of Detection Antibody Dilution

Buffer; use for the dilution of 100X 14-3-3 Gamma Detection Antibody. Ready to use.

100X HRP conjugated Anti-IgG Antibody: One vial containing 140 uL of HRP (horseradish peroxidase) conjugated anti-IgG antibody. Ready to use.

Conjugate Dilution Buffer: One bottle containing 12 mL of Conjugate Dilution Buffer; use for the dilution of 100X HRP conjugated Anti-IgG Antibody. Ready to use.

Substrate Reagent: One bottle containing 20 mL of the chromogenic substrate, tetra-methylbenzidine

(TMB). Ready to use.

Stop Solution: One bottle containing 20 mL of 1 N H

2

SO

4

. Ready to use.

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CircuLex

14-3-3 Gamma ELISA Kit

User’s Manual

For Research Use Only, Not for use in diagnostic procedures

Materials Required but not Provided

Pipettors: 2-20 µL, 20-200 µL and 200-1000 µL precision pipettors with disposable tips

Precision repeating pipettor

Microcentrifuge and tubes for sample preparation

Vortex mixer

Microplate washer: optional (Manual washing is possible but not preferable)

Plate reader: capable of measuring absorbance in 96-well plates at dual wavelengths of 450 nm/540 nm. Dual wavelengths of 450/550 or 450/595 nm can also be used. The plate can also be read at a single wavelength of 450 nm, which will give a somewhat higher reading.

Software package facilitating data generation and analysis :optional

500 or 1000 mL graduated cylinder

Reagent reservoirs

Deionized water of the highest quality

Disposable paper towels

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CircuLex

14-3-3 Gamma ELISA Kit

User’s Manual

For Research Use Only, Not for use in diagnostic procedures

Precautions and Recommendations

• Allow all the components to come to room temperature before use.

• All microplate strips that are not immediately required should be returned to the zip-lock pouch, which must be carefully resealed to avoid moisture absorption.

• Do not use kit components beyond the indicated kit expiration date.

• Use only the microtiter wells provided with the kit.

• Rinse all detergent residue from glassware.

• Use deionized water of the highest quality.

• Do not mix reagents from different kits.

• The buffers and reagents in this kit may contain preservatives or other chemicals. Care should be taken to avoid direct contact with these reagents.

• Do not mouth pipette or ingest any of the reagents.

• Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are handled.

• Dispose of tetra-methylbenzidine (TMB) containing solutions in compliance with local regulations.

• Avoid contact with the acidic Stop Solution and Substrate Solution, which contains hydrogen peroxide.

• Wear gloves and eye protection when handling immunodiagnostic materials and samples of rat origin, and these reagents. In case of contact with the Stop Solution and the Substrate Solution, wash skin thoroughly with water and seek medical attention, when necessary.

Biological samples may be contaminated with infectious agents. Do not ingest, expose to open

wounds or breathe aerosols. Wear protective gloves and dispose of biological samples properly.

CAUTION: Sulfuric Acid is a strong acid. Wear disposable gloves and eye protection when

handling Stop Solution.

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CircuLex

14-3-3 Gamma ELISA Kit

User’s Manual

For Research Use Only, Not for use in diagnostic procedures

Sample Collection and Storage

CSF (Cerebrospinal fluid): Centrifuge CSF samples at 4°C for 15 minutes at 10,000 x g to remove insoluble impurities. Assay immediately or store the samples on ice for a few hours before assaying.

Aliquots of the samples may also be stored at below -70°C for extended periods of time. Avoid repeated freeze-thaw cycles.

Cell lysates: Prepare cell lysates. Assay immediately or store the samples on ice for a few hours before assaying. Aliquots of the samples may also be stored at below -70°C for extended periods of time.

Avoid repeated freeze-thaw cycles. (See below.)

Other biological samples: Remove any particulates by centrifugation and assay immediately or aliquot and store samples at below -70°C. Avoid repeated freeze-thaw cycles. (See below.)

Note: Only CSF and some cell lysate samples been used and validated for the measurement with this kit. For the measurements of other biological samples, please perform on users’ own responsibility. Users must judge the accuracy, reliability, usability, qualification, and recency of the results for themselves.

Preparation of cell lysates and other biological samples:

Some and excessive concentrations of ingredients in cell lysis buffers or other buffers may influence the measurement results of the kit although not fully investigated.

• Limitation of concentrations in the samples before dilution step

(In the case of 10-fold dilution. See “Sample Preparation” in the “Detailed Protocol”.)

Salts: e.g. NaCl; 250 mM or less

Non-ionic detergents: e.g. NP-40; 1.0% or less

Ionic detergents: e.g. SDS; 0.1% or less

Reducing agents: e.g. DTT; 0.5 mM DTT or less

Divalent cations: e.g. MgCl2; 5.0 mM or less

Chelates: e.g. EDTA; 5 mM or less pH: 6.5 – 8.5

• Non-denaturing buffers, which don’t contain ionic detergents, are recommended.

e.g. Cell Lysis Buffer: 0.1%NP-40, 250mM NaCl, 50mM Hepes-KOH pH7.0, 5mM EDTA

• An example of preparation procedure of cell lysates

1. Suspend 1x10

7

cells (100 mm dish sub-confluent) into 1ml of Cell Lysis Buffer.

2. Keep on ice for 15 to 30 min.

3. Vortex for 30 second.

4. Centrifuge at 4°C for 15 minutes 15,000 x g.

5. Collect the supernatants as cell lysates.

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CircuLex

14-3-3 Gamma ELISA Kit

User’s Manual

For Research Use Only, Not for use in diagnostic procedures

Detailed Protocol

The CycLex Research Product CircuLex 14-3-3 Gamma ELISA Kit is provided with removable strips of wells so the assay can be carried out on separate occasions using only the number of strips required for the particular determination. Since experimental conditions may vary, an aliquot of the

14-3-3 Gamma Standard within the kit, should be included in each assay as a calibrator. Disposable pipette tips and reagent troughs should be used for all liquid transfers to avoid cross-contamination of reagents or samples.

Preparation of Working Solutions

All reagents need to be brought to room temperature prior to the assay. Assay reagents are supplied ready-to-use, with the exception of 10X Wash Buffer and 14-3-3 Gamma Standard.

1. Prepare a working solution of 1X Wash Buffer by adding 100 mL of the 10X Wash Buffer to 900 mL of deionized (distilled) water. Mix well. Store at 4°C for two weeks or -20°C for long-term storage.

2. Reconstitute 14-3-3 Gamma Standard with 1.0 mL of Sample/Standard Dilution Buffer. The concentration of the 14-3-3 Gamma should be 64,000 AU/mL, which is referred as the Master

Standard. Make aliquots and store at below -70°C. Avoid repeated freeze-thaw cycles.

3. Prepare working solutions of 1X 14-3-3 Gamma Detection Antibody by 100-fold diluting the 100X

14-3-3 Gamma Detection Antibody with the Detection Antibody Dilution Buffer, e.g. 10 µL of

100X 14-3-3 Gamma Detection Antibody + 990 µL of Detection Antibody Dilution Buffer and mix well, and 1X HRP conjugated Anti-IgG Antibody by 100-fold diluting the 100X HRP conjugated

Anti-IgG Antibody with the Conjugate Dilution Buffer in the same manner. Prepare the solutions just before using and discard unused one.

4. Prepare Standard Solutions as follows:

Use the Master Standard to produce a dilution series (below). Mix each tube thoroughly before the next transfer. The 16,000 AU/mL Standard Solution serves as the highest standard solution (Std.1).

The Dilution Buffer serves as the zero standard solution (Blank).

Standard

Solutions

Volume of Standard Dilution Buffer

Final

Concentration

Std.1 150 µL of Master Standard (64,000 AU/mL) 450 µL 16,000 AU/mL

Std.2 300 µL of Std. 1 (16,000 AU/mL) 300 µL 8,000 AU/mL

Std.3

Std.4

Std.5

Std.6

Std.7

Blank

300 µL of Std. 2 (8,000 AU/mL)

300 µL of Std. 3 (4,000 AU/mL)

300 µL of Std. 4 (2,000 AU/mL)

300 µL of Std. 5 (1,000 AU/mL)

300 µL of Std. 6 (500 AU/mL)

-

300 µL

300 µL

300 µL

300 µL

300 µL

300 µL

4,000 AU/mL

2,000 AU/mL

1,000 AU/mL

500 AU/mL

250 AU/mL

0 AU/mL

Note: Do not use a Repeating pipette. Change tips for every dilution. Wet tip with Dilution Buffer before dispensing. Unused portions of Master Standard should and Standard Solutions be aliquoted and stored at below -70°C immediately. Avoid multiple freeze and thaw cycles.

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CircuLex

14-3-3 Gamma ELISA Kit

User’s Manual

For Research Use Only, Not for use in diagnostic procedures

Sample Preparation

• CSF samples may be required 5- and 40-fold dilution with Dilution Buffer.

• Cell lysates and other biological samples might be required 10- to 1,000-fold dilution, though it depends on each sample, with Dilution Buffer.

Assay Procedure

1. Remove the appropriate number of microtiter wells (strips) from the foil pouch and place them into the well holder. Return any unused wells to the foil pouch, refold, seal with tape and store at 4°C.

2. Dilute samples with Dilution Buffer. (See “Sample Preparation” above.)

3. Pipette 100 µL of Standard Solutions (Std1-Std7, Blank) and diluted samples in duplicates, into the appropriate wells.

4. Incubate the wells at 25-30°C for 1 hour.

5. Wash 4-times by filling each well with 1X Wash Buffer (350 µL) using a squirt bottle, multi-channel pipette, manifold dispenser or microplate washer.

6. Add 100 µL of 1X 14-3-3 Gamma Detection Antibody into each well.

7. Incubate the wells at 25-30°C for 1 hour.

8. Wash 4-times by filling each well with 1X Wash Buffer (350 µL) using a squirt bottle, multi-channel pipette, manifold dispenser or microplate washer.

9. Add 100 µL of 1X HRP conjugated Anti-IgG Antibody into each well.

10. Incubate the wells at 25-30°C for 1 hour.

11. Wash 4-times by filling each well with 1X Wash Buffer (350 µL) using a squirt bottle, multi-channel pipette, manifold dispenser or microplate washer.

12. Add 100 µL of Substrate Reagent into each well. (Avoid exposing the well to direct sunlight.

Covering the plate with e.g. aluminum foil is recommended.) Return Substrate Reagent to 4°C immediately after the necessary volume is removed.

13. Incubate the wells at 25-30°C for 10-20 minutes. This incubation time may be extended up to 30 minutes if the reaction temperature is relatively low.

14. Add 100 µL of Stop Solution to each well in the same order as the previously added Substrate

Reagent.

15. Measure absorbance in each well using a spectrophotometric microplate reader at dual wavelengths of 450/540 nm. Dual wavelengths of 450/550 or 450/595 nm can also be used. Read the microplate at 450 nm if only a single wavelength can be used. Wells must be read within 30 minutes of adding the Stop Solution.

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14-3-3 Gamma ELISA Kit

User’s Manual

For Research Use Only, Not for use in diagnostic procedures

Note-1: Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.

Note-2: Reliable standard curves are obtained when either O.D. value doesn’t exceed 0.2 for the zero standard solution (Blank), or 2.8 for the highest standard solution (Std.1).

Note-3: If the microplate reader is not capable of reading absorbance greater than the absorbance of the highest standard, perform a second reading at 405 nm. A new standard curve, constructed using the values measured at 405 nm, is used to determine 14-3-3 Gamma concentration of off-scale samples. The readings at 405 nm should not replace the on-scale readings at 450 nm.

Calculations

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Plot the optical density for the standards versus the units of concentration of the standards and draw the best curve. To determine the units of 14-3-3 Gamma concentration in each sample, first find the absorbance value on the y-axis and extend a horizontal line to the standard curve.

At the point of intersection, extend a vertical line to the x-axis and read the corresponding units of

14-3-3 Gamma concentration. If the samples have been diluted, the units read from the standard curve must be multiplied by the dilution factor.

1. The dose-response curve of this assay fits best to a sigmoidal 4-parameter logistic equation. The results of unknown samples can be calculated with any computer program having a 4-parameter logistic function. It is important to make an appropriate mathematical adjustment to accommodate for the dilution factor.

2. Most microtiter plate readers perform automatic calculations of analyte concentration. The calibration curve is constructed by plotting the absorbance (Y) of calibrators versus log of the known concentration (X) of calibrators, using the 4-parameter function. Alternatively, the logit log function can be used to linearize the calibration curve (i.e. logit of absorbance (Y) is plotted versus log of the known concentration (X) of calibrators).

Measurement Range

The measurement range is 250 AU/mL to 16,000 AU/mL. Any sample reading higher than the highest standard should be diluted with Dilution Buffer in higher dilution and re-assayed. Dilution factors need to be taken into consideration in calculating the human 14-3-3 Gamma concentration.

Troubleshooting

1. The 14-3-3 Gamma Standards and samples should be run in duplicate, using the protocol described in the Detailed Protocol. Incubation times or temperatures significantly different from those specified may give erroneous results.

2. Poor duplicates, accompanied by elevated values for wells containing no sample, indicate insufficient washing. If all instructions in the Detailed Protocol were followed accurately, such results indicate a need for washer maintenance.

3. Overall low signal may indicate that desiccation of the wells has occurred between the final wash and addition of Substrate Reagent. Do not allow the wells to dry out. Add Substrate Reagent immediately after wash.

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14-3-3 Gamma ELISA Kit

User’s Manual

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Reagent Stability

All of the reagents included in the CycLex Research Product CircuLex 14-3-3 Gamma ELISA Kit have been tested for stability. Reagents should not be used beyond the stated expiration date. Upon receipt, kit reagents should be stored at 4°C, except the reconstituted 14-3-3 Gamma Standard (Master

Standard) must be stored at below -70°C. The Microplate should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack.

For Research Use Only, Not For Use in Diagnostic or Therapeutic procedures

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CircuLex

14-3-3 Gamma ELISA Kit

User’s Manual

For Research Use Only, Not for use in diagnostic procedures

Assay Characteristics

1. Sensitivity

Five assays were evaluated and the minimum detectable dose (MDD) of 14-3-3 Gamma.

The MDD (defined as such a concentration of 14-3-3 Gamma giving absorbance higher than mean absorbance of blank

(

*

)

plus three standard deviations of the absorbance of blank: A blank + 3*SD blank) is better than 250 AU/mL of sample.

(

*

)

Standard/Sample Dilution Buffer is pipetted into blank wells.

Typical standard curve

2.000

1.800

1.600

1.400

1.200

1.000

Standard Curve

0.800

0.600

0.400

0.200

0.000

0 2,000 4,000 6,000 8,000 10,000 12,000 14,000 16,000

AU/mL

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14-3-3 Gamma ELISA Kit

User’s Manual

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2. Precision

Intra-assay Precision (Precision within an assay)

Three samples of Raji (human Burkitt's lymphoma cell line) cell lysate known concentration were tested twelve times on one plate to assess intra-assay precision.

• Intra-assay (Within-Run, n=12); CV= 1.04 - 4.35 %

14-3-3 Gamma conc. (AU/mL)

1

2

3

4

Low

2,271.90

2,193.38

2,201.48

2,228.84

Middle

4,298.20

4,210.38

4,192.23

4,164.24

High

9,317.24

8,882.98

8,879.64

8,585.26

5

6

7

8

9

10

11

12

MEAN

S.D.

CV%

2,222.21

2,236.05

2,243.23

2,210.10

2,227.44

2,244.41

2,260.28

2,244.70

2,232.00

23.12

1.04%

4,151.33

4,233.14

4,650.08

4,579.00

4,553.39

4,634.24

4,398.29

4,404.98

4,372.46

190.37

4.35%

8,610.61

8,680.03

8,691.03

8,606.62

9,401.07

9,274.35

8,624.60

8,677.69

8,852.59

305.29

3.45%

Inter-assay Precision (Precision between assays)

Three samples of Raji (human Burkitt's lymphoma cell line) cell lysate known concentration were tested in five separate assays to assess inter-assay precision.

• Inter-assay (Run-to-Run, n=5); CV= 3.85 - 6.05 %

14-3-3 Gamma conc. (AU/mL)

1

2

3

4

5

MEAN

S.D.

CV%

Low

2,269.34

2,312.22

2,333.07

2,553.49

2,290.10

2,351.64

115.33

4.90%

Middle

4,255.06

4,371.21

4,666.39

4,563.30

4,608.39

4,492.87

173.04

3.85%

High

8,571.22

9,430.12

9,755.37

9,432.60

8,546.55

9,147.17

553.15

6.05%

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14-3-3 Gamma ELISA Kit

User’s Manual

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3. Linearity

To assess the linearity of the assay, Raji (human Burkitt's lymphoma cell line) cell lysate samples containing 14-3-3 Gamma were serially diluted with the Standard/Sample Dilution Buffer to produce samples with values within the dynamic range of the assay.

Linearity

10,000

9,000

Low

8,000

Middle

High

7,000

6,000

5,000

4,000

3,000

2,000

1,000

0

0 0.25

0.5

0.75

1

Dilution Ratio

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14-3-3 Gamma ELISA Kit

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Example of Test Results

Fig.1 Measurement of concentrations of 14-3-3 Gamma in CSF of patients with CJD

(

*

)

Units of 14-3-3 Gamma concentration in CSF from patients with CJD and non-CJD neurological disorders were measured by the CircuLex 14-3-3 Gamma ELISA Kit.

AU/mL

200,000

150,000

100,000

50,000

0

0

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14-3-3 Gamma ELISA Kit

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Fig.2 Detailed analysis of 14-3-3 Gamma level in CSF of CJD and other neurological disorders patients in the range of lower concentration

(

*

)

14-3-3 Gamma levels in CSF from CJD patients (n=124) and other neurological disorders patients

(n=99) were measured by the CircuLex 14-3-3 Gamma ELISA Kit and the result was shown at lower concentration.

AU/mL

10,000

87 Cases

8,000

6,000

4,000

2,000

0

0 2 4 6 8 10 12 14 16

(The numbers of each disorder are shown in parentheses.)

Abbreviations of disorders

CJD: Creutzfeldt-Jakob disease

DAT: Dementia of Alzheimer’s type

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14-3-3 Gamma ELISA Kit

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CVD: Cerebral Vascular Disorder

PD: Parkinson’s disease

PSP: progressive supranuclear palsy

FTLD: frontotemporal lobular degeneration

HD: Huntington’s disease

CBD: corticobasal degeneration

ALS: amyotrophic lateral sclerosis

MELAS: Mitochondrial myopathy, Encephalopathy, Lactic Acidosis, Stroke-like episodes

PCD: paraneoplastic cerebellar degeneration

LEMS: Lambert-Eaton myasthenic syndrome

MCI: mild cognitive impairment

(

*

)

The data in Fig.1 and Fig.2 were kindly provided by Dr. Yuki Matsui

1

and Dr. Katsuya Satoh

2

.

(

1

Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical Sciences,

Fukuoka University,

2

Department of Molecular Microbiology and Immunology,Graduate School of

Biomedical Science, Nagasaki University. See the reference (8) for details.)

References

1. The 14-3-3 brain protein in cerebrospinal fluid as a marker for transmissible spongiform encephalopathies. Hsich G, Kenney K, Gibbs CJ, Lee KH, Harrington MG. (1996) N Engl J Med.

335(13):924-30.

2. Isoform pattern of 14-3-3 proteins in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease. Wiltfang J, Otto M, Baxter HC, Bodemer M, Steinacker P, Bahn E, Zerr I, Kornhuber J,

Kretzschmar HA, Poser S, Rüther E, Aitken A. (1999) J Neurochem. 73(6): 2485-90.

3. An enzyme-linked immunosorbent assay to quantify 14-3-3 proteins in the cerebrospinal fluid of suspected Creutzfeldt-Jakob disease patients. Kenney K, Brechtel C, Takahashi H, Kurohara K,

Anderson P, Gibbs CJ Jr. (2000) Ann Neurol. 48(3):395-8.

4. Determination of 14-3-3 protein levels in cerebrospinal fluid from Creutzfeldt-Jakob patients by a highly sensitive capture assay. Peoc'h K, Schröder HC, Laplanche J, Ramljak S, Müller WE. (2001)

Neurosci Lett. 301(3):167-70.

5. CSF tests in the differential diagnosis of Creutzfeldt-Jakob disease. Sanchez-Juan P, Green A,

Ladogana A, Cuadrado-Corrales N, Sáanchez-Valle R, Mitrováa E, Stoeck K, Sklaviadis T,

Kulczycki J, Hess K, Bodemer M, Slivarichová D, Saiz A, Calero M, Ingrosso L, Knight R,

Janssens AC, van Duijn CM, Zerr I. (2006) Neurology. 67(4):637-43.

6. Total tau protein in cerebrospinal fluid and diffusion-weighted MRI as an early diagnostic marker for Creutzfeldt-Jakob disease. Satoh K, Shirabe S, Tsujino A, Eguchi H, Motomura M, Honda H,

Tomita I, Satoh A, Tsujihata M, Matsuo H, Nakagawa M, Eguchi K. (2007) Dement Geriatr Cogn

Disord. 24(3):207-12. (Epub 2007 Aug 10.)

7. The role of cerebrospinal fluid proteins as early diagnostic markers for sporadic Creutzfeldt-Jakob disease. Pennington C, Chohan G, Mackenzie J, Andrews M, Will R, Knight R, Green A. (2009)

Neurosci Lett. 455(1):56-9. (Epub 2009 Mar 5.)

8. High sensitivity of an ELISA kit for detection of the gamma-isoform of 14-3-3 proteins: usefulness

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For Research Use Only, Not for use in diagnostic procedures

in laboratory diagnosis of human prion disease. Matsui Y, Satoh K, Miyazaki T, Shirabe S, Atarashi

R, Mutsukura K, Satoh A, Kataoka Y, Nishida N. (2011) BMC Neurol. 11: 120.

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This kit was developed and produced by CycLex Co., Ltd., Nagano, Japan, under license from

Nagasaki University, Nagasaki, Japan.

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