CN1378077A

CN1378077A - Method for detecting total bile acid and detecting reagent

Info

Publication number: CN1378077A
Application number: CN 02119402
Authority: CN
Inventors: 肖洪武
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-05-10
Filing date: 2002-05-10
Publication date: 2002-11-06

Abstract

The present invention provides a detecting reagent and detecting method of total bile acid. Bile acid is first oxidized specifically with 3 alpha-hydroxy steroid dehydrogenase and Thio-NAD to produce 3-ketosteroid, NADH and Thio-NADH, and then bile acid and NAD are produced in the presence of the produced 3-ketosteroid and 3 alpha-hydroxy steriod dehydrogenase, and so on, the amount of bile acid is amplified and great amount of water soluble yellow non-dye Thio-NADH is produced. Finally, the absorbency variation of the produced Thio-NADH is measured as bile acid value. The said method has high stability and high sensitiivty and may be used in clinical determination of bile acid.

Description

A kind of assay method of TBA and mensuration reagent

The present invention relates to a kind of assay method and mensuration reagent that is used for the detection technique, particularly a kind of TBA of external quantitative measurement serum serum tolal bile acid content.

Norway Ke Ming company in 1974 adopts the 3 α-hydroxysteroid dehydrogenase of pure system, and (3 α-HSD) successfully produce first generation enzyme process bile acid reagent make the mensuration of bile acid really enter clinical field.After this, bright and Japanese first chemistry of Norway section improves reagent components with first generation reagent, adopts to start reagent, has developed second generation bile acid reagent.Afterwards, (3 α-HSD) third generation bile acid test kit is developed in coupling to Japanese first chemistry 3-oxygen-5 β-steroid-Δ 4 dehydrogenasas (Δ 4DH) of purifying out from pseudomonas with 3 α-hydroxysteroid dehydrogenase.

Above-mentioned these a few class reagent indication principles have all adopted diaphorase and the blue salt of tetrazolium to generate formazan dye, thus, third generation reagent has improved the pigment generation exponentially, thereby improved extinction coefficient, solved the shortcoming of first generation reagent poor repeatability when the low value sample test, but, because generating, increases pigment, causing instrument to pollute increases, simultaneously, such reagent reacting is subject to reducing substances and enzyme interference in the blood, as the disclosed a kind of serum tolal bile acid determination reagent of Chinese patent CN-1281983, because it is by containing nitro tetrazolium orchid, oxidized coenzyme, diaphorase, Sodium Pyruvate, the reagent I that the solution of phosphate buffer constitutes; Form with the reagent II that constitutes by the solution that contains 3 α-hydroxysteroid dehydrogenase, human serum albumins, EDTA, phosphate buffer, its test philosophy is to utilize 3 α-hydroxysteroid dehydrogenase, the hydrogen that diaphorase is taken off the bile acid molecule is delivered to acceptor molecule nitro tetrazolium orchid, generate blue look compound after the latter reduces, detect for colorimetric analysis.At this, adopt the blue combination of diaphorase and nitro tetrazolium, easily pollute cuvette and stop up the biochemical instrument pipeline, it is also on the low side that it measures sensitivity, is subjected to reducing substances and enzyme serious interference in the blood.

The objective of the invention is to provide for the deficiency that solves above-mentioned technology a kind of easy to use, good stability, highly sensitive, remove and disturb obviously and to the assay method of the free of contamination a kind of TBA of biochemical instrument pipeline and measure reagent.

In order to achieve the above object, the assay method of a kind of TBA that the present invention is designed, it comprises adopts 3 α-hydroxysteroid dehydrogenase (3 α-HSD), it is characterized in that utilizing 3 α-hydroxysteroid dehydrogenase and Thionicotinamide-NAD (Thio-NAD) specific oxidation bile acid, generate the 3-ketosteroid after the oxidation, reduced coenzyme (NADH) and ketosteroid Thionicotinamide-NAD (Thio-NADH), at this, the 3-ketosteroid that generates is in the presence of 3 α-hydroxysteroid dehydrogenase and reduced coenzyme (NADH), generate bile acid and oxidized coenzyme (NAD) again, every circulation primary doubles sensitivity than original reaction, so reaction cycle is reciprocal, thereby amplify the bile acid amount of trace, produce a large amount of non-dyestuff ketosteroid of water-soluble yellow Thionicotinamide-NADs (Thio-NADH), the absorbance of measuring the Thio-NADH that generates at last changes, and promptly records bile acid level.

Assay method by above-mentioned TBA, the mensuration reagent of TBA provided by the invention, it comprises 3 α-hydroxysteroid dehydrogenase of 0.5KU/L-100KU/L, the damping fluid of 5-500mmol/L, the surfactant of 0.005%-5% and the stabilizing agent of 0.005%-50% (dissimilar stabilizing agent dosage differ greatly), it is characterized in that it contains the Thionicotinamide-NAD of 0.01mmol/L-10mmol/L (Thio-NAD).The reduced coenzyme that also can contain 0.01-10mmol/L.In order to reduce the interference of protein in the mensuration process most effectively, described surfactant can be selected the polyoxyethylene surfactant especially for use, as Brij polyoxyethylene lauryl ether series, anhydrous sorbitol polyoxyethylene series, polyoxyethylene alkyl ether series, polyoxyethylene nonylphenol ether series etc.Certainly, also can select zwitterionic surfactant for use, as empgen BB (Empigem BB).

In addition,, in order reducing cost, and to improve the precision that it measures sensitivity and mensuration better, in above-mentioned composition, also can to contain the glucose-6-phosphate dehydrogenase (G6PD) of 0.1KU-50 KU/L and G-6-P and circulate and generate NADPH because NADPH costs an arm and a leg; Its described reduced coenzyme also can be NADH, Thio-NADH or NADPH.

In addition, damping fluid described herein can be a Good ' s damping fluid (trade name, read by transliteration), phosphate buffer, pyrophosphate damping fluid, glycine buffer, citrate buffer, diethanolamine, triethanolamine class damping fluid, carbonate buffer solution, Tris class damping fluid or boric acid class damping fluid, its pH value can be 3.0 to 11.0, the buffer capacity 0.05-1M of damping fluid.

Described stabilizing agent can be Mg ion, NaCl, KCl, CaCl2, bovine serum albumin(BSA), human serum albumins, microbiotic (chloromycetin, erythromycin, penicillin, gentamicin), intercalating agent EDTA class, polyglycol, disaccharides (sorbierite, sweet mellow wine, sucrose, trehalose etc.), polyvalent alcohol (ethylene glycol, glycerine, propylene glycol etc.), glycylglycine and antioxidant.

In addition, in reagent, can also add agent interfering (Sodium Pyruvate, oxalic acid and Oxalates and oxamic acid)

Adopt the assay method of a kind of TBA of the present invention to measure TBA, easy to use, good stability, removing are disturbed obviously, sensitivity compared with prior art can improve about 20 times, pollution-free to the biochemical instrument pipeline, make it to have in the clinical assays accurately and rapidly to bile acid very active operation significance, particularly bile acid determination has important value to the clinical diagnosis of acute liver disease, chronic liver disease, cirrhosis, cholestasia, gastric ulcer, colonic diseases etc. accurately.

The invention will be further described in conjunction with the accompanying drawings below by embodiment.

Embodiment 1:

The mensuration reagent of a kind of TBA that present embodiment is described is divided into two components: reagent A and reagent B: reagent A:

PH4.0 damping fluid MES (2-N-morphine ethyl sulfonic acid) 5mM;

Thionicotinamide-NAD (Thio-NAD) 0.95g/L;

Antiseptic NaN3 0.9g/L;

Intercalating agent EDTA.2Na 0.5mmol/L

Surfactant B rij35 0.5g/L

Remove agent interfering Sodium oxamate 2mmol/L reagent B:

PH9.0 phosphate buffer 200Mm

3α-HSD???????????????????15KU/L

NADH??????????????????????6g/L

MgCl2?????????????????????2mmol/L

Antiseptic NaN3 0.6g/L

BSA???????????????????????2g/L

Intercalating agent EDTA.2Na 0.5mmol/L

Its assay method is to adopt the automatic biochemistry analyzer with double reagent function, operates as follows:

Admixture	Blank pipe	Standard pipe	Measure pipe
Admixture	Blank pipe	Standard pipe	Measure pipe	Reagent A	????240μl	????240μl	????240μl
Distilled water	????10μl			Reagent A	????240μl	????240μl	????240μl
Distilled water	????10μl			Titer		????10μl
Sample			????10μl	Titer		????10μl
Sample			????10μl	Mix, put 37 ℃ of preincubates 3 to 5 minutes
Reagent B	????80μl	????80μl	????80μl	Mix, put 37 ℃ of preincubates 3 to 5 minutes
Reagent B	????80μl	????80μl	????80μl	Mixing is put 37 ℃ and was hatched 1 minute, reads under the auxiliary wavelength 660nm of predominant wavelength 405nm/ and respectively manages absorbance A 1; Hatched 4 to 5 minutes, and read and respectively manage absorbance A 2; Calculate Δ A=A2-A1

Calculate: bile acid concentration (μ mol/L)=C standard ^*(Δ A sample-Δ A blank)/Δ A sample ^*Δ A blank

Embodiment 2:

The mensuration reagent of a kind of TBA that present embodiment is described, be divided into two components: reagent A and reagent B, at this, other is to adopt alternative different damping fluids, surfactant and select different stabilizing agents to add in order to obtain better reagent stability with embodiment 1 phase region.And the raising of surfactant promotion enzyme activity, thereby enzyme dosage is reduced.Reagent A:

PH4.0 glycine buffer 5mM;

Thionicotinamide-NAD (Thio-NAD) 0.95g/L;

Antiseptic NaN3 0.9g/L;

Intercalating agent EDTA.2Na 0.5mmol/L

Surfactant soil temperature 80 0.5g/L

Remove agent interfering Sodium oxamate 2mmol/L reagent B:

PH9.0 Tris damping fluid 200Mm

3α-HSD??????????????????????10KU/L

NADH?????????????????????????6g/L

MgCl2????????????????????????2mmol/L

Antiseptic NaN3 0.6g/L

BSA??????????????????????????2g/L

Intercalating agent EDTA.2Na 0.5mmol/L

Stabilizing agent glycylglycine 50Mm

Sweet mellow wine 2%

Polyglycol 1%

Method of testing is identical with embodiment 1.

Embodiment 3:

In order to improve its precision of measuring sensitivity and mensuration better and to reduce cost, the mensuration reagent of a kind of TBA that present embodiment is described, be divided into two components: reagent A and reagent B, wherein in component, be provided with glucose-6-phosphate dehydrogenase (G6PD), G-6-P, reduced coenzyme is selected NADPH for use, corresponding surface or property agent, stabilizing agent, goes agent interfering also to make corresponding different choice.Reagent A:

PH4.0 glycine buffer 5mM;

Thionicotinamide-NAD (Thio-NAD) 1.5g/L;

Antiseptic NaN3 0.9g/L;

Intercalating agent EDTA.2Na 0.5mmol/L

Surfactant soil temperature 20 0.5g/L

Empgen BB (Empigem BB) 2ml/L

Remove agent interfering oxamic acid potassium 2mmol/L reagent B:

PH8.75 Tris damping fluid 200Mm

3α-HSD????????????????????????????????15KU/L

Glucose-6-phosphate dehydrogenase (G6PD) 10KU/L

G-6-P 16mmol/L

NADPH?????????????????????120μmol/L

MgCl2?????????????????????5mmol/L

Antiseptic NaN3 0.6g/L

BSA???????????????????????2g/L

Intercalating agent EDTA.2Na 0.5mmol/L

Embodiment 4:

In order to improve the stability of reagent, add antioxidant and stabilizing agent at reagent A, B: reagent A:

PH4.0 damping fluid MES (2-N-morphine ethyl sulfonic acid) 5mM;

Thionicotinamide-NAD (Thio-NAD) 0.95g/L;

Antiseptic NaN3 0.9g/L;

Intercalating agent EDTA.2Na 0.5mmol/L

Surfactant B rij35 0.5g/L

Remove agent interfering Sodium oxamate 2mmol/L

Ethylene glycol 20% reagent B:

PH9.0 AMP damping fluid 500mM

3α-HSD?????????????????????????????????15KU/L

NADH????????????????????????????????????6g/L

MgCl2???????????????????????????????????2mmol/L

Antiseptic NaN3 0.6g/L

BSA?????????????????????????????????????2g/L

Intercalating agent EDTA.2Na 0.5mmol/L

Antioxidation 2 mercapto ethanol 1mM (0.1-200mmol/L)

Claims

1, a kind of assay method of TBA, it comprises adopts 3 α-hydroxysteroid dehydrogenase, it is characterized in that utilizing 3 α-hydroxysteroid dehydrogenase and Thionicotinamide-NAD (Thio-NAD) specific oxidation bile acid, generate the 3-ketosteroid after the oxidation, reduced coenzyme (NADH) and ketosteroid Thionicotinamide-NAD (Thio-NADH), at this, the 3-ketosteroid that generates is in the presence of 3 α-hydroxysteroid dehydrogenase and reduced coenzyme (NADH), generate bile acid and oxidized coenzyme (NAD) again, so reaction cycle is reciprocal, thereby amplify the bile acid amount of trace, produce a large amount of non-dyestuff ketosteroid of water-soluble yellow Thionicotinamide-NADs (Thio-NADH), the absorbance of measuring the Thio-NADH that generates at last changes, and promptly records bile acid level.

2, a kind of mensuration reagent of TBA, it comprises 3 α-hydroxysteroid dehydrogenase of 0.5 KU/L-100 KU/L, the damping fluid of 5-500mmol/L, the surfactant of 0.005%-5% and the stabilizing agent of 0.005%-50%, it is characterized in that it contains the Thionicotinamide-NAD of 0.01mmol/L-10mmol/L (Thio-NAD).

3, the mensuration reagent of a kind of TBA according to claim 2 is characterized in that it contains the reduced coenzyme of 0.01-10mmol/L.

4,, it is characterized in that described surfactant selects the polyoxyethylene surfactant especially for use according to the mensuration reagent of claim 2 or 3 described a kind of TBAs.

5,, it is characterized in that it also contains the glucose-6-phosphate dehydrogenase (G6PD) of X to Y part according to the mensuration reagent of claim 2 or 3 described a kind of TBAs.

6, the mensuration reagent of a kind of TBA according to claim 4 is characterized in that it also contains glucose-6-phosphate dehydrogenase (G6PD) and the G-6-P of 0.1KU-50 KU/L.

7, the mensuration reagent of a kind of TBA according to claim 3 is characterized in that described reduced coenzyme can be NADH, NADP or NADPH.

8, the mensuration reagent of a kind of TBA according to claim 4 is characterized in that described reduced coenzyme can be NADH, NADP or NADPH.

9, the mensuration reagent of a kind of TBA according to claim 5 is characterized in that described reduced coenzyme can be NADH, NADP or NADPH.

10, the mensuration reagent of a kind of TBA according to claim 6 is characterized in that described reduced coenzyme can be NADH, NADP or NADPH.