CN101055271A - Enzyme method reagent kit for detecting DBil - Google Patents

Abstract

The invention discloses an enzyme method detection direct bilirubin kit, which accelerates reaction of the bilirubin by adding enzyme composite accelerant, ensures specificity of reaction by selecting composite inhibitor, and increases stability of reagent by adding enzyme protectant. The kit possesses better clinic application future.

Description

Enzyme method reagent kit for detecting DBil

Technical field:

The invention belongs to the biological reagent technical field.Be specifically related to a kind of enzyme method reagent kit for detecting DBil.

Background technology:

Cholerythrin is an important evidence of judging jaundice clinically, also is the important indicator of liver function.The cholerythrin total amount increases, indirect bilirubin increases and shows: hemolytic anemia, incompatible blood transfusion, malignant disease, icterus neonatorum etc.Directly all increase with indirect bilirubin and show: acute icteric hepatitis, chronic active hepatitis, cirrhosis, toxic hepatitis can cause that the cholerythrin total amount increases, direct and indirect bilirubin all increases; The cholerythrin total amount increases, bilirubin direct increases and shows: intrahepatic and extrahepatic obstructive jaundice, cancer of pancreas, bile capillaries type hepatitis and other bile stasis of bloods syndrome that stagnates.

Normal serum total bilirubin concentration is 1.7～17.1 μ mol/L, and wherein bilirubin direct is lower than 3.4 μ mol/L.When total bilirubin reaches 34 μ mol/L, can find jaundice clinically; As the serum total bilirubin overrun and naked eyes are not seen jaundice, then be called jaundice occult, jaundice is most commonly in disease in the liver and gallbladder, but the other system disease also can occur.

Cholerythrin (bilirubin) is the metabolic product of protoheme, mainly transforms, drains through combination in liver; It can exist with three kinds of forms in serum: unconjugated bilirubin (BU, i.e. unconjugated bilirubin), combined with bilirubin (Bc: single glucuronic acid Bcm and two bilirubin glucuronide Bcc sum) and cholerythrin (the covalently bound cholerythrin of albumin).Cholerythrin is a kind of effective anti-oxidants, has the ability of catching oxygen radical, can protect lipid and lipoprotein not oxidized.Synergy is educated by it and interior other anti-oxidative defense system of human body; with the myocyte position that is present in ventricle after the albumin bound; stop this position to produce oxygen radical, scalable cardiac muscle cell's cholerythrin antioxidation activity, the protection ventricular muscle cell is not damaged by oxygen radical.Therefore when bilirubin concentration reduced, the danger of coronary heart disease increased.

The bilirubin direct method for measuring mainly contains diazonium method, enzyme process and vanadate oxidizing process.Diazo reagent method history at most, and is still most widely used so far.Most popular have J-G method and a M-E method.The former makes it to become green by redness under alkali condition at the diazonium cholerythrin of cholerythrin and diazo reagent reaction back generation.The transfer of spectrum has increased the sensitivity and the specificity of reaction, do not disturb substantially in green environment, but this change has increased the difficulty of robotization.Therefore adopt this method often have to manual operations.The M-E method generates back direct color comparison (redness) at the diazonium cholerythrin, and formality is simple, and being easy to robotization is advantage, but poor specificity, the result of haemolysis sample is seriously disturbed.Because the diazonium method is with a long history, medically relevant bilirubinic various argumentations are all based on this law.But there is defective in it: first diazo reagent instability, and it must be generated by sodium nitrite and aminobenzenesulfonic acid temporarily, at most can only be with 2 days (general 6 hours) after the generation; It two is that the atopic of diazo reagent and a courage and straight courage is not good, surveys straight courage under certain condition, and courage participates in reaction between a part, if the reaction time prolongs, participation cause the inaccurate of measured value more.At above-mentioned 2 points, people did countless improvement, but did not still have satisfied result so far.And the operation of this method is complicated, reagent is unstable, the reaction is not single-minded.

In addition, chemical oxidization method promptly uses chemical oxidization method to survey bilirubinic origin very early, and the cholerythrin when just adopting the seventies potassium ferricyanide to remove titrimetry survey calcium disturbs, and also once has report to survey total courage with potassium ferricyanide method later on.But also all only rest in the methodological discussion both at home and abroad, and do not have actual application value.Japanese scholar To-kuda in 1993 delivers the vanadic acid oxidizing process and surveys serum mesobilirubin.This kit in 1998 has developed numerous users through clinical practice is very fast, and well received, thinks that linearity, specificity and enzyme process relevant of this method all reaches more satisfactory level.Bilirubinic tetrapyrrol(e) structure has reductibility, various theoretically low molecular inorganic oxidizer and have the various transition metals of oxidisability under certain condition can the oxidation cholerythrin, thereby can be used for measuring cholerythrin.But must guarantee the selectivity of reaction, also will prevent interference and subsidiary reaction that early, middle and late phase of oxidation reaction may occur hand and foot.This compounds also has benzylhydroperoxide sodium, sodium peroxydisulfate, manganese acetate (III), copper sulphate, iron sulfate etc. except that vanadic acid and sodium nitrite.Therefore developing these class methods also has wide space.The practicality of these class methods has obtained proof, but makes little progress.

From relatively above-mentioned, the enzymatic assays cholerythrin has simple, special characteristics.The enzyme process oxidizing process is better, linear broad, precision height, disturbed less.Ot-suji in 1988 reports a kind of new survey total, straight courage enzyme process, and the total courage of its effect is also passable, and straight courage is measured pH is dropped to 3.7, and uses CuSO4 to do the promoter of enzyme, but this method is not promoted yet.Nineteen ninety lhara etc. is reported in during straight courage after neonate's phototherapy measures, and enzyme process is the result obviously raise than diazonium method.Reason is that the photobilirubin enzyme process that produces after the illumination can decompose by analysis, thereby measured value is higher, and the diazonium rule is not.This false rising take place chaotic when may cause differentiating neonatal physiology or pathological jaundice clinically.

Thereby, though at present enzyme process is surveyed the cholerythrin technology and can be entered clinical practice. go back imperfection, in the measuring process of bilirubin direct, exist reaction velocity slow excessively, prevent that the interference of indirect bilirubin is poor, the activity of enzyme fast or the like the defective that descends.

Summary of the invention:

Technical matters to be solved by this invention is to overcome above-mentioned weak point, and a kind of acceleration bilirubin direct reaction is provided, and the enzyme process that improves reagent stability detects the reagent of DBil.

The present invention is based on following principle:

(1) select for use effective substances to quicken the reaction of bilirubin direct.Wherein the compound accelerant of enzyme is HgCl2, Cetyl Chloride potassium ammonia, TrionX-405 (surfactant);

(2) inhibitor of the effective indirect bilirubin of increase is guaranteed the specificity of reacting.Available composite inhibitor is: potassium chloride, reduced diphosphopyridine nucleotide, the potassium ferricyanide etc.;

(3), added special enzymatic protective reagent, sweet mellow wine and Na for the stability of guarantee reagent ₂CO ₃

The invention provides a kind of enzyme method reagent kit for detecting DBil, this kit is made up of following reagent 1 and reagent 2:

Reagent 1:

Damping fluid 10-200mmol/L

HgCl ₂ 10-100mmol/L

Triton x-405 0.01-20ml/L

Cetyl Chloride potassium ammonia 20-100mmol/L

Reagent 2:

Na ₂CO ₃ 10-200mmol/L

NaHCO ₃ 10-200mmol/L

Bilirubin oxidase 2000-4000U/L

Reduced diphosphopyridine nucleotide 10-200mmol/L

KCl 10-500mmol/L

Sweet mellow wine 10-200mmol/L

proclin300 0.01-0.5ml/L

Potassium ferricyanide 10-200mmol/L

Damping fluid described in the mentioned reagent is: sodium citrate-lactic acid buffer; Glycocoll-hydrochloride buffer; Acetate-sodium acetate buffer.

The preparation method:

1. elder generation adds 80% deionized water;

2. add different agent dissolves in water;

3. reagent 1 mixes with 3: 1 ratios with 2, regulates required pH value;

4. add enzyme at last, add remaining water.

It is as follows that kit of the present invention detects effect: sodium citrate-lactic acid buffer; Glycocoll-hydrochloride buffer; Acetate-sodium acetate buffer;

(1) 0.1M HgCl ₂Cetyl Chloride potassium ammonia, Triton x-405 are as the influence of compounding activation agent to reaction:

Activator is to the influence of reaction

Concentration	Do not add activator and reach the reaction end required time	Add the compounding activation agent and reach the required time of reaction end
			19.1μmol/L	5min	1.8min
31.4μmol/L	13min	2.1min

58μmol/L	24min	3.2min
			80μmol/L	30min	3.9min

(2) influence of inhibitor to reacting: the potassium chloride of 0.1M, reduced diphosphopyridine nucleotide, the potassium ferricyanide suppress bilirubin direct as composite inhibitor

Inhibitor is to the influence of reaction

	Concentration	DB concentration (not inhibiting)	DB concentration (inhibiting)
				Sample one	Straight courage 19.1 μ mol/L	24.2μmol/L	19.2μmol/L
Total courage 28.3 μ mol/L
	Sample two	Straight courage 31.4 μ mol/L	42.3μmol/L		31.2μmol/L
Total courage 88.9 μ mol/L
		Sample two		Straight courage 19.1 μ mol/L		28.2μmol/L	19.1μmol/L
Total courage 60.3 μ mol/L
	Sample three		Straight courage 21.4 μ mol/L	40.3μmol/L	21.4μmol/L
Total courage 70.9 μ mol/L
		Sample four	Straight courage 21.4 μ mol/L			32.3μmol/L	21.2μmol/L
62.3μmol/L
	Sample five		Straight courage 28.4 μ mol/L	37.3μmol/L	29.2μmol/L
70.3μmol/L
		Sample six	Straight courage 16.4 μ mol/L			24.3μmol/L	17.2μmol/L
38.2μmol/L

(3) different enzymatic protective reagents are to the influence of bilirubin oxidase: the sweet mellow wine and the Na that add 0.1M ₂CO ₃Protective effect to enzyme

Time	Live with the remaining enzyme of protective agent	Add 0.1mol/L sweet mellow wine 0.1mol/L Na 2CO 3Remaining enzyme is lived
			0	100％	100％
1 month	95％	98％
			3 months	80％	95％

6 months	50％	88％
			12 months	20％	77％

Above-mentioned testing result shows that kit of the present invention has very strong protective effect to bilirubin oxidase; As time goes on; protective effect meeting to enzyme is more obvious, adds protectant reagent after 12 months and does not compare with not adding protectant reagent, and remaining enzyme motility rate raises 57%.

Description of drawings:

Fig. 1 be clinical relevance ratio.Ordinate is the diazonium method, and abscissa is an enzyme process.

Embodiment:

Example 1

Reagent 1

Sodium citrate 300mmol/L

Lactic acid 50mmol/L

HgCl ₂ 50mmol/L

Triton x-405 5ml/L

Cetyl Chloride potassium ammonia 50mmol/L

Reagent 2

Na ₂CO ₃ 200mmol/L

NaHCO ₃ 200mmol/L

Bilirubin oxidase 4000U/L

Reduced diphosphopyridine nucleotide 100mmol/L

KCl 20mmol/L

Sweet mellow wine 20mmol/L

proclin300 0.3ml/L

Potassium ferricyanide 50mmol/L

This example kit has been made clinical correlation relatively: the clinical sample of getting 30 routine variable concentrations is with 3 kinds of parallel detections of method

3 kinds of method test results

Sample number	Diazonium method (μ mol/L)	Oxidation enzyme process (μ mol/L)	Vanadate method (μ mol/L)
				1 2 3 4 5 6 7 8 9 10 11 12 13 14 15	1.74 5.67 3.02 6.89 4.12 8.62 8.54 2.14 7.38 3.13 2.14 8.64 2.14 5.43 2.14	1.75 5.72 3.05 34.2 42.8 58.3 8.55 2.16 7.39 3.11 2.13 8.65 2.16 5.45 2.15	1.82 6.32 3.75 8.12 5.23 9.31 8.92 2.24 7.10 3.29 2.19 8.29 2.01 5.26 2.17

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

5.21 3.26 4.64 9.03 7.84 15.4 10.2 17.4 6.56 7.87 20.4 8.59 4.23 2.26 5.79

5.21 3.27 4.67 9.05 7.82 15.2 10.3 17.6 6.58 7.88 20.5 8.56 4.25 2.25 5.76

5.29 3.58 4.39 8.25 7.61 14.9 9.2 16.5 6.32 7.22 20.9 7.32 4.10 2.13 5.26

Example 2

Reagent 1

Glycocoll 100mmol/L

Hydrochloric acid 50mmol/L

HgCl2 50mmol/L

Triton x-405 5ml/L

Cetyl Chloride potassium ammonia 50mmol/L

Reagent 2

Na ₂CO ₃ 200mmol/L

NaHCO ₃ 200mmol/L

Bilirubin oxidase 4000U/L

Reduced diphosphopyridine nucleotide 100mmol/L

KCl 20mmol/L

Sweet mellow wine 20mmol/L

proclin300 0.3ml/L

Potassium ferricyanide 50mmol/L

Example 3

Reagent 1

Phthalic acid 100mmol/L

Hydrochloric acid 50mmol/L

HgCl2 50mmol/L

Triton x-405 5ml/L

Cetyl Chloride potassium ammonia 50mmol/L

Reagent 2

Na ₂CO ₃ 100mmol/L

NaHCO ₃ 100mmol/L

Bilirubin oxidase 4000U/L

Reduced diphosphopyridine nucleotide 100mmol/L

KCl 20mmol/L

Sweet mellow wine 20mmol/L

proclin300 0.4ml/L

Potassium ferricyanide 50mmol/L

Example 4

Reagent 1

Acetate 100mmol/L

Sodium acetate 100mmol/L

HgCl2 50mmol/L

Triton x-405 5ml/L

Cetyl Chloride potassium ammonia 50mmol/L

Reagent 2

Na ₂CO ₃ 200mmol/L

NaHCO ₃ 200mmol/L

Bilirubin oxidase 4000U/L

Reduced diphosphopyridine nucleotide 100mmol/L

KCl 20mmol/L

Sweet mellow wine 20mmol/L

proclin300 0.3ml/L

Potassium ferricyanide 50mmol/L

Example 5

Reagent 1

Acetate 200mmol/L

Sodium acetate 150mmol/L

HgCl2 50mmol/L

Triton x-405 5ml/L

Cetyl Chloride potassium ammonia 50mmol/L

Reagent 2:

Na ₂CO ₃ 100mmol/L

NaHCO ₃ 100mmol/L

Bilirubin oxidase 4000U/L

Reduced diphosphopyridine nucleotide 50mmol/L

KCl 20mmol/L

Sweet mellow wine 20mmol/L

proclin300 0.2ml/L

Potassium ferricyanide 50mmol/L

Claims (3)

1, a kind of enzyme method reagent kit for detecting DBil is characterized in that this kit is made up of following reagent 1 and reagent 2:

Reagent 1:

Damping fluid 10-200mmol/L

HgCl ₂ 10-100mmol/L

Triton x-405 0.01-20ml/L

Cetyl Chloride potassium ammonia 20-100mmol/L

Reagent 2:

Na ₂CO ₃ 10-200mmol/L

NaHCO ₃ 10-200mmol/L

Bilirubin oxidase 2000-4000U/L

Reduced diphosphopyridine nucleotide 10-200mmol/L

KCl 10-500mmol/L

Sweet mellow wine 10-200mmol/L

proclin300 0.01-0.5ml/L

Potassium ferricyanide 10-200mmol/L

2, a kind of enzyme method reagent kit for detecting DBil according to claim 1 is characterized in that wherein said damping fluid is: sodium citrate-lactic acid buffer; Glycocoll-hydrochloride buffer; Acetate-sodium acetate buffer; Phthalic acid-hydrochloric acid.

3, a kind of enzyme method reagent kit for detecting DBil according to claim 1, the blending ratio that it is characterized in that wherein said reagent 1 and reagent 2 is 3: 1.

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