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DISCRETPAK™ REAGENTS - Bile Acids

Intended Use

For IN VITRO diagnostic use in the quantitative determination of Bile Acids in serum using manual or automated applications.

Method Principle

In the Catachem Bile Acids procedure, 3α-hydroxy Bile Acids are converted to corresponding 3-keto hydroxy Bile Acids by the action of the enzyme 3α-Hydroxysteroid Dehydrogenase (3α-HSDH) with concomitant reduction of NAD+ to NADH. The NADH thus produced is subsequently oxidized to NAD+ in a diaphorase-catalyzed reaction where Nitrotetrazolium Blue (NBT) is reduced to form a Formazan dye, which has an absorption maximum at  540 nm. The intensity of the color produced is directly proportional to the concentration of Bile Acids in the serum sample. The reaction scheme below illustrates the reactions that take place in this Bile Acids procedure.

3α-HSDH
            3α-hydroxy Bile Acid + NAD ─────>3-keto hydroxy Bile Acids + NADH

Diaphorase
         NADH + NBT ──────────> NAD+ + Formazan

 

Reagent Storage And Stability

Catachem Bile Acids Enzyme Color Reagent (R1) and Activator Reagent (R2) are stored at  2-8°C. When stored as directed these reagents are stable until expiration date stated on the label.

Working Reagent Preparation

Catachem Bile Acids are packaged in ready-to-use form. No preparation is required. Upon opening the reagent containers, These Working Reagents (R1) and (R2) are stored at 2-8°C. When stored as directed, the Working Reagent R1 and R2 are stable for 60 days.

Method Performance Characteristics

Sensitivity: Using a path length of 1 cm, a D-absorbance of 0.0016-0.0024 per μmol/L should be obtained.
Linearity: In this procedure there is not significant nonlinearity over the range of 0-250 mmol/L.
Precision: Precision data was obtained using three levels of protein based controls and following the NCCLS EP5-T2 procedure (9). The following results were observed.

Bile Acids

Within-Run Precision

Total Precision

MEAN

SD

CV

SD

CV

µmol/L

µmol/L

%

µmol/L

%

25

0.77

3.00

1.23

5.01

150

2.84

1.86

7.30

5.08

250

2.21

0.88

11.67

4.56

 

Accuracy

Correlation studies were carried out between this automated Bile Acids method (Y) and a reference automated Bile Acids procedure based on the 3α-HSDH and Diaphorase reactions (X). Canine serum samples were assayed and the results compared by the least square regression. The following statistics were observed:

Correlation Data

Parameter

Data Observed

N

12

Range

4.1-471

Mean Y

41.88

Mean X

43.84

Regression

Y  = 0.959x - 0.16

Correlation

r = 0.978

Sy,x

11.29