UMA CO., LTD.

MEASURE TBA
2-19-6 Yokosuka Reagent for determination of Total Bile Acid
Matsudo, Chiba, Japan ThioNAD/NADH

 2 ~ 8 °C IVD In vitro Diagnostics Packages

R1 1  60 mL R2 1  20 mL
 DO NOT freeze  12 months/block from light R1 1  45 mL R2 1  15 mL

1. PURPOSE OF USE not be analyzed soon, store sample 2 ~ 8 C and analyze

In vitro determination of Total Bile Acid in serum, plasma. within 3 days.

2. GENERAL INSTRUCTION 6. MEASUREMENT PRINCIPLE

1. For in vitro diagnostics use only. Bile Acid in samples is specifically oxidized by
2. Diagnosis should be made in a comprehensive manner, 3α-hydroxysteroid dehydrogenase (3α-HSD) and
in accordance with other related test results and clinical β-thionicotinamide adenine dinucleotide, oxidized form
symptoms by the doctor in attendance. (thio-NAD) and generates 3-ketosteroid and β-nicotinamide
3. For guaranteed results, usage of this product must adenine dinucleotide, reduced-form (thio-NADH).
comply with the instruction in this manual. Generated 3-ketosteroid is reduced by 3α-HSD and
4. For automatic analyzers: follow their instructions. β-nicotinamide adenine dinucleotide, reduced-form
5. This product contains sodium azide as preservative. If (thio-NADH) and generates bile acid and
eye, mouth or skin comes in contact with this product, take β-thionicotinamide adenine dinucleotide, oxidized form
emergency procedures (for example, wash away with (thio-NAD). By repeating these oxidization and reduction,
plenty of water), and consult with a doctor if necessary. bile acid in samples is converted into thio-NADH several
fold, eventually bile acid can be determined with high
3. MATERIALS REQUIRED BUT NOT INCLUDED
sensitivity by measuring increase ratio of thio-NADH.
- Saline 0.9 % and high grade purified water
3𝛼−𝐻𝑆𝐷
- Micropipet and other basic laboratory equipment. Bile acid + thio-NAD → 3-Ketosteroid + thio-NADH

- Calibrators and Controls (separatedly sold) 3-ketosteroid + thio-NADH → 3-Ketosteroid + NADH

4. REAGENT COMPOSITION & PREPARATION 3-Ketosteroid + NADH → Bile Acid + NAD

- Reagent R-1: β-thionicotinamide adenine dinucleotide,
Bile Acid + NAD → Bile acid + thio-NAD
oxidized form (thio-NAD)
- Reagent R-2: β-nicotinamide adenine dinucleotide, 7. STANDARD MEASUREMENT OPERATION
reduced-form(thio-NADH); 3α-hydroxysteroid Specimen Calibrator Blank
Dehydrogenase (3α-HSD) (S) (Std) (B)
- Calibrator (separatedly sold): Ready for use
Specimen (L) 15 - -
- Controls Lyo-1 & Lyo-2 (separatedly sold): Put 1 mL of - 15 -
Calibrator (L)
purified water to the vials of controls (L, H); leave at room
Saline (L) - - 15
temparature for 30 minutes before use. After reconstituted, 1500 1500 1500
R-1 (L)
controls can be use without dilution.
Incubate at 37 C in 5 minutes
5. SAMPLE PREPARATION & STORAGE R-2 (L) 500 500 500
- Serum: Wait until sample completly coagulated. Take Mix well; incubate at 37 C for 5 minutes; measure
the supernatant to use as specimen. absorbance at 405/660 nm from 6 ~ 9 minute
- Plasma: Treat sample by anticoagulant; leave sample to Note: See sample preparation for details of specimen
stand for 3 hours or centrifuge at 2000 rpm for 2 minutes;
8. CALCULATION & UNIT CONVERSION
take the plasma layer (supernatant) and use as specimen.
Calculation
- Analyze sample soon after collection. In case, it could

1/2 Revision 05/2013

 - Calculate ∆Abs of specimen & standards vs blank 13. HANDLING, USAGE & DISPOSAL
- Plot a calibration curve TBA (μmol/L) = f(∆Abs) Handling
- Calculate TBA concentration in specimen using the 1. Specimen can be potentially positive for infectious agents
curve (doing same procedure for Controls) including hepatitis B virus and HIV. Wear glove and goggle
Unit conversion when needed.
1 μmol/L = 0.405 μg/mL 2. In case reagents got into skin, eye or mouth by mistake,
wash it immediately with plenty of water and consult the
9. PERFORMANCE & CORRELATION TEST
doctor if needed.
Performance
3. If reagents are spilled, dilute with water and wipe it out. If
- Sensitivity: When using purified water, absorbance change
is 0.000 ~ 0.020; when using standard solution (bile acid 50 specimen is spilled, spray 80% of alcohol over the
specimen and wipe it out.
μmol/L), absorbance change is in 0.0150 ~ 0.045.
Usage
- Specificity test:  10 %.
1. Store reagents under specified condition. Do not use
- Reproducibility Test: When identical specimens are
measured ten times simultaneously, the within-run cv is after expiration date.
2. Do not use the container and auxiliaries included in this
below 3.0%.
- Measuring range: 1 ~ 180 μmol/L. kit for other purposes.
3. Do not mix reagents of different lot for use.
Correlation test
Same measuring principle 4. Do not add to the reagent being used even if it is the
same lot number.
Regression equation Y = 1.049X+0.443 (n=100)
Correlation coefficient R = 0.999 Disposal
1. All specimens, as well as all instruments (e.g. test tubes)
(Y: value obtained from using UMA reagent)
that come in contact with the specimens, must be treated by
10. EXPECTED VALUES the following methods, or they must be treated according to
Normal reference range the manual for infectious medical waste provided in each
- Less than 10 μmol/L facility.
Reference range should be established at each facility and ・ Sterilize with an autoclave, subjecting them to high
judgement should base on measurement results in a pressure saturated steam at 121 °C for more than 20
comprehensive manner together with clinical symptoms minutes. Do not process waste containing sodium
and other measurement results. hypochlorite solution with an autoclave.
11. INTERFERENCES ・Immerse at least one hour in sodium hypochlorite solution
- Not available for common interferences (active chloride concentration of over 1000 ppm).
2. This reagent contains sodium azide. Sodium azide can
12. INFORMATION FOR AUTOANALYZERS react with lead pipe and/or steel pipe and can generate
Calculation Method Rate explosive metal azide. Make sure to use plenty of water at
Temperature 37 C disposal. Concentration of sodium azide in R-2 is 0.05%.

Specimen 2.1
14. OTHER INSTRUCTIONS AND CAUTION
Volume (μL) R1 210
- Results may differ depending on the sample/reagent
R2 70
ratio. Adjust parameters for different analyzer.
Main 405
Wavelength (nm) - Prepare the calibration curve on the day of
Sub- 660
Point 1 10 determination.
Measurement
Point 2 22
(cycle) Point 3 30

Calibration type Linear

Unit μmol/L

2/2 Revision 05/2013