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磷脂試劑盒
Phospholipids C （reagent kit） - COD DAOS method

Summary and explanation of the test:
The Wako phopholipids C is an enzymatic colorimetric method using N-Ethyl-N-（2-hydroxy-3-sulfopropyl）-3,5-dimethoxyaniline （DAOS）. This is reagent has high specificity and is not interfered with ascorbic acid and bilirubin in serum.
Principle of the method:
As shown in the chemical reactions below, Phospholipids in serum （lecithin, lysolecithin and sphingomyelin） are hydrolyzed to free choline by phospholipase D. The liberated choline is subsequently oxidized to betaine by choline oxidase with the simultaneous production of hydrogen peroxide. The hydrogen peroxide which is produced quantitatively, oxidatively couples 4-aminoantipyrine and DAOS to yield a chromogen with maximum absorption at λ=600nm

Reagents 　　　　　　　　　　　　　　　　　　　　　　　　　　（For 120 tests）
（1） Buffer　　　Good's buffer （pH 7.5）　　　50 mmol / L　　　　　8 x 50 mL
（2） Color Reagent　　　　　　　　?。╳hen reconstituted）　　　　　8 x for 50 mL
　　Phospholipase D　　　　　　　　　　　0.47 U/mL
　　Choline Oxidase　　　　　　　　　　　　2.0 U/mL
　　Peroxidase 　　　　　　　　　　　　　　4.2 U/mL
　　DAOS 　　　　　　　　　　　　　　　　0.77 mmol/L
　　4-Aminoantipyrine 　　　　　　　　　　0.24 mmol/L
　　Ascorbic acid oxidase 　　　　　　　　　3.9 U/mL
（3） Standard Solution 2 x 10 mL
　　（Corresponding to 300 mg/dL of phospholipids contents）
　　Choline Chloride 　　　　　　　　　　　　54 mg/dL

Warnings and Precautions
For In Vitro Diagnostic Use
Not to be used internally in humans or animals
Do not mix or use the reagents from one test unit with those of another test unit which has a different lot number.
Do not use reagents pat the expiration date stated on each reagent container label.

Specimen Collection
Serum
Ascorbic acid. Bilirubin and hemolysis do not affect the phospholipids measurement.
The Wako method measures the 3 kinds of phospholipids in serum （lectithin, lysolecithin and sphingomyelin） but does not measure cephalin.
Materials required but not supplied
Test tubes
Pipettes
Water bath, capable of maintaining 37 degree C
Spectrophotometer or Colorimeter, capable of measuring absorbance at 600 nm.
Preparation of reagent
COLOR REAGENT SOLUTION
Dissolve the contents of one vial （for 50mL） of Color Reagent in one bottle （50 mL） of Buffer. This solution is stable for 1 week at 2-10 degree C.
Procedure
Sample （S） Standard （Std） Blank （Bl）
Pipette into a test tube
Sample Sample 0.02 mL Standard 0.02 mL - *1
COLOR REAGENTSOLUTION 3.0 mL 3.0 mL 3.0 mL
Mix incubate for 5 minutes at 37 degree C and measure the absorbance of S and Std against Bl at 600 nm.
Absorbance （600nm） A S A Std Blank
*1: The omission of 0.02 mL of water does not significantly affect the absorbance measured.
*2: When measure with two wavelength: λ1/λ2 = 600/700 nm.

CAUTION
The color reaction finishes in about 3 minutes and the 5 minutes of incubation at 37 degree C is enough for the reaction. The extended incubation will cause the color deterioration. Please avoid keeping the test tube warm longer than 15 minutes. The color is stable for 1 hour in room temperature.

Results
（Calculation） A S: Absorbance of sample
Phospholipids （mg/dL） = （A S / A Std ） x 300 A Std: Absorbance of standard
Limitations of the procedure
The calibration curve is linear up to 1000 mg/dL of phospholipids content.
Expected Values
Serum: 145 - 257 mg/dL
Since expected values are affected by age, sex, diet, geographical location and other factors, each laboratory should establish it own expected values for this procedure.
Additional recommended products
Code No. 410-00101 Control Serum I （Normal） 10 x for 5 mL
Code No. 416-00201 Control Serum II （Abnormal） 10 x for 5 mL
Reference
Takayama M., Itoh S., Nagasaki T. and Tanimizu I.: Clin. Chim. Acta. 79, 93-98 （1977）
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