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ANALIS

Isoamyl™ kit for Beckman Coulter Paragon® Electrophoresis - GB: Instructions for use

QUOTATION
INFORMATION
QUOTATION
INFORMATION
DESCRIPTION TECHNICAL DESCR.
DESCRIPTION

REVISION 1

1. General
2. Intended use
3. Principle of the test
4. Contents
5. Configuration of instrument
6. Other reagent and material needed
7. Training
8. Reagent preparation and storage
9. Type of specimen
10. Instrument preparation
11. Sample preparation
12. Operations
13. Calibration
14. Analysis of results
15. Calculation
16. Imprecision and sensitivity
17. Reference range
18. Interference
19. Quality Control
20. References

1. General

1. Date: september-03 - Revision: 01

EURO PN 844111001
PN 10-004050
100 tests per kit

figure 2

2. Intended use

For the separation of α-amylase isoenzymes in serum by agarose gel electrophoresis.

The Isoamyl kit provides a borate buffer at pH 6.95 for the electrophoretic separation on agarose gels. A Phadebas® (Pharmacia Diagnostics) reagent is used as substrate to obtain the stained fractions.

The substrate is a water-insoluble cross-linked starch polymer carrying a blue dye. It is hydrolyzed by α-amylase to form water soluble blue fragments.

4. Contents

Electrophoresis Buffer (TRIS-borate 1.5 mol/L) 2x200mL (PN 10-004051)

Equilibration Buffer (TRIS-borate 0.6 mol/L) 1x200mL (PN 10-004052)

Isoamyl Substrate(Phadebas® Pharmacia Diagnostics) 30 tablets (PN 10-004053)

Agarose gels : 1x10

Measure 25 mL 1x

Plastic pliers 1x

Liquipipette Graduat (3mL) 1x

Sample Template (purple) 1x10

Template blotters 1x10 (19x126mm)

Gel Blotters 20 (83 x 101 mm)

5. Configuration of instrument

-Beckman Coulter's Paragon® Electrophoresis System.

6. Other reagent and material needed

  • Pipettes.
  • Graduated cylinder.
  • Deionised water.
  • Beckman Coulter's Paragon® Electrophoresis System.

INSTRUMENT (OPTIONAL)

A densitometer capable of scanning three inch by four inch film at 600 nm. Refer to manufacturer's instructions for operation and calibration procedures.

7. Training

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

8. Reagent preparation and storage

Electrophoresis Buffer Mix 400mL of buffer with 600mL of deionised water.

Buffer is stable in closed container for 60 days.

Substrate: 3 tablets of Phadebas® in 6 mL deionised water.

9. Type of specimen

Serum samples should be collected in the manner normally used for any laboratory test. Freshly drawn serum from a fasting individual is preferred, but samples may be stored at 4°C for 72 hours for later analysis. Refrigerated samples should be reactivated by incubation at 37°C for 30 minutes prior to analysis. Samples showing evidence of hemolysis should not be used.

10. Instrument preparation

See Paragon® Electrophoresis System.

REUSABLE FOR 10 GELS

11. Sample preparation

Serum samples are not diluted.

12. Operations

Equilibration

  • Blot gel
  • Pour 20 mL equilibration buffer in Gel Tray (ready to use)
  • Place the gel, agarose down on the liquid.
  • Wait for 30 minutes (don't close the container)

Application

  • Blot gel
    AVOID BRINGING AMYLASE ON THE GEL WITH THE FINGERS OR SALIVA.
  • Locate sample template
    Refer to the picture for template alignment

figure 3

  • Dispense sample 5 µL
    (samples with amylase activity higher than twice the upper normal value have to be applied for a shorter time: 1 to 3 min.)
  • Wait for 5 minutes
  • Gently blot template with 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 for 40 minutes at 150 volts

Detection

  • Place gel in incubation box
  • Distribute evenly on top of the gel with the liquipette the suspension of 3 tablets of substrate in 6 mL of deionised water.
  • Cover the box
  • Incubate for one hour at 45°C without moving the box
  • Rinse gel under tap water.

13. Calibration

The densitometer's performance should be verified according to the manufacturer's instructions.

14. Analysis of results

The amylase isoenzyme pattern may be visually interpreted on the wet gel or may be scanned wet on a densitometer at 600 nm to get the relative percentage of each amylase isoenzyme.

15. Calculation

Fractions can be recalculated from the total amylase activity.

For example:

Total activity 35 U/L

P2 fraction of 12% gives 4U/L

S2 fraction of 58% gives 20U/L

S2 fraction of 27% gives 10U/L

S4 fraction of 3% gives 1U/L

16. Imprecision and sensitivity

For N=10 and amylase= 84 U/L

Within gel

Between gel

U/L

%CV

U/L

%CV

P2

18.1

1.4

18.4

3.8

S2

46.4

2.1

46.6

2.9

S3

16.1

3.5

15.9

9.1

S4

3.3

14.6

3.3

20.3

Sensitivity allows the detection of fractions with activities as low as 1 U/L.

17. Reference range

Each laboratory should establish its own reference range to conform to patient population.

Reference values established from 1300 healthy individuals:

Total amylase activity (*)

24 U/L

72 U/L

P2

9

32

P3

0

2

S2

7

42

S3+S4

1

6

(*) total amylase activity was determined at 25°C with Wako reagents.

18. Interference

None known.

19. Quality Control

It is recommended that fresh normal sera or commercially available quality control sera be included in each electrophoretic procedure.

20. References

Bibliography.

Van Hoof V, Michielsen P, Roelandt R et al.

Evaluation of the Isoamyl system and its application in a prospective study of serum amylase after endoscopy. Biologie Prospective. Comptes rendus du 7ième colloque de Pont-à-Mousson,1989:261-4

Van Hoof V, Geeraerts L, Van Meirvenne H, Van Mullem M, Chevigné R, Van Campenhout C, Lepoutre L.

Reference Values for Amylase Isoenzymes Determined by Agarose Electrophoresis. Clin Chem 1990;36:1151-2.

Michielsen P, Van Hoof V, Lepoutre L, Van Maercke Y.

Klinische Toepassingen van de Isoamylasenbepaling. Tijdschr. voor Geneeskunde 1990;46:1251-6

Analis Brochures:

B 53 E9702 α-Amylase Isoenzymes separation by agarose gel electrophoresis.

B 54 D9703 Trennung der α-Amylase-Isoenzyme auf Agarosegelen.

TECHNICAL DESCR.
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