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ANALIS

Protur™ B.J. kit for Beckman Coulter Paragon® Electrophoresis - GB: Instructions for use

QUOTATION
INFORMATION
QUOTATION
INFORMATION
DESCRIPTION
DESCRIPTION

1. General

Date: 4 sep-03 - Revision: 01
EURO PN 844111005
PN 10-004080
20 tests per kit

figure 1

2. Intended use

The kit is intended for the immunofixation of proteins and Bence Jones proteins in non concentrated urine.

3. Principle of the test

Proteins in an electric field migrate toward one of the electrode poles at varying rates. Migration separates the proteins. When specific antisera combine with specific proteins, antigen-antibody precipitation will occur.

The Protur B.J.™ kit contains components for the electrophoretic separation of proteins in non concentrated urine in a buffered agarose gel.

After electrophoresis, antiserum is applied.

4. Contents

Equilibration Buffer(0.44 mol/L TRIS-borate buffer) 1x200mL (PN 10-004061)

Electrophoresis Buffer (2.22mol/L TRIS-borate buffer) 1x200mL (PN 10-004062)

Protur Dye 1x100mL (PN 10-004064)

Protur Plus antiserum (Anti- IgG,A,M - kappa, -lambda) 3x1.6mL (PN 10-004081)

Contains Sodium Azide

Agarose gels : 10

Sample template 10

Antiserum template 10

Measure (25mL) 1x

Template blotters 10 (19 x 126 mm)

Gel blotters 50 (83x101mm)

Drying blottesr 40 (76 x 101 mm)

5. Configuration of instrument

- See Beckman Coulter's Paragon® Electrophoresis System.

6. Other reagent and material needed

- Pipettes.

- Graduated cylinder.

- Distilled water.

- Acid-alcohol solution

- Saline solution

- Beckman Coulter's Paragon® Electrophoresis System.

7. Training

The operator should be familiar with Beckman Coulter's Paragon Electrophoresis System.

8. Reagent preparation and storage

Agarose Gels: Just prior to use, carefully remove a Gel from the foil package. Gels should be stored at room temperature, 8°C to 30°C, until expiration date. Improper temperature or storage conditions can result in atypical migrations. DO NOT STORE GELS NEAR HEAT EMITTING DEVICES. For example: CRT, computer, or table top instrument. DO NOT REFRIGERATE OR FREEZE.

Protur Plus™ Equilibration Buffer: READY FOR USE.

Protur Plus™ Electrophoresis Buffer Mix 200mL of buffer with 800mL of deionised water.

Buffer is stable in closed container for 60 days.

Protus Plus™ Staining Solution:

  • prepare acid-alcohol solution: 500 mL deionised water, 300 mL methanol, 200 mL acetic acid.
  • mix 100 mL Dye with 200 mL acid-alcohol solution.

Saline solution:

8.5 g NaCl in 1 L deionised water.

Store reagents at room temperature

9. Type of specimen


Use fresh unconcentrated urine.

10. Instrument preparation

See Paragon® Electrophoresis System.

Place in wet processor:

  • container 1: dye solution

  • container 2: deionised water

  • container 3: acid alcohol solution

  • container 4: acid alcohol solution

  • container 5: deionised water

REUSABLE FOR 10 GELS

11. Sample preparation

Fresh urine sample.

12. Operations

Equilibration

- Blot gel

- pour 20 mL equilibration buffer in Gel Tray.

- Place the gel, agarose down on the liquid.

- Wait for 30 minutes (don't close the container)

Application

- Blot gel

- Locate sample template

- Dispense sample 5 µL

- Wait 5 minutes

- Gently press fingertip across template blotter

- Remove and discard blotter and template

Electrophoresis

- Pour 45 mL electrophoresis buffer in each compartment of the cell

- Locate gel on bridge

- Separate 35 minutes at 100 volts

Detection

- Blot gel

- Locate Antiserum template

- Apply 80 µL of antiserum

- Place gel in incubation box

- Close box and incubate at room temperature for 30 minutes

- Remove the gel place in gel frame and rinse in saline solution for 10 minutes

- Place gel on press dryer base (agarose side up)

- Place a second gel blotter, moistened with saline, onto gel surface followed by 2 drying drying blotter and the press dryer top plate. Press dry for 10 minutes.

- repeat previous step

- Place gel in gel frame and wipe excess solution

- Place gel in drying oven until dry (maximum 90°C)

- Process gel in

o stain solution 3 min.

o deionised water 2 min.

o acid-alcohol solution 1 min.

o acid-alcohol solution 15 sec. (or longer until a clear background is obtained)

o deionised water 5 sec..

o Wipe excess solution

- Place gel in drying oven until dry (maximum 90°C)

- Evaluate gel

13. Calibration

Not applicable.

14. Analysis of results

figure 2

Evaluate gels for precipitations.

15. Calculation

No calculations are required.

16. Imprecision

Not applicable.

17. Reference range

Not applicable.

18. Interference

None known.

19. Quality Control

Use control urine.

20. References

1. Chopin N. Exploration biologique des protéinuries. Classification, méthodes d'investigation et interprétation. Spectra Biologie 1993;1:49-53
2. Chopin N, Le Carrer D. Etude de la sélectivité des protéinuries : description d'un nouvel index. Rev Fr Lab 1994;269:103-7
3. Chopin N, Le Carrer D. Exploration biologique des tubulopathies : mise à jour. Rev Fr Lab 1994;269:109-12
4. Le Carrer D, Nicolas A, Ducasse L. L'analyse des protéinuries au laboratoire de biologie en 1992. Rev Fr Lab 1992;245:41-7
5. Le Carrer D, Chopin N. Profil protéique urinaire : proposition d'un protocole d'exploration biologique des protéinuries. Rev Fr Lab 1994;269:29-37
6. Pourignaux F, Brohet M, Louis P, Chevigné R. News methods for detecting and identifying monoclonal and BJ proteins in urine without concentration. Sixth Int Symp of Nephrology, Montecatini Terme, Italy, June 1989
7. Van Hoof V, Chevigné R, Van Campenhout C, Lepoutre L. Evaluation of the Protur/Microprotur system  Beckman-Analis-Belgium for the protein electrophoresis of non-concentrated urine samples. 7ieme Coll Biologie Prospective, Pont à Mousson, 1988

Brochures:

B 25 E92 Analysis of Urinary Proteins by Electrophoresis without Concentration.

B 25a E97

B 46 D95 Urinproteinelektrophorese von nicht einkonzentrierten Urinproben.

B 38 F94 L'analyse des protéines urinaires par électrophorèse sans concentration.

B 39 E94 Urinary Proteins analysis by electrophoresis without concentration.

B 39a E96

B 45 D95 Elektrophoretische Urinproteinanalyse ohne Einkonzentrierung.

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