CN101597636A - Measure the method for target enzymic activity with the maximum instantaneous velocity of conjugate enzyme reaction - Google Patents
Measure the method for target enzymic activity with the maximum instantaneous velocity of conjugate enzyme reaction Download PDFInfo
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Abstract
The present invention is the data analysing method that is used for biomedicine field conjugate enzyme reaction assay target enzymic activity; Obtain sampling interval no longer than the 10s response data to write down conjugate enzyme reaction process or interpolation at interval no longer than 10s after postponing to be no more than 30s, the corresponding V-bar of adjacent four data points of regression analysis is first momentary velocity; Analyze the variation that momentary velocity carries out with reaction, with fitting of parabola from first momentary velocity after reaction 1.0min and crossed vertex and dropped to data the momentary velocity suitable with first momentary velocity, represent the target enzymic activity with the maximum instantaneous velocity on para-curve summit; This data analysing method is used for conjugate enzyme reaction assay target enzymic activity and can significantly improves the linear response upper limit or reduce the toolenzyme consumption, and this data analysing method advantage is more obvious at most more for the coupling toolenzyme; Be used to analyze with the identical experiment condition of classical initial velocity method under gpt coupling serum lactic dehydrogenase response curve can improve the linear response upper limit more than 1 times.
Description
TECHNICAL FIELD OF THE INVENTION
The present invention mainly reaches stable state required time of lag of effect according to conjugate enzyme reaction, thereby measures the method for target enzymic activity with the maximum instantaneous velocity of conjugate enzyme reaction, can be used for fields such as biological medicine fundamental research, Clinical Laboratory, environmental health monitoring and surveilliance.
The background technology of invention
When the used substrate of the active enzyme of required mensuration and product all are difficult to directly measure, utilize toolenzyme to act on corresponding product so that the speed that the easy quantitative amount of product of available conventional equipment generates is a kind of practical approach, be called the coupling enzyme process.Coupling toolenzyme commonly used comprises the enzyme of desaturase or other production colour developing product, so that by the effect to target enzyme product such as desaturase, make the indication substrate or the indication product NADH (NADPH) of desaturase change, but the activity of target enzyme to be measured is calculated in the variation of continuous monitoring NADH or NADPH absorption like this.Also can use the final coloured product that generates of peroxidase determination in the coupling enzyme process, but its reaction is not easy to continuous monitoring usually.The conjugate enzyme determination techniques is widely used in biological medicine fundamental research and Clinical Laboratory, for example coupling serum lactic dehydrogenase (LDH) is measured gpt (ALT) activity, coupling malate dehydrogenase (malic acid dehydrogenase) (MDH) is measured the glutamic-oxal(o)acetic transaminase activity, and two kinds of toolenzymes of coupling glucokinase and glucose-6-phosphate dehydrogenase are measured alpha-amylase activity etc. simultaneously.
Analyze the basic theories of conjugate enzyme reaction assay target enzymic activity according to classical initial velocity method, for the signal pace of change that makes toolenzyme act on can directly to measure material to cause is directly proportional with reaction system target enzyme reaction to be determined initial velocity, be that the conjugate enzyme reaction system reaches stable state, the active of used tool enzyme usually need be very high, could shorten the conjugate enzyme reaction system and reach the needed time of stable state, reduce reaction system and reach the consumption of indication substrate before the stable state and ensure that can to survey the upper limit high as far as possible.
When using single toolenzyme, institute's target enzymic activity of surveying linearity upper limit is no more than 1% of toolenzyme maximum activity usually, makes that the consumption of toolenzyme is very big usually and cost is very high just can reach needed linearity range.When using a plurality of toolenzyme, cost is just more obvious.Therefore, how to reduce the consumption of toolenzyme and ensure even widen linearity range, the especially linear upper limit is used conjugate enzyme technical measurement target enzymic activity to routine and is had important value.
The present invention reaches the stable state phenomenon of required time of lag according to the conjugate enzyme reaction process, propose to analyze the momentary velocity of whole process, the method of representing the target enzymic activity with maximum instantaneous velocity, thereby under the same tool enzyme dosage, significantly improve the conjugate enzyme determination of activity upper limit, perhaps reduce the toolenzyme consumption and obtain identical linearity range with corresponding cost.
But data analysing method of the present invention only is applicable to the conjugate enzyme reaction system of continuous monitoring reaction process, can be used for the fundamental research of Clinical Laboratory, biological medicine etc.
Summary of the invention
The present invention utilizes the conjugate enzyme reaction process to reach stable state need be than the phenomenon of long delay time, shorten the logging interval of continuous monitoring reaction process or the sampling interval that interpolation shortens analyzed data, thereby determine the initial velocity (activity) of target enzyme to be measured by the maximum instantaneous velocity in the analytical reaction process, thereby the toolenzyme with same amount significantly improves the linear upper limit that the conjugate enzyme reaction system can be surveyed the target enzymic activity, perhaps significantly reduces the consumption of toolenzyme and cost and keeps the linear upper limit of the suitable target surveyed enzymic activity simultaneously; Its advantage is obvious more when the toolenzyme Michaelis-Menton constant is high more, and the toolenzyme of use is obvious more more at most.
Analysis mode conjugate enzyme response data is found, when in the raw data during no outlier point in the conjugate enzyme reaction process momentary velocity fall after rising, the centre is kept stable, be that reaction system is in stable state and momentary velocity is constant substantially, so adopt parabolic equation match momentary velocity in stable state or determine the maximum instantaneous velocity of para-curve summit correspondence near the variation tendency between steady-state zone.
Core of the present invention is a data analysis technique, and except the sampling interval that requires analyzed data was short, all the other can be identical with classical initial velocity method.But after using this method to ensure required linearity range, the consumption of toolenzyme can be optimized minimizing.For example, for measure gpt then use with the identical experiment condition of classical initial velocity method to improve the survey line upper limit; Then can reduce the toolenzyme consumption during for the mensuration glutamic-oxal(o)acetic transaminase also reduces cost; Then can use identical experiment condition also to significantly improve the linear upper limit that to survey the target enzymic activity when measuring alpha-amylase activity for a plurality of toolenzymes of coupling.
Representative applications process of the present invention comprises the steps:
(1) according to preparing the reaction system of conjugate enzyme, perhaps designs coupling toolenzyme consumption voluntarily to reduce the toolenzyme cost with the identical condition of classical initial velocity method;
(2) start in the reaction 30s with no longer than 30s continuous recording reaction process (accompanying drawing 1 and accompanying drawing 5) at interval; But 10s interval sampling when analyzing one by one on the automatic biochemistry analyzer, parallel analysis can be with 30s or longer interval;
(3) the interior reaction process data of 5.0min are arrived in continuous monitoring 3.0 altogether;
(4) judge whether sampling interval meets the requirements, if sampling interval is no longer than 10s then do not need to carry out interpolation processing, if be longer than 10s then need to carry out interpolation processing to shorten sampling interval;
(5) corresponding four the consecutive number strong points of regression analysis starting point obtain corresponding momentary velocity (analyzing as accompanying drawing 2 with Excel);
(6) successively to adjacent four data point regression analyses, judge whether the coefficient of determination of regression analysis meets the requirements, obtain regression analysis satisfactory reaction process momentary velocity changing trend diagram (accompanying drawing 3 and accompanying drawing 6);
(7) behind the selective reaction 1.0min and to cross momentary velocity suitable with original speed in the maximum instantaneous velocity decline process be stop, fitting of parabola is determined the maximum instantaneous velocity (accompanying drawing 3) of summit correspondence;
(8) above-mentioned analytic process available dedicated program or in common softwares such as Excel, realize all.
Description of drawings
340nm absorbs and changes in Fig. 1 purifying ALT coupling 1200U/LLDH reaction process
Fig. 2 tabulates with the data-switching of the reaction process that writes down in the Excel analysis chart 1
The momentary velocity variation tendency of data correspondence and fitting of parabola figure in Fig. 3 analysis chart 1
Fig. 4 measures the purifying ALT amount response curve of the initial velocity of purifying ALT to the reaction system demarcation
340nm absorption variation in the ALT coupling 1200U/L LDH reaction process in the clinical serum specimen of Fig. 5
The momentary velocity variation tendency of data correspondence and fitting of parabola figure in Fig. 6 analysis chart 4
Specifically execute routine mode
Embodiment 1: analyze purifying ALT coupling LDH reaction process and measure the ALT activity
1. test the record of response curve
(1) adopts identical experiment condition with classical initial velocity method analysis ALT coupling LDH, comprise temperature, substrate, toolenzyme and pre-reaction eliminate may disturbing of assorted enzyme etc. (details referring to: leaf should be lovely, Wang Yusan. " national Clinical Laboratory working specification ". second edition. Jiangsu: press of Southeast China University, 1997, pp204-206);
(2) 30s began after the adding α-Tong Wuersuan started the ALT reaction, with the variation of 340nm absorption in the 5 interval continuous monitoring reaction process; The reaction process that writes down at interval in the 5.0min with 5s absorbs variation (accompanying drawing 1).
2. the data analysis of reaction process
(1) as with monitoring reaction course at interval more than the 10s, then by the cubic polynomial interpolation, per three source recording points are carried out interpolation, and to reach logging interval be 5s;
(2) with logging interval be the data of 5s, obtain momentary velocity since four consecutive point of second data point regression analysis;
(3) analyze the momentary velocity (accompanying drawing 2) of per four consecutive point with Excel;
(4) be not less than 0.95 momentary velocity with the regression analysis coefficient of determination and make trend map, drop to suitable point to it from first and use fitting of parabola, represent ALT activity (accompanying drawing 3) with the maximum instantaneous velocity of para-curve summit correspondence;
(5) measure gained ALT initial velocity to purifying ALT amount response curve (accompanying drawing 4, the linear upper limit can reach 80U/L).
Embodiment 2: analyze ALT coupling LDH reaction process mensuration ALT activity in the clinical serum specimen
1. test the record of response curve
(1) adopts and the identical experiment reaction conditions of purifying ALT; May disturbing of assorted enzyme eliminated in pre-reaction;
(2) 30s began after the adding α-Tong Wuersuan started the ALT reaction, with the variation (accompanying drawing 5) of 340nm absorption in the 5s interval continuous monitoring 5.0min reaction process.
2. the data analysis of reaction process
(1) obtains momentary velocity since four consecutive point of second data point regression analysis;
(3) with reference to purifying ALT reaction process analytical procedure, analyze the momentary velocity of per four consecutive point correspondences successively, the coefficient of determination of affirmation regression analysis is not less than 0.95 momentary velocity and is used in subsequent analysis;
(2) put it from first momentary velocity and drop between suitable point and use fitting of parabola, represent ALT activity (accompanying drawing 6) with the maximum instantaneous velocity of para-curve summit correspondence.
Claims (7)
1, a kind of data analysing method is used for determining that by the short conjugate enzyme experiment reaction process data of regression analysis sampling interval maximum instantaneous velocity represents conjugate enzyme reaction system target enzymic activity;
2, described according to claim 1, this data analysing method is characterised in that:
(1) is applicable to the conjugate enzyme reaction process of analyzing the one or more toolenzymes of coupling;
(2) this type of its reaction process of conjugate enzyme reaction needed applicatory continuous monitoring;
(3) used data analysing method mainly is the maximum value that interpolation, regression analysis and fitting of parabola are asked the summit correspondence;
(4) analyzed conjugate enzyme reaction process sampling interval should be shorter, then needs to obtain the short response data of sampling interval by interpolation with longer sampling interval and analyze with this method;
3, described according to claim 1 and claim 2, this data analysing method is characterised in that:
(1) but the experiment reaction process data logging of direct analysis at interval should be no longer than 10s;
(2) after 10s is longer than at the interval of write down experiment reaction process data, obtain sampling interval by modes such as batten difference, polynomial interpolations to analyze with this method again no longer than the reaction process data of 10s;
(3) the corresponding original experimental data sampling interval of this data analysing method should not be longer than 30s, otherwise advantage can weaken;
4, described according to claim 1 and claim 2, this data analysing method is characterised in that:
(1) be applicable to the enzyme reaction process data analysis of coupling toolenzyme, its quantity to the coupling toolenzyme does not have significant limitation, but the coupling toolenzyme write down the total time of reaction process more for a long time should be long more, otherwise its advantage in using can weaken; Usually record total time arrives 6.0min at 3.0min;
(2) the necessary continuous monitoring of the suitable conjugate enzyme reaction process of institute is so use desaturase always in institute's coupling toolenzyme so that detect substrate NADH (or NADPH) or product NADH (or NADPH) tracking reaction process;
(3) also available other of suitable conjugate enzyme reaction process be convenient to the toolenzyme that continuous detecting product or substrate change;
(4) available absorption, fluorescence, luminous, electrochemistry etc. can obtain the technical monitoring reaction process that reliable signal changes;
5, described to claim 3 according to claim 1, this data analysing method is characterised in that:
(1) continuous recording conjugate enzyme reaction process should not be longer than 30s time of lag, can start the reaction 20s after with regard to the opening entry reaction process; As write down and be longer than the advantage that 30s can reduce this data analysing method time of lag;
(2) can analyze since the 3rd record data point usually; As being longer than 30s the time of lag of continuous recording reaction process and logging interval is longer than 10s, analyze since second record data point;
(3) to the experiment gained or by the reaction process data of interpolation gained sampling interval no longer than 10s, from aforesaid analyzed starting point, determine that by regression analysis the corresponding V-bar of adjacent four record data points is as the corresponding momentary velocity of first point in these four consecutive number strong points; The momentary velocity of the whole process of analysis that the rest may be inferred; Last three data points are not analyzed in the analyzed conjugate enzyme reaction process data;
(4) coefficient of determination of any adjacent four data of regression analysis needs greater than 0.95, otherwise needs to confirm whether the corresponding raw data of analyzed data point be outlier (outlier); If outlier point then this momentary velocity determine should be omitted in the maximum instantaneous velocity process at fitting of parabola subsequently;
(5) maximum instantaneous velocity is determined in the variation of fitting of parabola momentary velocity; The fitting of parabola scope be from initial momentary velocity to 1.0min after and momentary velocity drop to and the initial suitable position of momentary velocity, make to be become the bell jar type to change substantially by the momentary velocity in the fitted area; Obtain its equation of change with this section of fitting of parabola bell jar type data, determine that the corresponding maximum instantaneous velocity in para-curve summit represents the target enzymic activity; This fitting of parabola momentary velocity variation tendency can be eliminated the influence that the outlier point brings in the measuring data;
6, described to claim 5 according to claim 1, the characteristic application process of this data analysing method is:
(1) according to preparing the reaction system of conjugate enzyme, perhaps reduces the toolenzyme consumption and ensure identical linearity range to reduce cost with the identical condition of classical initial velocity method;
(2) start reaction 30s with interior beginning with no longer than 30s continuous recording reaction process at interval; Can use the 10s sampling interval when on automatic biochemistry analyzer, analyzing one by one, parallel record 30s then commonly used or longer interval; At the interval sampling about available 5s on the common spectrophotometer;
(3) write down 3.0 to 6.0min altogether; If sampling interval no longer than 10s then do not need to carry out interpolation processing, obtains the data that sampling interval is no more than 10s if be longer than 10s then need to carry out interpolation processing;
(4) from deciding starting point, the regression analysis data obtain momentary velocity in the reaction process, judge the coefficient of determination;
(5) determine the fitting of parabola scope, represent the target enzymic activity with the maximum instantaneous velocity of para-curve summit correspondence;
7, described to claim 3 according to claim 1, the application characteristic of this data analysing method comprises:
(1) need carry out interpolation when longer except logging interval and obtain desired data and need the specific routine, other data analysis process available dedicated program also can realize in as common softwares such as Excel.
(2) this data analysing method is used to analyze the conjugate enzyme reaction process and can significantly reduces the toolenzyme consumption and improve the linear upper limit of target enzyme assay; The linear upper limit of target enzymic activity can reach 10% of toolenzyme during usually with single toolenzyme, and toolenzyme is to the avidity of middle substrate low more (being that its Michaelis-Menton constant is high more), and then advantage is obvious more; Advantage is also obvious more more at most for the toolenzyme that uses.
(3) this method can be used for analyzing the Clinical Laboratory field to measure coupling desaturases such as serum glutamic pyruvic transminase, glutamic-oxal(o)acetic transaminase, α-Dian Fenmei is the conjugate enzyme reaction system of toolenzyme, measures the linear upper limit of target enzymic activity and keeps the linear lower limit suitable with classical initial velocity method thereby significantly improve this class conjugate enzyme reaction system.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104991056A (en) * | 2015-08-05 | 2015-10-21 | 武汉林勉生物技术有限公司 | Method for serological test and quantitative analysis |
| CN106323963A (en) * | 2016-08-19 | 2017-01-11 | 基蛋生物科技股份有限公司 | Multilayer-film dry chemical reagent strip for measuring glutamic-pyruvic transaminase by using enzyme coupled continuous monitoring assay |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1641041A (en) * | 2004-05-18 | 2005-07-20 | 王尔中 | Adenosin-deaminase diagnostic kit and adenosine deaminease activity determining method |
| CN101173889B (en) * | 2007-11-30 | 2012-07-11 | 重庆医科大学 | Method for measuring enzymatic activity by integration method and initial rate method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104991056A (en) * | 2015-08-05 | 2015-10-21 | 武汉林勉生物技术有限公司 | Method for serological test and quantitative analysis |
| CN106323963A (en) * | 2016-08-19 | 2017-01-11 | 基蛋生物科技股份有限公司 | Multilayer-film dry chemical reagent strip for measuring glutamic-pyruvic transaminase by using enzyme coupled continuous monitoring assay |
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