Instructions for use
a-Amylase Saliva
SA P-6900
α-Amylase Saliva
Colorimetric test for the quantitative determination of α-Amylase in saliva
1.
Precautions
Warning notices and general information
Do not use any components of the kit if they are past the expiration date. Do not mix test reagents of
different charges! Avoid microbial contamination of the test reagents and washing water. Observe the
incubation periods and washing instructions. Never pipette by mouth and avoid contact of reagents and
specimens with skin. No smoking, eating or drinking in areas where samples or kit test tubes are handled.
When working with kit components or samples, always wear protective gloves and wash your hand
horoughly as soon as you have finished the work. Avoid spraying of any kind! All in the protocol indicated
volumes (pipetting volumes and pretreatment steps) has to be performed according to the protocol.
Optimal test results are only obtained when using calibrated pipettes.
2.
Principle of the test
The human α-Amylase hydrolyses the 2-chloro-4 nitrophenyl-α-maltotrioside (CNP-G3) in glucose
polymers and short-chain p-nitrophenyl-oligosaccharide with formation of 2-chloro-4-nitrophenol (CNP).
The increase of the extinction is evaluated spectrophotometrically at 405 nm and is proportional to αamylase activity in the sample.
3.
Clinical significance
Amylase is the name given to enzymes that break down starch. They are classified as saccharidases,
enzymes that cleave polysaccharides.
Although the amylases are designated by different greek letters, they all act on α-1,4 glycosidic bonds.
The α-amylases are calcium metalloenzymes, completely unable to function in the absence of calcium.
The α-amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from
amylose, or maltose, glucose and "limit dextrin" from amylopectin. In animals, it is a major digestive
enzyme.
Although found in many tissues, the α-amylase is most prominent in pancreatic juice and saliva which
each have their own isoform.
Salivary α-amylase breaks starch down into maltose and dextrin. This form is also called ptyalin. Ptyalin
will break large, insoluble starch molecules into soluble starches (amylodextrin, erythrodextrin,
achrodextrin) producing successively smaller starches and ultimately maltose. Ptyalin acts on linear
α(1,4) glucosidic linkages, but compound hydrolysis requires an enzyme which acts on branched
products. Salivary amylase is inactivated in the stomach by gastric acid.
Pancreatic α-amylase randomly cleaves the α(1-4)glycosidic linkages of amylose to yield dextrin, maltose
or glucose molecules.
4.
Storage and stability
Store the reagents at 2 - 8 °C until expiration date. Do not use components beyond the expiration date
shown on the kit labels. Do not mix various lots of any kit component within an individual assay.
5.
Sample collection and storage
For sample collection is advised to use glass or no recycled polypropylene tubes and straws.
Do not use polysterene tubes or commercially available devices for the saliva collection to avoid false
results.
Let the saliva flow down through the straw into the tube, the samples should be stored at -20 °C.
5.
Contents of the kit
SA P-6913
SA P-6926
BA D-0032
6.
Assay Buffer
Concentrate (5x)
Release Buffer
Microtiter Plate
1 x 50 ml
concentrate, dilute content with dist. water
to a final volume of 250 ml
1 x 32 ml
ready for use
1 x 96 wells 12 strips, 8 wells each, break apart
Additional materials and equipment required but not provided in the kit
- Calibrated variable precision micropipettes (e.g. 10-100 µl / 100-1,000µl)
- Microtiter plate washing device
- EIA reader capable of reading absorbance at 450 nm
- Centrifuge capable of at least 3.000 x g
- Absorbent material for blotting the strips
- Distilled water
- Vortex mixer
Revision date: 30-Oct-2009
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7.
Test procedure
Allow reagents and samples to reach room temperature.
7.1
Preparation of acylation solution
Assay Buffer
Dilute the 50 mL Assay Buffer Concentrate with distilled water to a final volume of 250 mL.
Storage: until expiration date (see vial label).
7.2
Sample preparation
1.
Thaw the frozen saliva samples and centrifuge for 15 minute at 3000 x g.
2.
Dilute 10 µl of supernatant in 1 ml of diluted Assay Buffer.
3.
Shake 5 min at RT (18-25°C) on an orbital shaker (600 - 900 rpm).
4.
Use 10 µl of supernatant for the colour reaction.
7.3
Colour reaction
1.
Pipette 10 µl of diluted samples into the respective wells of the Microtiter Plate.
2.
Pipette 10 µl of distilled water into the wells with blank.
3.
Add 300 µl of Release Buffer to all wells.
4.
Incubate 3 min at 37°C.
If you should manually dispense a high number of samples, it is advised to use to the maximum four
strips for each tests.
5.
Read the absorbance of the solution in the wells two times, first after 1 minute and second after 5
minutes using a microplate reader set to 405 nm.
8.
Calculation of results
Calculate ∆A/min.= [(A5 min –A1 min) / 4]
Calcul: α-Amylase (U/mL) = ∆A/min. x 253
8.1
Quality control
Each laboratory should assay controls at normal, high and low levels range of α-amylase for monitoring
assay performance. These controls should be treated as unknowns and values determined in every test
procedure performed. 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 be consistent 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.
8.2
Expected values
The reference ranges given below should only be taken as a guideline. It is recommended that each
laboratory should establish its own normal values.
Adults (Normal)
80.0 U/mL
Absolute Range:
4 – 400 U/mL
10.
Assay characteristics
Analytical Sensitivity (Limit of Detection)
The lowest detectable concentration of α-amylase that can be distinguished from the zero standard is 2,5 U/mL
at the 95 % confidence limit.
Precision
Intra-Assay
3 different levels of controls CV (<1.5 %) n = 16
Revision date: 30-Oct-2009
Inter-Assay
3 different saliva samples in 2 different lots
CV (<1.5 %)
3/4
100
90
%B/Bo
80
70 11.
60
Limitation of Procedure
Assay Performance
It is important that the time of reaction in each well is held constant for reproducible results. Plate
readers measure the OD vertically. Do not touch the bottom of the wells.
Interpretation of results
If computer controlled data reduction is used to calculate the results of the test, it is imperative that the
predicted values for the calibrators fall within 10% of the assigned concentrations.
12.
Suggestions for possible errors
No colorimetric reaction
- no Reagent A pipetted
- contamination of Reagent A
- errors in performing the assay procedure (e.g. accidental pipetting of reagents from the wrong vial,
etc.)
Too low reaction (too low ODs)
- incorrect Reagent A (e.g. not from original kit)
- incubation time too short, incubation temperature too low
Too high reaction (too high ODs)
- incorrect Reagent A (e.g. not from original kit)
- incubation time too long, incubation temperature too high
Unexplainable outliers
-
contamination of pipettes, tips or containers, CV% too high within-run
reagents and/or strips not pre-warmed to room temperature prior to use
incubation conditions not constant (time, temperature), CV % too high between-run
too long dispense time
person-related variation
13.
Waste Management
Reagents must be disposed off in accordance with local regulations.
14.
Literature
Tietz NW. Clinical Guide to Laboratory Tests, 3rd ed. Philadelphia: WB Saunders Company: 46-49 (1995)
(IFCC) “Approved Recommendation on IFCC Methods for the measurement of catalytic concentration of
Enzymes” _Part 9
Clin Chem Lab Med 36(3): 185 –203 (1998)
Symbols:
Storage
temperature
Manufacturer
Contains sufficient for
<n> tests
Expiry date
Batch code
For in-vitro diagnostic
use only!
Consult instructions
for use
Content
CE labelled
Caution
Catalogue
number
For research use
only!
Revision date: 30-Oct-2009
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