CN1912580A - Serum starch enzyme reagent kit and preparation method thereof - Google Patents

Abstract

A serum amylase kit is prepared as containing NaCI content of 70mmol /L, CaCI2 content of 1.0mmol /L and HEPES content of 50mmol /L in buffer liquid; containing EPS-GT content of 3.5mmol /L in substrate liquid; containing a-AMY content of 60U /ml and human serum albumin content of 20g /L in enzyme storing liquid; applying temperature of 37deg.c, buffer liquid pH of 7.15 and enzyme content of 7.1U /ml as testing conditions of kit when kit is used to carry out test.

Description

Serum starch enzyme reagent kit and preparation method thereof

Technical field

The preparation and the method that the invention belongs to measuring body inner blood characteristic particularly relate to serum starch enzyme reagent kit and preparation method

Background technology

As far back as nineteen twenty-nine, Elman has just established the value of serum amylase (Amy) on acute pancreatitis diagnosis.Measure the method for gross activity and divide four classes, the first kind is measured the consumption of substrate starch: by viscosimetry, nephelometry, iodine-starch method, and this class methods poor accuracy, susceptibility is low; The second class sugared method of making a living is measured product glucose; The 3rd class is a chromogen substrate decomposition method, and dyestuff and insoluble starch are combined into chromogen substrate (being commonly called as staining starch), along with the degraded of the starch dyestuff that dissociates, surveys the content of dyestuff under Amy catalysis, and these class methods need specific apparatus, use wideless.The 4th class is an enzyme coupling method: with forming diastase substrate and auxiliary enzymes and the indicator enzyme system detection diastase of determining, can improve the stoichiometric relationship of reaction, help realizing continuous detecting and use international unit.α-Amy hydrolysis micromolecule oligosaccharides products therefrom is more more definite than hydrolyzed starch.Domestic Ni Xing loyalty etc. has been reported employing NaN ₃Direct determination method for activator.Be published in clinical examination magazine in 1996 on the 6th phase, the method is a substrate with chloronitrobenzene maltose, because of reasons such as its cross pollutions, false positive is increased.1998 international clinical chemistry association (IFCC) to have ratified with 4-nitrophenols-α-D-maltotriose be that substrate detects α-Amy method, this method has good accuracy, precision.In the IFCC reference method:

A: substrate is 4-nitrophenols-α-D-arsenic glucopyranoside glycosides enzyme, wako company kit, and temperature T is 30 ℃, PH is 7.1.The product α of B:Boehringer Manheim company-Amy kit, substrate are ethylidene-[4-nitrophenols (G1]-α-D-Fructus Hordei Germinatus seven glucosides, coenzyme, and alpha-glucosidase, temperature T is 30 ℃, PH is 7.1.

Ethylidene-4-the nitrophenols-1 of the Klaus Lorentz application protection of setting up in addition, 4-α-D-Fructus Hordei Germinatus seven glucosides (EPS_-G7) detect in the method for α-Amy: temperature T is 30 ℃, and PH is 7.0, and the enzyme working fluid contains enzyme 6.2mmol/L.

Because of dissociating of 4-nitrophenols (4-NP) raises with temperature, but probe temperature is 30 ℃ now, and lower, so influence the catalytic degradation reaction of α-Amy, lag time is long, and linear range is narrow, the incompatibility conventional analysis.

Summary of the invention

The present invention provides a kind of serum starch enzyme reagent kit and preparation method thereof for solving the technical matters that exists in the prior art.

The technical scheme that the present invention takes for the technical matters that exists in the solution prior art is:

A kind of serum starch enzyme reagent kit, it comprises damping fluid, substrate solution and enzyme stock solution, NaCl content is 70mmol/L in its damping fluid, Cacl ₂Content is 1.0mmol/L, N-2 hydroxyethyl piperazine-N '-ethylsulfonic acid (HEPES) content is 50mmol/L, substrate is ethylidene-4-nitrophenols-1 in the substrate solution, 4-α-D-Fructus Hordei Germinatus seven glucosides (EPS-G7) content is that alpha-glucosidase content is 60U/ml in 3.5mmol/L, the enzyme stock solution, and human serum albumins content is 20g/L.

Described serum starch enzyme reagent kit, temperature is 37 ℃ during its kit test, and buffer solution ph is 7.15, and enzyme content is 7.1U/ml in the enzyme working fluid.

The preparation method of reagent in the described serum starch enzyme reagent kit:

A. the preparation method of damping fluid: 1.02gNaCl, 2.98gN-2 hydroxyethyl croak piperazine-N-ethylsulfonic acid (HEPES) are dissolved in about 220ml distilled water, add 0.07ml 258mmol/L CaCl ₂, regulate pH value to 7.15 with about 24ml 0.2mmol/L NaOH down, accurately add water to 250ml for 37 ℃;

B. the preparation method of substrate solution: 157mgEPS-G7 is dissolved in 1 liquid, accurately adds to 100ml;

C. the preparation method of enzyme stock solution: with content 25U/mg, 37 ℃ alpha-glucosidase: 10mg and 80mg human serum albumins are dissolved to 4.2ml with 50mmol/L PH 7.15 HEPES damping fluids;

D. the preparation method of enzyme working fluid: get enzyme stock solution 2.4ml and be added in the 17.6ml PH7.15 HEPES damping fluid.

In order to understand essence of the present invention better, describe below by experiment and result.

Experimental principle: EPS-G7 and alpha-glucosidase decompose all reactions equably, generate 4-nitrophenols and glucose.

Experimental apparatus: SPS-100 automatic clinical chemistry analyzer (Dutch Vital company, 6～1795); The digital acidometer of pH-3B type (Jiangsu Electrical Analysis Instrument Factory, B-42); Analytical balance (ten thousand/, Shanghai Changjiang River scientific instrument factory, TG328A).

Reagent: EPS-G7 (German CENTRONIC) lot number is U0150; Alpha-glucosidase is from saccharomycete, and specific activity is 25U/mg, and lot number is B-174207 (Boehringer); N-2 hydroxyethyl piperazine-N '-ethylsulfonic acid (HEPES), the pancreatic amylase standard items, lot number is P 9903, (reference substance 476 that authenticates by the American National biological chemistry standard and the Quality Control council), and the human serum diastase that is higher than 15 times of the reference value upper limits, lot number P 196561 (Roch), cholerythrin (analyzing pure SIGMAGRADE) lot number 57F 0111; The α of Boehringer company-Amy kit, lot number are B 1125.To satisfy the pure standard of regulations such as Bower, other chemical reagent is analytical reagent to 4-nitrophenols (4-NP) through three crystallizations.

Experiment parameter: wavelength 405nm, 37 ℃ of temperature, enzyme working fluid 500 μ L, specimen amount blood 20 μ L or urinate 10 μ L, substrate solution 100 μ L, time delay 120s, Measuring Time 120s.

Computing formula U/L=Δ A/min * V _T/ ε/L/V _S

Urine U/L=Δ A/min * 1200.5

Blood U/L=Δ A/min * 1120

Experimental result

1.4-NP determining of molar absorptivity according to the Lorentz method, at the 1.0cm optical path, wavelength 405nm place, pH is respectively 7.00,7.05, and 7.10,7.15,7.20 the time measure the absorbance of 100 μ mol/L4-NP, and calculate ε and be respectively 1051.6,1101.9,1151.8,1200.5,1245.3mol/L, ε 1200.5mol/L when this law is selected pH7.15 for use.

Damping fluid be chosen in PH, when concentration of substrate is constant, choose HEPES, phosphate and three kinds of damping fluids of hydroxyethyl methane (BTP) respectively detectable concentration be the amylopsin standard items of 49.3U/L, n=20 the results are shown in Table 1.By finding out in the table, enzyme is active in the BTP damping fluid to reduce 14%, active decline 6% in phosphate buffer, and HEPES damping fluid did not influence.Therefore select the buffer system of HEPES damping fluid for use as this experiment.

3. select the suitableeest concentration of substrate when other experiment condition is constant,, detect of the influence of the substrate of variable concentrations, draw concentration of substrate and enzyme reaction rate curve reaction rate at the 405nm place.See Fig. 1.Draw Linewear-Burk figure, see Fig. 2.Getting regression equation according to data is y=24.39+4.37x, draws with ESP-G ₇For the Km=0.179mmol/L of the α-AMY of substrate in view of the above this law to select concentration of substrate for use be 3.5mmol/L.

4. the selection of optimal pH is respectively 6.9,7.0 with PH, the ESP-G of 7.1,7.15,7.2,7.3 HEPES damping fluid preparation 3.5mmol/L ₇Substrate solution, the 405nm place measures α-Amy activity, the results are shown in Figure 3, learn that from Fig. 3 the optimum PH of diastase is 6.9-7.0, AMY is active when PH7.15 keeps 98% of maximum activity, because when active PH is higher than optimum, the absorptance of 4-NP increases, compensated the loss of AMY activity, therefore selected 7.15, so that detect more perfect.

The linear response time and the range of linearity determine get the amylopsin standard items active for 49.3U/L on SPS-100, will be set in 12min the reaction time, the reaction time curve.See Fig. 4.With people's blood amylase of high vigor be diluted to 200,400,800,1200,1400,1600U/L, survey enzyme activity respectively, the range of linearity of enzyme.See Fig. 5.From figure, learnt the expanded range of detection, can one-time detection come out, and guaranteed precision, surpassed and survey again after the 1500U/L sample dilutes with physiological saline from 1000-1500U/L.

6. precision experiment gets that high, medium and low value is respectively 398,118, the standard items of 49.3U/L, surveys continuously 20 times, obtains withinrun precision.Every part of serum is surveyed once, surveys 20d continuously, gets betweenrun precision.The results are shown in Table 2.CV is 1.4-2.6% in its batch, and CV is 1.9-2.8% between batch

7. reclaiming experiment is in the proenzyme liquid of 49.3U/L in activity, and the adding vigor is 5.1,3.1 respectively, detects enzyme activity after the titer of 2.5U/L, the results are shown in Table 3.

8. optimal reaction temperature is chosen in other condition when constant, when being respectively 25 ℃, 30 ℃, 35 ℃, 37 ℃, 40 ℃, temperature of reaction measures enzyme reaction rate Δ A/min, the results are shown in Figure 6, because the ionization of chromogen increases with the rising of temperature, the molar absorptivity of 4-NP is high by about 8% in the time of 37 ℃ than at 30 ℃ the time, so select 37 ℃ of reaction systems.

9. respectively that concentration is different haemoglobin, cholerythrin, glucose, the protein of interference experiment joins actively in the amylopsin standard items of 49.3U/L, the results are shown in Table 4, table 5, table 6, table 7.Peace is shone 100 ± 3% recovery test standard, and we find 29.5g/L haemoglobin, 610mmol/L, and 100mmol/L glucose, 70mmol/L protein are to reacting noiseless.

10. the correlativity experiment is measured the amylopsin standard items respectively with this law and IFCC approval Boehringer company's kit (substrate is a 4-nitrophenols-1,4-α-D-maltotriosides).Recording dependent equation is y=10.9+1.445x, γ=0.994, n=20.Because related coefficient＜0.975, so the method for this method and IFCC recommendation has good correlativity.

11. after the preparation of reagent stability observation work reagent, A=0.187 when measuring 405nm immediately; 5 ℃ of 4 week back A=0.206; 5 week back A=0.396; Experimental results show that A＞0.40 o'clock, measurement result is on the low side.So it was 3 weeks that 5 ℃ of substrates that do not contain adjuvant limit storage period, it was 5 weeks that enzyme solutions limits storage period.

The Preliminary Clinical situation

Research object is grown up 80 at my institute's Physical Examination, each 40 of men and women, and 41.4 years old mean age, meet following condition, ultrasound diagnosis: liver, courage, pancreas, spleen, kidney are all normal.Blood test: total protein is 62～75g/L, empty stomach GLU＜6.1mmol/L, Cr＜96mmol/L, γ-GT＜20U/L, ALT＜40U/L is divided into five groups with them, per 8 male sex, 8 women are one group, between 20～69 years old, per 10 years old is an age group, all stays urine, blood sampling in the morning on an empty stomach.Acute pancreatitis 42 examples, chronic pancreatitis 4 examples, mumps 1 example.

Measurement result normal person does not have significant difference for masculinity and femininity, in age groups, in all contrast experiments, P 〉=0.9 does not have significant difference yet, therefore, by the detection to 80 routine health adults, the result meets normal distribution, set up the term of reference of 0.95 credibility interval: blood 33.9～96.2U/L, urine 65.4～187.6U/L; 42 routine acute pancreatitis detect: blood 296-1252U/L, urine 506.3～1954.7U/L; Chronic pancreatitis is more acute on the low side: blood 138-504U/L, urine 238.5～429.6U/L; Mumps: blood 269U/L, urine 197.6U/L.

Advantage and good effect that the present invention has are:

All continuous detecting α-Amy detection method, use the 4-nitrophenols-1 of ethylidene sealing, 4-α-D-Fructus Hordei Germinatus seven glucosides are substrate and new saccharomycetic alpha-glucosidase, decompose all reactions equably, generate 4-nitrophenols and glucose, do not need complicated pretreatment and specific apparatus, reagent has advantages such as enough stability and easy operating, the required sample volume of conventionally test program is reduced, shorten time delay, reduced the interference of some situation of matrix, changed the reaction conditions of IFCC recommend method equally, change probe temperature into 37 ℃ by 30 ℃, PH changes 7.15 into by 6.9-7.0.Enzyme concentration changes 7.1mmol/L into by 6.2mmol/L, 100% dissociates so that reduce, and has increased release property, has prolonged the dependent linearity scope, and by the evaluation analysis to these results, this detection method is the method for a kind of detection α-Amy that more becomes perfect.

Description of drawings

Fig. 1 is concentration of substrate and enzyme reaction rate profile;

Fig. 2 is Linewear-Burk figure;

Fig. 3 is the influence figure of PH to enzymatic activity;

Fig. 4 is the time of enzymatic reacting curve map;

Fig. 5 is the linear range figure of enzyme;

Fig. 6 is the influence figure of temperature to reaction velocity.

Embodiment

1. damping fluid:

1.02gNaCl, 2.98gHEPES are dissolved in about 220ml distilled water, add 0.07ml258mmol/L CaCl ₂, regulate PH to 7.15 with about 24ml 0.2mmol/L NaOH down, accurately add water to 250ml for 37 ℃.

2. substrate solution:

157mgEPS-G7 is dissolved in 1 liquid, accurately adds to 100ml.

3. enzyme stock solution:

With content 25U/mg, 37 ℃ alpha-glucosidase: 10mg and 80mg human serum albumins are dissolved to 4.2ml with 50mmol/L PH 7.15 HEPES damping fluids.

4. enzyme working fluid:

Get enzyme stock solution 2.4ml and be added in the 17.6ml PH7.15 HEPES damping fluid, allow it contain enzyme 7.1U/ml.

5.4-nitrophenols (4-NP):

Take by weighing 1.38mg 4-NP to volumetric flask with analytical balance, accurately constant volume 100ml.

The selection of table 1. damping fluid

Damping fluid	Enzymatic activity x (U/L)	Relative activity
			HEPES BTP phosphate	49.2 42.5 44.5	1 0.86 0.94

Table 2. precision experimental result n=20

	Withinrun precision			Betweenrun precision
							x	s	cv％	x	s	cv％
	The high value of low value intermediate value	49.3 118 398	1.28 1.97 5.78	2.6 1.7 1.4	49.3 119 400	1.36 2.41 7.47							2.8 2.0 1.9

Table 3. reclaims experimental result

Numbering	1	2	3
				The stoste enzymatic activity adds enzymatic activity actual measurement enzymatic activity and reclaims enzymatic activity recovery %	42.6 5.1 47.6 5.0 96.1	42.6 3.04 45.5 2.9 96.8	42.6 1.52 44.1 1.5 96

α-the Amy specific activity after table 4. added haemoglobin

Measurement result	1	2	3	4	5	6	7
								Hgb mg/L α-Amy U/L	0 42.6	92.2 41.9	184.4 42.8	368.8 41.8	737.5 42.5	1475 42.7	2950 42.5

α-the Amy specific activity after table 5. added cholerythrin

Measurement result	1	2	3	4	5	6
							Cholerythrin mmol/L α-Amy U/L	0 42. 6	10. 7 42. 8	21. 3 42. 4	42. 7 42. 9	85. 5 42. 2	171 42. 5

α-the Amy specific activity after table 6. added glucose

Measurement result	1	2	3	4	5	6	7
								GLU mmol/L α-Amy U/L	0 42.6	5.0 42.9	10.0 42.7	15.0 42.8	20.0 42.2	25.0 42.7	50.0 42.6

α-the Amy specific activity after table 7. added protein

Measurement result	1	2	3	4	5	6
							Protein g/L α-Amy U/L	0 42.6	4.4 41.9	8.7 42.4	17.5 42.6	35 42.3	70 42.7

Claims (3)

1. serum starch enzyme reagent kit, it comprises damping fluid, substrate solution and enzyme stock solution, it is characterized in that NaCl content is 70mmol/L in the damping fluid, Cacl ₂Content is 1.0mmol/L, and N-2 hydroxyethyl piperazine-N '-ethylsulfonic acid (HEPES) content is 50mmol/L; Substrate is ethylidene-4-nitrophenols-1 in the substrate solution, and 4-α-D-Fructus Hordei Germinatus seven glucosides (EPS-G7) content is 3.5mmol/L; Alpha-glucosidase in the enzyme stock solution (a-AMY) content is 60U/ml, and human serum albumins content is 20g/L.

2. serum starch enzyme reagent kit according to claim 1, temperature is 37 ℃ when it is characterized in that this kit test, and buffer solution ph is 7.15, and enzyme content is 7.1U/ml in the enzyme working fluid.

3. the preparation method of reagent in the serum starch enzyme reagent kit according to claim 1 is characterized in that:

A. the preparation method of damping fluid: 1.02gNaCl, 2.98gHEPES are dissolved in about 220ml distilled water, add 0.07ml 258mmol/L CaCl ₂, regulate pH value to 7.15 with about 24ml 0.2mmol/L NaOH down, accurately add water to 250ml for 37 ℃;

B. the preparation method of substrate solution: 157mgEPS-G7 is dissolved in 1 liquid, accurately adds 1 liquid to 100ml;

C. the preparation method of enzyme stock solution: with content 25U/mg, 37 ℃ alpha-glucosidase 10mg and 80mg human serum albumins are dissolved to 4.2ml with 50mmol/L PH7.15 HEPES damping fluid;

D. the preparation method of enzyme working fluid: get enzyme stock solution 2.4ml and be added in the 17.6ml PH7.15 HEPES damping fluid.

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