CN1217006C - Method for quantitative determination of biochemical substance by enzyme analysis via predicting background - Google Patents
Method for quantitative determination of biochemical substance by enzyme analysis via predicting background Download PDFInfo
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- CN1217006C CN1217006C CN 03135649 CN03135649A CN1217006C CN 1217006 C CN1217006 C CN 1217006C CN 03135649 CN03135649 CN 03135649 CN 03135649 A CN03135649 A CN 03135649A CN 1217006 C CN1217006 C CN 1217006C
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Abstract
The present invention relates to a method for quantitative measurement of biochemical substances by enzymatic analysis through predicting background. The present invention has the technical scheme that a buffer solution and a tool enzyme are added to a sample solution to be measured; measurement light absorption changes by following reaction time, and an integral rate equation to fit and analyze of the descending reaction curve of light absorption consumed by substrates is used for predicting the background, instead of directly measuring the background. The present invention has the advantages of high analysis speed, low cost, no sensitivity on enzyme activity variation, resistance of interference factor influencing enzyme activity, high maximum measurement limit to reach five times of uricase michaelis constant, and high measuring precision; none of liquid samples needs diluting.
Description
Technical field
The present invention relates to field such as Clinical Laboratory and measure the method for uric acid content in the body fluid, especially carry out the method for uric acid content in the enzymatic analysis quantitative assay body fluid by the prediction background.
Background technology
Enzymatic analysis is measured content of predetermined substance and is had very high selectivity, is used for measuring the specific biochemical substances of mixture and has important value.The existing enzymatic analysis method of measuring specific biochemical substances content in the mixture mainly contains two classes: kinetics methodology and terminal point null readings.The terminal point null readings needs the analysis of high density with toolenzyme and very long reaction times, could guarantee that substrate is transformed into product fully, measures the variation of respective detection index again.The testing index that this method need be measured before and after the enzyme reaction changes, and could eliminate the interference of background.This method cost height, analysis efficiency is low, is unfavorable for automated analysis.Current can the conventional kinetics methodology of using mainly be to utilize concentration of substrate to be lower than enzyme K
m10% o'clock initial velocity and enzyme substrates concentration be directly proportional, infer original amount of substrate by the initial velocity of assaying reaction system.Opposite with the terminal point null readings, this kinetics methodology analysis speed is fast, and cost is low, and is insensitive to background.But this method is all very sensitive to all factors that influence enzymic activity, and the mensuration upper limit is lower than enzyme K
m10%.Competitive inhibitor at present commonly used increases the K that analyzes with toolenzyme
mImprove the mensuration upper limit, or eliminate this KINETIC METHOD by various rate equation least square fitting enzyme analysis response curves the enzymic activity influence factor is disturbed.But no matter use the differential rate equation, it is the Michaelis-Menton equation of enzyme, still predict that with integrating rate equation model response curve initial amount of substrate still is maximum product amount, all can't guarantee the reasonable accuracy of measurement result simultaneously, insensitive to background again, insensitive to the interfering factors that influences enzymic activity, and have the ideal mensuration upper limit.Therefore, all current kinetics methodologys still have certain defective.
For example, measuring the monitoring that uric acid content in serum and the urine is widely used in gout and renal function at present, is the conventional project of Clinical Laboratory.The method of mensuration uric acid has a variety of, uses uriKoxidase to carry out enzymatic analysis and just has selectivity and specificity.According to the quantitative object of used instrument, can be divided into and utilize 293nm uv-absorbing direct quantitative substrate and measure product quantitative two classes indirectly in the practice.At present the method for measuring the substrate direct quantitative can only be carried out enzymatic analysis with the terminal point null readings, needs with the reaction of high density uriKoxidase for a long time, independently measuring background and eliminate the interference of background.Very sensitive to background during the direct quantitative substrate with kinetics methodology, and it is too low and do not have a practical significance to measure the upper limit.Measure the indirect quantitative methods usable power of product method, but cost height and interfering factors are many; As very sensitive to background again, and need the very long reaction times with the terminal point null readings, can't automatic assay.Therefore, need to improve kinetics methodology and carry out enzymatic analysis and measure the upper limit of uric acid content and measure precision, and synchronism eliminates is to the susceptibility of background, but just can become the method for the conventional practice of easy automatization.
Summary of the invention
The objective of the invention is to improve and carry out the upper limit and the precision that enzymatic analysis is measured uric acid content in the body fluid, eliminate simultaneously to background and to influencing the susceptibility of the active interfering factors of uriKoxidase with KINETIC METHOD.
Technical scheme of the present invention is: carry out the method for uric acid content in the enzymatic analysis quantitative assay body fluid by predicting background, it is characterized in that carrying out as follows:
(1), getting a certain amount of humoral sample adds in vitro;
(2), getting a certain amount of needed damping fluid adds in vitro identical;
(3), with the abundant mixing of above-mentioned solution;
(4), above-mentioned system is selected certain temperature constant temperature 5～10 minutes between 10～37 ℃;
(5), at the above-mentioned required quantitative analysis uriKoxidase solution that in vitro adds, mixing immediately, and with the moment that adds uriKoxidase be that reaction starting point picks up counting, rapidly solution is shifted entering cuvette simultaneously;
(6), pick up counting, beginning after 15 seconds with the specified time between 5～60 seconds serves as that recording light absorbs and changes at interval, continues record 6 minutes, obtains the two-dimensional array Af (tf that reaction times and photoabsorption constitute from reaction starting point
i, Af
i);
(7), absorb A with corresponding linear regression Calculation machine program prediction initial light
0, f, the photoabsorption that has just begun in first minute according to reaction changes, and carries out linear regression analysis and obtains regression equation A
i=a+b * t, wherein, a is the intersection point of regression equation at the longitudinal axis, b is the linear response coefficient of regression equation and is negative value that the response delay time t1 that determines according to experiment is extrapolated to reaction starting point, calculates the initial substrate photoabsorption A of this reaction starting point correspondence
0, f
(8), the data filter pre-treatment removes the data point of no significant difference, get to start 40 seconds later some data points of reaction and be first point of another new array At (ts
1, At
1), search the second point (ts of the decline of photoabsorption thereafter greater than the new array of closest approach conduct of certain boundary
2, At
2); By that analogy, with the gained data storing be new array At (t
i, At
i), the data point of this new array must be no less than 5, this boundary is selected between 0.002～0.005, to given curve, this boundary is a determined value, and different curves can be different, differ by more than more than 0.600 to last point that is write down from first that writes down, then selecting this boundary is 0.005, if be 0.002 from first of record to differing less than 0.100 then select this boundary last point that is write down, the rest may be inferred by analogy for it;
(9), data fitting prediction background A
B, f, after the data-switching with corresponding be the integrating rate equation model response curve of independent variable(s) with reaction times, promptly with disregarding reversed reaction list substrates enzymes Michaelis-Menton kinetics equation integrated form ln[(A
I, 0-A
B, f)/(A
T, i-A
B, f)]+(A
I, 0-A
T, i)/(ε * K
m)=(V
m/ K
m) * ts
i, A in the equation
I, 0Be the photoabsorption of first point of the new array At behind the data filter on the analyzed curve, A
B, fBackground for the corresponding reaction system of analyzed curve with the new array At of this integrating rate equation nonlinear least square fitting, carries out data-switching and match in the corresponding computer program, its process comprises:
The first layer circulation, constant step size is progressively regulated nonlinear parameter A downwards
B, f:
Regulating A
B, fStep-length constant be 0.001 progressively to regulate downwards at given range, regulate A
B, fStarting point for absorb for a short time by 0.002 than analyzed corresponding minimum light, terminal point is difference with the difference of body fluid sample;
As add in the humoral sample afterreaction system uric acid concentration to be determined greater than used analysis with uriKoxidase Michaelis-Menton constant K
m1.5 times, and contain competitive inhibitor or the noncompetitive inhibitor of analyzing with uriKoxidase, then K in the system
mBe parameter, enter second layer circulation, otherwise do not enter this second layer circulation; Enter this layer and circulate then that constant step size is enzyme K
m2% to 10%, progressively raise K
mTo its 3 times of K
m
If do not enter this second layer circulation, then K
mBe default known constant, but its K of different uriKoxidase
mDifferent;
Molar extinction coefficient (ε) is selected generally acknowledged reference value for use, is used for data-switching and response curve match;
Given A
B, fAnd K
mAfter be calculated as follows y
i
y
i＝ln(A
i，0-A
b，f)+A
i，0/(ε×K
m)-ln(A
t，i-A
b，f)-A
t，i/ε×K
m
Can determine y by the propagation of error rule
iError is:
σ
y＝-σ×(ε×K
m+A
t，i-A
b，f)/[(A
t，i-A
b，f)×ε×K
m]
Wherein the random error of σ for measuring is generally 0.001, can get y by this formula
iError, with y
iThe inverse of square-error is corresponding weighting factor, presses y
i=a+b * ts
iCarry out conventional linear least square fitting response curve, calculate the match corresponding variance and analyze the F value;
Regulate K if enter
mSecond layer circulation, then at given A
B, fK is regulated in the back in setting range
mThe maximum F value of gained is returned the first layer circulation, as given A
B, fThe F value of gained; And then A is progressively regulated in circulation
B, f, relatively gained F value is big or small, seeks the parameter A of corresponding maximum F value
B, fAnd K
mCombination;
Do not regulate K if enter
mSecond layer circulation, then directly with the known constant K that sets
mRegulate the A that provides with circulation
B, fCalculate the F value of corresponding match; Relatively gained F value is big or small, seeks the parameter A of corresponding maximum F value
B, f
(10), according to the A that promptly analyzes same reaction curve gained from the same reaction mixture system
0, fAnd A
B, fCalculate its difference Δ A,, calculate the uric acid concentration to be measured of reaction system, calculate the concentration of uric acid to be measured according to the amount of the contained humoral sample of reaction system by the material optical extinction coefficient of respective standard curve or setting.
Be the curve of base consumption in the integrating rate equation least square fitting enzyme reaction process of independent variable(s) in order to the reaction times in the step of the present invention (nine), the background when thereby pre-measured reaction reaches complete, body fluid is urine among the present invention, the amount that then adds urine is 6 μ l, and analyzing with uriKoxidase solution in the step (five) is 24 μ l, concentration is the Cndida Utilis uriKoxidase solution of 0.8-1.0U/ml, the timed interval in the step (six) is 30 seconds, gets 40 seconds data point of startup reaction in the step (eight) and is first point of another new array At (ts
1, At
1); If body fluid is blood plasma, the amount that then adds blood plasma is 20 μ l, and analyzing with uriKoxidase solution in the step (five) is 10 μ l, concentration is the Cndida Utilis uriKoxidase solution of 0.8-1.0U/ml, the timed interval in the step (six) is 15 seconds, gets 45 seconds data point of startup reaction in the step (eight) and is first point of another new array At (ts
1, At
1).Damping fluid is that concentration is 50mmol/L in the step (two), contains 50 μ mol/L iron ion complexing agent diethyl triamino pentaacetic acids, and pH is 9.2 borate buffer 1.17ml, is 25 ℃ of constant temperature water baths 10 minutes in the step (four).
Present method is also measured other enzymatic analysis system of background applicable to needs.
The present invention is used for testing uric acid, and just can directly to measure uric acid quantitative in the uv-absorbing of 293nm, determines that according to the initial velocity linear extrapolation initial light absorbs A during manual operations
0, f, independently measure the preceding A of enzyme-added startup reaction when automated analysis is measured
0, f, be the integrating rate equation model homeostatic reaction curve prediction background A of independent variable(s) in order to the reaction times
B, f, with A
0, fWith A
B, fDifference represent the photoabsorption of uric acid in the reaction system, determine uric acid concentration according to the typical curve of Lambert-Beer law or measuring.This law is predicted background with integrating rate equation model enzyme analysis response curve, rather than directly measures background.Simultaneously, prediction starting point substrate photoabsorption but not directly measure initial substrate photoabsorption, i.e. same response curve prediction starting point photoabsorption and terminal point photoabsorption from being write down.Obviously, there is tangible covariance between starting point of being predicted and the terminal point.The difference that this method is measured starting point and terminal point photoabsorption with the low activity uriKoxidase is represented amount of substrate, and correspondence is measured ratio of precision terminal point null readings height, and the upper limit that the mensuration upper limit is measured uric acid method in animal body fluids or the tissue extract than all that report at present is all high.
The most significant advantage of the present invention is: cost is lower than all methods of using now, to the existing method height of the ratio of precision of lower concentration humoral determination, directly measuring uric acid absorbs quantitatively, so the accuracy height belongs to kinetics methodology, so the analysis efficiency height, the uriKoxidase activity changes does not within the specific limits have obvious influence to the result, to because the background variation that cuvette cleaning efficiency difference causes is also insensitive, measure the upper limit than the method height of using at present, can the conventional practice application.
Description of drawings
Fig. 1 is the embodiment of the invention 1 flow chart of steps;
Fig. 2 is the embodiment of the invention 2 flow chart of steps;
Fig. 3 is a type reaction graphic representation of the present invention;
Fig. 4 is a reaction starting point enlarged view among Fig. 3;
Fig. 5 is the present invention Δ A
293Response curve to uric acid content in the reaction system.
Embodiment
Embodiment 1: referring to Fig. 1, Fig. 3, Fig. 4 and Fig. 5, carry out the method that enzymatic analysis is measured uric acid content in the urine sample by the prediction background, it is characterized in that carrying out as follows:
(1), getting urine sample 6 μ l adds in vitro;
(2), get 1.17ml, concentration is 50mmol/L, contains 50 μ mol/L iron ion complexing agent diethyl triamino pentaacetic acids (DETAPA), the borate buffer of pH9.2 adds in vitro identical;
(3), above-mentioned invisible spectro mixed solution vortex is shaken abundant mixing;
(4), with above-mentioned invisible spectro mixed solution 25 ℃ of constant temperature water baths 10 minutes;
(5), in vitro adding 24 μ l concentration is the Cndida Utilis uriKoxidase solution of 0.8-1.0U/ml, also available Bacillus fastidasus uriKoxidase; Mixing immediately, and with the moment that adds enzyme be that reaction starting point picks up counting, shift entering the quartz cuvette that capacity is 2.0ml simultaneously rapidly;
(6), pick up counting, begin after 15 seconds to continue record 6 minutes, obtain the array Af (tf that reaction times and photoabsorption constitute 30 seconds to be that timed interval recording light absorbs and changes from reaction starting point
i, Af
i);
(7), absorb A with corresponding linear regression Calculation machine program prediction initial light
0, f: according to changing, carry out regression analysis and obtain regression equation A in the photoabsorption of reacting in incipient first minute
i=a+b*t, wherein, a is the intersection point of regression equation at the longitudinal axis, b is the linear response coefficient of regression equation and is negative value that the response delay time that manual operations is set is 9 seconds, the initial substrate photoabsorption A of the corresponding reaction starting point of extrapolation calculating
0, f
(8), data filter pre-treatment: get 40 seconds data point of startup reaction and be first point of another new array At (ts
1, At
1), the difference of searching photoabsorption thereafter is greater than 0.002 closest approach, the second point (ts as new array
2, At
2); By that analogy, with the gained data storing be new array At (ts
i, At
i);
(9), least square fitting prediction background A after the data-switching
B, f: after the data-switching with corresponding be the integrating rate equation of independent variable(s) with reaction times, promptly disregard the integrated form ln[(A of the single substrates enzymes Michaelis-Menton kinetics of reversed reaction equation
I, 0-A
B, f)/(A
T, i-A
B, f)]+(A
I, 0-A
T, i)/(ε * K
m)=(V
m/ K
m) * ts
i, A in the equation
I, 0Be selected new array Ab on the analyzed curve
iFirst put corresponding photoabsorption, A
B, fBackground for the corresponding reaction system of analyzed curve; With the new array At of this integrating rate equation nonlinear least square fitting
i, in the corresponding computer program, carry out data-switching and match, its process comprises:
The first layer circulation, constant step size is regulated nonlinear parameter A
B, f:
Regulating A
B, fStep-length constant be 0.001 progressively to regulate downwards at given range, regulate A
B, fStarting point for to absorb little by 0.002 than analyzed corresponding minimum light;
Second layer circulation, constant step size is 0.5 μ mol/L, progressively raises K since 12.5 μ mol/L
mTo 37 μ mol/L; Molar extinction coefficient (ε) is 11.5 (mmol/L * cm)
-1, be used for data-switching and response curve match;
Determine predefined A
B, fAnd K
mAfter be calculated as follows y
i
y
i＝ln(A
i，0-A
b，f)+A
i，0/ε×K
m-ln(A
t，i-A
b，f)-A
t，i/ε×K
m
Determine y by the propagation of error rule
iError is:
σ
y＝-σ×(ε×K
m+A
t，i-A
b，f)/((A
t，i-A
b，f)×ε×K
m)
Wherein σ gets 0.001 for measuring random error, calculates Y by this formula
iError, with y
iThe inverse of square-error is corresponding weighting factor, presses y
i=a+b * ts
iCarry out conventional linear least square fitting response curve, calculate the F statistic of match correspondence;
In above-mentioned scope, regulating A
B, fThe maximum F value of gained is returned the last layer circulation and is compared, at A
B, fAnd K
mGiven range search the parameter A of corresponding maximum F value
B, f, K
mAnd V
m/ K
mCombination;
(9), according to A
0, fAnd A
B, fCalculate its difference Δ A; Calculate the uric acid concentration of above-mentioned reaction system by its typical curve; Multiply by 200 uric acid concentrations that promptly obtain in the urine sample.
Embodiment 2: referring to Fig. 2, Fig. 3, Fig. 4 and Fig. 5, carry out the method that enzymatic analysis is measured uric acid content in the plasma sample by the prediction background, it is characterized in that carrying out as follows:
(1), getting plasma sample 20 μ l adds in vitro;
(2), get 1.17ml, concentration is the borate buffer of 50mmol/L, contains 50 μ mol/L iron ion complexing agent diethyl triamino pentaacetic acids (DETAPA), pH9.2 adds in vitro identical;
(3), above-mentioned invisible spectro mixed solution vortex is shaken abundant mixing;
(4), with above-mentioned test tube 25 ℃ of constant temperature water baths 10 minutes;
(5), in vitro adding 10 μ l concentration is the Cndida Utilis uriKoxidase solution of 0.8-1.0U/ml, also available Bacillus fastidiosus uriKoxidase; Mixing immediately, and with the moment that adds enzyme be that reaction starting point picks up counting, shift entering the quartz cuvette that capacity is 2.0ml simultaneously rapidly;
(6), pick up counting, begin after 15 seconds to continue record 6 minutes, obtain the array Af (tf that reaction times and photoabsorption constitute 15 seconds to be that timed interval recording light absorbs and changes from reaction starting point
i, Af
i);
(7), absorb A with corresponding linear regression Calculation machine program prediction initial light
0, f: according to changing, carry out regression analysis and obtain regression equation A in the photoabsorption of reacting in incipient first minute
i=a+b*t, wherein, a is the intersection point of regression equation at the longitudinal axis, the linear response coefficient of b regression equation is by the initial substrate photoabsorption A of the corresponding reaction starting point of setting of response delay time extrapolation calculating
0, f
(8), data filter pre-treatment: get 45 seconds data point of startup reaction and be first point of another new array At (t
1, At
1), the difference of searching photoabsorption thereafter is greater than 0.002 closest approach, second B as new array
2(tl
2, At
2); By that analogy, be new array At with the gained data storing
i(t
i, At
i);
(9), least square fitting prediction background A after the data-switching
B, f: after the data-switching with corresponding be the integrating rate equation of independent variable(s) with reaction times, promptly disregard the integrated form ln[(A of the single substrates enzymes Michaelis-Menton kinetics of reversed reaction equation
I, 0-A
B, f)/(A
T, i-A
B, f)]+(A
I, 0-A
T, i)/(ε * K
m)=(V
m/ K
m) * ts
i, A in the equation
I, 0For first of selected new array At on the analyzed curve put corresponding photoabsorption, A
B, fBackground for the corresponding reaction system of analyzed curve; With the new array At of this integrating rate equation nonlinear least square fitting
i, in the corresponding computer program, carry out data-switching and match, its process comprises:
Nonlinear parameter A is regulated in the constant step size circulation
B, f:
Regulating A
B, fStep-length constant be 0.001 progressively to regulate downwards at given range, regulate A
B, fStarting point little by 0.002 for absorbing than analyzed corresponding minimum light, terminal point is different and different with sample;
Get uric acid molar extinction coefficient (ε) and be 11.5 (mmol/L * cm)
-1Constant K
mBe 12.5 μ mol/L, be used for data-switching and response curve match; If with Bacillus fastidiosus uriKoxidase then constant K
mBe 100 μ mol/L;
Determine predefined A
B, fAnd K
mAfter be calculated as follows y
i
y
i＝ln(A
i，0-A
b，f)+A
i，0/ε×K
m-ln(A
t，i-A
b，f)-A
t，i/ε×K
m
Determine y by the propagation of error rule
iError is:
σ
y＝-σ×(ε×K
m+At，
i-A
b，f)/((At，
i-A
b，f)×ε×K
m)
Wherein σ gets 0.001 for measuring random error, calculates Y by this formula
iError, with Y
iThe inverse of square-error is corresponding weighting factor, presses y
i=a+b * ts
iCarry out conventional linear least square fitting response curve, calculate the F statistic of match correspondence;
In above-mentioned scope, regulating A
B, fThe maximum F value of gained is returned and is compared, at A
B, fAnd K
mGiven range search the parameter A of corresponding maximum F value
B, fAnd V
m/ K
mCombination;
(9), according to A
0, fAnd A
B, fCalculate its difference Δ A; Calculate the uric acid concentration of above-mentioned reaction system by the optical extinction coefficient of its typical curve or setting; Multiply by 60 uric acid concentrations that promptly obtain in the plasma sample.
After adopting the present invention, analyze the photoabsorption decline response curve of base consumption by the integrating rate equation model and predict background, belong to kinetics methodology, so analysis speed is fast.The present invention only needs uriKoxidase as instrument, do not need other toolenzyme, and needed uriKoxidase amount is all low when measuring the body fluid uric acid than existing all kinetics methodologys or terminal point null readings, so this invention is existing all lower with method than all with cost of conventional practice; Enzymatic analysis kinetics methodology of the present invention changes insensitive to enzymic activity, the interfering factors that influences enzymic activity is had resistance; By the direct assaying reaction curve of 293nm uv-absorbing, but the background of protein etc. is noiseless to the result; It is very high to measure the upper limit, can reach more than five times of uriKoxidase Michaelis-Menton constant; Humoral sample does not need dilution just can directly measure, and adds iron ion complexing agent (DETAPA) in the mensuration system, has guaranteed the stability of uriKoxidase in the reaction process, thereby can use integrating rate equation enzyme analysis reaction process location parameter.The method of the invention is not limited to the quantitative assay to uric acid content in the body fluid, also can be used in the mixtures such as tissue extract the quantitative assay to uric acid or other specific biochemical substances.
Claims (5)
1. one kind is carried out the method for uric acid content in the enzymatic analysis quantitative assay body fluid by the prediction background, it is characterized in that carrying out as follows:
(1), getting a certain amount of humoral sample adds in vitro;
(2), getting a certain amount of needed damping fluid adds in vitro identical;
(3), with the abundant mixing of above-mentioned solution;
(4), above-mentioned system is selected certain temperature constant temperature 5～10 minutes between 10～37 ℃;
(5), at the above-mentioned uriKoxidase solution that in vitro adds requirement, mixing immediately, and with the moment that adds uriKoxidase be that reaction starting point picks up counting, rapidly solution is shifted entering cuvette simultaneously;
(6), pick up counting, beginning after 15 seconds with the specified time between 5～60 seconds serves as that recording light absorbs and changes at interval, continues record 6 minutes, obtains the two-dimensional array Af (tf that reaction times and photoabsorption constitute from reaction starting point
i, Af
i);
(7), absorb A with corresponding linear regression Calculation machine program prediction initial light
0, f, the photoabsorption that has just begun in first minute according to reaction changes, and carries out linear regression analysis and obtains regression equation A
i=a+b * t, wherein, a is the intersection point of regression equation at the longitudinal axis, b is the linear response coefficient of regression equation and is negative value that the response delay time t1 that determines according to experiment is extrapolated to reaction starting point, calculates the initial substrate photoabsorption A of this reaction starting point correspondence
0, f
(8), the data filter pre-treatment removes the data point of no significant difference, get to start 40 seconds later some data points of reaction and be first point of another new array At (ts
1, At
1), search the second point (ts of the decline of photoabsorption thereafter greater than the new array of closest approach conduct of certain boundary
2, At
2); By that analogy, with the gained data storing be new array At (t
i, At
i), the data point of this new array must be no less than 5, this boundary is selected between 0.002～0.005, to given curve, this boundary is a determined value, and different curves can be different, differ by more than more than 0.600 to last point that is write down from first that writes down, then selecting this boundary is 0.005, if be 0.002 from first of record to differing less than 0.100 then select this boundary last point that is write down, the rest may be inferred by analogy for it;
(9), data fitting prediction background A
B, r, after the data-switching with corresponding be the integrating rate equation model response curve of independent variable(s) with reaction times, promptly with disregarding reversed reaction list substrates enzymes Michaelis-Menton kinetics equation integrated form ln[(A
I, 0-A
B, f)/(A
I, i-A
B, f)]+(A
I, 0-A
T, i)/(ε * K
m)=(V
m/ K
m) * ts
i, A in the equation
I, 0Be the photoabsorption of first point of the new array At behind the data filter on the analyzed curve, A
B, fBackground for the corresponding reaction system of analyzed curve with the new array At of this integrating rate equation nonlinear least square fitting, carries out data-switching and match in the corresponding computer program, its process comprises:
The first layer circulation, constant step size is progressively regulated nonlinear parameter A downwards
B, f:
Regulating A
B, fStep-length constant be 0.001 progressively to regulate downwards at given range, regulate A
B, fStarting point for absorb for a short time by 0.002 than analyzed corresponding minimum light, terminal point is difference with the difference of body fluid sample;
As uric acid concentration to be determined in the humoral sample afterreaction system greater than used uriKoxidase Michaelis-Menton constant K
m1.5 times, and contain competitive inhibitor or the noncompetitive inhibitor of analyzing with uriKoxidase, then K in the system
mBe parameter, enter second layer circulation, otherwise do not enter this second layer circulation; Enter this layer and circulate then that constant step size is enzyme K
m2% to 10%, progressively raise K
mTo its 3 times of K
m
If do not enter this second layer circulation, then K
mBe default known constant, but its K of different uriKoxidase
mDifferent;
Molar extinction coefficient (ε) is selected generally acknowledged reference value for use, is used for data-switching and response curve match;
Given A
B, fAnd K
mAfter be calculated as follows y
i
y
i＝ln(A
i，0-A
b，f)+A
i，0/(ε×K
m)-ln(A
t，i-A
b，f)-A
t，i/ε×K
m
Can determine y by the propagation of error rule
iError is:
σ
y＝-σ×(ε×K
m+A
t，i-A
b，f)/[(A
t，i-A
b，f)×ε×K
m]
Wherein the random error of σ for measuring is generally 0.001, can get y by this formula
iError, with y
iThe inverse of square-error is corresponding weighting factor, presses y
i=a+b * ts
iCarry out conventional linear least square fitting response curve, calculate the match corresponding variance and analyze the F value;
Regulate K if enter
mSecond layer circulation, then at given A
B, fK is regulated in the back in setting range
mThe maximum F value of gained is returned the first layer circulation, as given A
B, fThe F value of gained; And then A is progressively regulated in circulation
B, f, relatively gained F value is big or small, seeks the parameter A of corresponding maximum F value
B, fAnd K
mCombination;
Do not regulate K if enter
mSecond layer circulation, then directly with the known constant K that sets
mRegulate the A that provides with circulation
B, fCalculate the F value of corresponding match; Relatively gained F value is big or small, seeks the parameter A of corresponding maximum F value
B, f
(10), according to the A that promptly analyzes same reaction curve gained from the same reaction mixture system
0, fAnd A
B, fCalculate its difference Δ A,, calculate the uric acid concentration to be measured of reaction system, calculate uric acid concentration to be measured according to the amount of the contained body fluid of reaction system by the material optical extinction coefficient of respective standard curve or setting.
2. method of carrying out uric acid content in the enzymatic analysis quantitative assay body fluid by the prediction background according to claim 1, it is characterized in that: be the curve of base consumption in the integrating rate equation least square fitting enzyme reaction process of independent variable(s) in order to the reaction times in the step (nine), thus the background when pre-measured reaction reaches complete.
3. method of carrying out uric acid content in the enzymatic analysis quantitative assay body fluid by the prediction background according to claim 1, it is characterized in that: the uric acid that is used for measuring urine, body fluid is the urine of 6 μ l described in the step (), analyzing with uriKoxidase solution in the step (five) is that 24 μ l concentration are the Cndida Utilis uriKoxidase solution of 0.8-1.0U/ml, the timed interval in the step (six) is 30 seconds, gets 40 seconds data point of startup reaction in the step (eight) and is first point of another new array At (ts
1, At
1).
4. method of carrying out uric acid content in the enzymatic analysis quantitative assay body fluid by the prediction background according to claim 1, it is characterized in that: the uric acid that is used for measuring blood plasma, body fluid is the blood plasma of 20 μ l described in the step (), analyzing with uriKoxidase solution in the step (five) is that 10 μ l concentration are the Cndida Utilis uriKoxidase solution of 0.8-1.0U/ml, the timed interval in the step (six) is 15 seconds, gets 45 seconds data point of startup reaction in the step (eight) and is first point of another new array At (ts
1, At
1).
5. according to claim 3 or 4 described methods of carrying out uric acid in the enzymatic analysis quantitative assay body fluid by the prediction background, it is characterized in that: damping fluid is that concentration is 50mmol/L in the step (two), contain 50 μ mol/L iron ion complexing agent diethyl triamino pentaacetic acids, pH is 9.2 borate buffer 1.17ml, is 25 ℃ of constant temperature water baths 10 minutes in the step (four).
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| CN102507470B (en) * | 2011-10-20 | 2014-09-24 | 重庆医科大学 | Method for measuring enzyme substrate amount by combining enzyme reaction process method and terminal balance method |
| CN103837487A (en) * | 2014-03-19 | 2014-06-04 | 潍坊鑫泽生物科技有限公司 | Uric acid detection method and detection kit |
| CN105466927B (en) * | 2014-07-02 | 2020-04-10 | 深圳迈瑞生物医疗电子股份有限公司 | Method for identifying, correcting and alarming abnormal reaction curve of turbidimetry |
| CN116793976B (en) * | 2023-06-29 | 2024-05-10 | 徐詹程 | Method for analyzing substrate concentration fitting standard curve through enzymatic reaction |
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