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　　　　　　　　　(when 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

*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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β-葡聚糖试剂盒
Manufacturer: Wako Pure Chemical Industries, Ltd.
BGSTAR A Kit Product Code:639-01051 Package:20tests Storage:keep at 2-10 degree C
BGSTAR B Kit Product Code:636-01061 Package:20tests Storage:keep at 2-10 degree C

Summary
This product is for research use only. Do not administer it to human.
Amebocyte lysate of horseshoe crabs is highly reactive to endotoxin and has been used as an endotoxin detecting reagent in various fields. The lysate is also reactive to beta-glucan. A purified fraction is prepared when the endotoxin reactive factors and coagulogen are removed from the lysate. The main reagent in this kit is the purified fraction added with the chromogenic synthetic substrate. This reagent is not reactive to endotoxin and specifically detects beta-glucan.

Principle of the test
The factor G in the purified fraction of the lysate reacts with (1->3)-beta-D-glucan. Then the factor G cascade reactions are activated. The clotting enzyme produced in the cascade reactions reacts with the substrate to release p-nitroaniline. The released p-nitroaniline is coupled with the diazocoupling reagent. Then the absorbance of the reaction mixture is measured. The amount of the p-nitroaniline is quantitatively determined.
BGSTAR can be used on a microplate reader by the endpoint method or the kinetic method. In the endpoint method, higher sensitivity can be obtained as the released p-nitroaniline is coupled with the diazo reagent and the absorptivity is enhanced. In the kinetic method, an operator can leave after starting the reaction and the measurable range is wider.

Ordering information
639-01051 BGSTAR A KIT
Contents: (1) Main Reagent, 1 vial x 2.2 mL, (2) Buffer Solution, 1 vial x 3.0 mL, (3) Standard Curdlan, 1 vial x approx. 1,000 pg, (4) beta-Glucan-Free Water, 1 vial x 15 mL, (5) Diazocoupling Reagent, 1 vial x 4.8 mg, (6) Solvent for Diazocoupling Reagent 1, 1 vial x 12 mL, (7) Diazocoupling Reagent 2, 1 vial x 36 mg, (8) Diazocoupling Reagent 3, 1 vial x 8.4 mg

636-01061 BGSTAR B KIT
Contents: (1) Main Reagent, 1 vial x 2.2 mL, (2) Buffer Solution, 1 x 3.0 mL

Note: With one vial of Main Reagent, 40 tests can be performed on a microplate reader. For the kinetic method, Diazocoupling Reagents are not used.

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Immunology - Monoclonal Antibodies
A. Monoclonal Antibodies
Anti Human COX-1, MAb [Clone: hPES 01]
012-18511 500ug (500uL)
-20 degree C, D/I, Liquid
Prepare in PBS solution contqaining no preservative.
Subclass: IgG
Specificity: Reacts with COX-1 of human and sheep.
Working Dilution: Westernblot 1 : 200

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Anti Human COX, Rabbit
015-18501 for 100uL
-20 degree C, Lyophilized
Reconstitute in 100uL distilled water to make solution
containing 3% BSA in 0.9% sodium chloride.
Specificity: Reacts with COX-1 of human, rat, mouse, bovine
and sheep, and COX-2 of human, rat and mouse.
Working Dilution: Westernblot 1 : 1,000 ~ 1 : 2,000

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Anti Soluble Guanylate Cyclase (sGC), MAb
[Clone: mAB3221]
019-17801 20ug (40uL)
-20 degree C, D/I, Liquid
Prepared from culture supernatant and prepared in glycine-
Tris solution (pH 7.4). Contains no preservatives and stabilizers.
Isotyper: IgG1
Specifically reacts with rat, bovine and human sGC, and
strengens in the reactivity on activation of sGC by NO,
probably, due to the conformational changes of the enzyme
and its associated antibody-antigen complex.
Working Dilution:
Westernblot 1 : 5,000
Immunofuluorescence 1 : 250

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Anti soluble Guanylate Cyclase (sGC), MAb,
NO insensitive [Clone: mAB28131]
017-18201 20ug (40uL)
-20 degree C, D/I, Liquid
Prepared from culture supernatant and prepared in glycine-
Tris solution (pH 7.4). Contains no preservatives and stabilizers.
Isotype: IgG1
Spercifically reacts with rat, bovine and human beta-subunit of
sGC, but not strengthened in the reactivity on activation of
sGC by NO.
Working Dilution:
Westernblot 1 : 5,000
Immunofluorescence 1 : 250

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Anti Rat iNOS, MAb [Clone: A2]
012-18631 for 1mL (2ug/vial)
-20 dgree C, Lyophilized
Lyophilized form in 20 mmol/L HEPES solution (pH 7.3) containing
0.1% BSA as a stabilizer.
Subclass: IgG1
Spercificity: Reactive with iNOS of rat and mouse.
Working Dilution:
Westernblot 1 : 1,000 ~ 1 : 3,000
Immunofluorescence 1 : 1

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Anti Rat nNOS, MAb [Clone: C7]
019-18641 for 1mL (40ug/vial)
-20 dgree C, Lyophilized
Lyophilized form in 20 mmol/L HEPES solution (pH 7.3) containing
0.1% BSA as a stabilizer.
Subclass: IgG1
Specificity: Specific for rat nNOS. Does not react with eNOS and iNOS
Working Dilution:
Westernblot 1 : 500
Immunofluorescence 1 : 10