CN101597636B

CN101597636B - Method for measuring target enzyme activity by using maximum instantaneous velocity of coupling enzyme reaction

Info

Publication number: CN101597636B
Application number: CN200910104280XA
Authority: CN
Inventors: 廖飞; 赵运胜; 杨晓兰; 桑宇; 谢燕玲; 廖娟; 刘北忠; 于明安
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2009-07-08
Filing date: 2009-07-08
Publication date: 2012-10-31
Anticipated expiration: 2029-07-08
Also published as: CN101597636A

Abstract

The invention discloses a data analysis method used for measuring target enzyme activity by using coupling enzyme reaction in the field of biological medicaments. The method comprises the following steps: recording a coupling enzyme reaction process in an interval of not more than 10 seconds after the time delay of not more than 30 seconds or interpolating to acquire reaction data in a sampling interval of not more than 10 seconds, and analyzing a corresponding average velocity of adjacent four data points as a first point instantaneous velocity through regression; and analyzing the change of the instantaneous velocity along with the reaction, fitting the data between the first instantaneous velocity to an instantaneous velocity which surpasses the maximum and falls to be equivalent with the first instantaneous velocity after 1.0 minute of the reaction by using a parabola, and using the maximum instantaneous velocity at the vertex of the parabola to express the target enzyme activity. The data analysis method used for measuring the target enzyme activity by using the coupling enzyme reaction can remarkably improve the upper limit of linear response or reduce the using amount of tool enzymes; if the coupling tool enzymes are more, the advantage of the data analysis method is more obvious; and the data analysis method used for analyzing a glutamic-pyruvic transaminase coupling lactate dehydrogenase reaction curve under the same experimental condition as the classical initial velocity method can improve the upper limit of the linear response by more than one time.

Description

Measure the method for target enzymic activity with the maximum instantaneous velocity of conjugate enzyme reaction

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 through 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; It is active that coupling MDH (MDH) is measured glutamic-oxal(o)acetic transaminase, and two kinds of toolenzymes of coupling gk 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; It is very high that used tool enzyme active needs usually, could shorten the conjugate enzyme reaction system and reach the needed time of stable state, and the minimizing reaction system reaches the consumption of the preceding indication substrate of stable state and ensures 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, make toolenzyme consumption usually very greatly 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; The logging interval or the interpolation that shorten the continuous monitoring reaction process shortened by the SI of analytical data; Thereby confirm the initial velocity (activity) of target enzyme to be measured through 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 consumption and the cost of toolenzyme 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 basically, so adopt parabolic equation match momentary velocity to confirm the maximum instantaneous velocity that the para-curve summit is corresponding in stable state or near the variation tendency between steady-state zone.

Core of the present invention is a data analysis technique, except require by the SI of analytical data 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 property 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 the SI meet the requirements, if the SI is no longer than 10s then need not carry out interpolation processing, if be longer than 10s then need carry out interpolation processing to shorten the SI;

(5) corresponding four the consecutive number strong points of regression analysis starting point obtain corresponding momentary velocity (analyzing like accompanying drawing 2 with Excel);

(6) successively to adjacent four data points regression analyses, judge whether the coefficient of determination of regression analysis meets the requirements, obtain the satisfactory reaction process momentary velocity of regression analysis 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 confirmed the maximum instantaneous velocity (accompanying drawing 3) that the summit is corresponding;

(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 is with the data-switching tabulation of the reaction process that writes down in the Excel analysis 1

Corresponding momentary velocity variation tendency and the fitting of parabola figure of data in Fig. 3 analysis 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

Corresponding momentary velocity variation tendency and the fitting of parabola figure of data in Fig. 6 analysis 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; But comprise temperature, substrate, toolenzyme and pre-reaction eliminate the interfere 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 through the cubic polynomial interpolation, per three source recording points are carried out interpolation, and to reach logging interval be 5s;

(2) use the data of logging interval, obtain momentary velocity since four consecutive point of the second data points regression analysis as 5s;

(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 active (accompanying drawing 3) with the corresponding maximum instantaneous velocity in para-curve summit;

(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; But the interfere of assorted enzyme is 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 the second data points regression analysis;

(3) with reference to purifying ALT reaction process analytical procedure, analyze the corresponding momentary velocity of per four consecutive point 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 active (accompanying drawing 6) with the corresponding maximum instantaneous velocity in para-curve summit.

Claims

1. a conjugate enzyme of lacking through the regression analysis SI is tested the method that reaction process is confirmed maximum instantaneous velocity mensuration conjugate enzyme reaction system target enzymic activity, it is characterized in that:

(1) according to preparing the reaction system of conjugate enzyme, perhaps reduces the toolenzyme consumption and keep identical linear response 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, utilization can obtain absorption, fluorescence, the luminous or electrochemical techniques monitoring reaction course that reliable signal changes; On automatic biochemistry analyzer, use the 10s SI during analytic sample one by one, and use the 30s SI during the different sample of parallel record; On common spectrophotometer, use the SI of 5s;

(3) write down 3.0 to 6.0min altogether; If the SI is no longer than 10s then need not carry out interpolation processing, analyze with this method again no longer than the reaction process data of 10s if be longer than 10s then need obtain the SI through batten difference or polynomial interpolation mode;

(4) analyze since the 3rd record data point; Be longer than 10s then since second record data point analysis as being longer than 30s and logging interval the time of lag of continuous recording reaction process;

(5) to the experiment gained or through the reaction process data of interpolation gained SI no longer than 10s; The record data point beginning that quilt from step (4) is analyzed confirms that through 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; Do not analyzed by last three data in the analysis conjugate enzyme reaction process data;

(6) coefficient of determination of any adjacent four data of regression analysis needs greater than 0.95, otherwise needs to confirm whether put corresponding raw data by analytical data be outlier; If outlier then this momentary velocity confirm to be omitted in the maximum instantaneous velocity process at fitting of parabola subsequently;

(7) maximum instantaneous velocity is confirmed in the variation of fitting of parabola momentary velocity; The scope of fitting of parabola 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 basically by the momentary velocity in the fitted area; Obtain its equation of change with this section of fitting of parabola bell jar type data, confirm that the corresponding maximum instantaneous velocity in para-curve summit representes the target enzymic activity.

2. claim 1 is described tests the method that reaction process is confirmed maximum instantaneous velocity mensuration conjugate enzyme reaction system target enzymic activity through short conjugate enzyme of regression analysis SI, it is characterized in that:

(1) this method is applicable to the conjugate enzyme reaction process of analyzing the one or more toolenzymes of coupling;

(2) data analysis process can use Excel software to realize;

(3) this data analysing method is used to analyze the conjugate enzyme reaction process, can significantly reduce the toolenzyme consumption and improves the linear response upper limit of target enzyme assay; Toolenzyme is low more to the avidity of middle substrate, and then the advantage of this data analysing method raising mensuration target enzymic activity linear response upper limit is obvious more; The toolenzyme that uses more at most this data analysing method to improve the advantage of measuring the target enzymic activity linear response upper limit also obvious more;

(4) this method can be used for analyzing that the Clinical Laboratory field measures with the coupling desaturase is the conjugate enzyme reaction system of serum glutamic pyruvic transminase, glutamic-oxal(o)acetic transaminase or the AMS of toolenzyme, measures the linear response upper limit of target enzymic activity and keeps the linear response lower limit suitable with initial velocity method thereby significantly improve this type conjugate enzyme reaction system.