CN110426518A - A kind of method of DPP-4 enzymatic activity in quantitative detection human plasma - Google Patents
A kind of method of DPP-4 enzymatic activity in quantitative detection human plasma Download PDFInfo
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Abstract
The present invention relates to technical field of analysis and detection, in particular to the method for DPP-4 enzymatic activity in a kind of quantitative detection human plasma, mainly include equalized temperature, add quality-control sample and sample to be tested and sealing plate is incubated for, adds the standard sample simultaneously operating procedure of sealing plate incubation plus Gly-Pro -7- amino -4- methylcoumarin hydrobromate and sealing plate incubation, twice read plate, has the characteristics that detection cycle is short, it is convenient to operate, it is accurate sensitive to detect.
Description
Technical field
The present invention relates to technical field of analysis and detection, in particular to DPP-4 enzymatic activity in a kind of quantitative detection human plasma
Method.
Background technique
Show according to Chinese chronic diseases in 2010 and its risk factor monitoring report, the glycosuria of China 18 years old or more adult
Sick illness rate was 10.7% up to 11.6%, 2013 year, thus speculated that maturity-onset diabetes patient numbers in China's are 1.14 hundred million, it has also become
The most country of diabetic's number in the world.China's diabetes prevalence increases rapidly from lower than 1% to only more than 10%
With 30 years, and increase trend will be presented whithin a period of time.With the improvement of people's living standard in our country, urbanization degree
Raising and aging of population aggravation, the illness rate of diabetes can also further increase.Bring diabetes itself therefrom
And its height of complication is disabled the harm such as lethality, can not only bring serious health problem, but also will increase corresponding
Medical expense, aggravate the burden of health care and social economy, it will choose as China's public health service is the severeest
War.
According to International Diabetes Federation parting scheme in 1997, diabetes can be divided into: type-1 diabetes mellitus, type-2 diabetes mellitus,
And other special patients with type Ⅰ DM.Wherein type-2 diabetes mellitus (hereinafter referred to as T2It D is) since the resistant function to insulin causes
's.When body dietary intake causes Glucose in Blood by Cyclic to increase, human body can secrete glucagon-like-peptide-1 (GLP-1,
Hereinafter referred to as GLP-1) etc. hormones.These hormones and corresponding receptor combine, and synthesis and secretion insulin inhibits pancreas hyperglycemia
The release of element, reduces the synthesis of liver glucose, to reduce blood sugar concentration.
Dipeptidyl peptidase IV (DPP-4 enzyme, hereinafter referred to as DPP-4 enzyme) is a kind of multifunctional protein, with T cell activation antigen
CD26 and adenosine deaminase binding protein homotype are expressed in blood plasma with soluble form and epithelial cell, endothelial cell, lymph are thin
The various kinds of cell such as born of the same parents surface.The catalysis of DPP-4 enzyme comes from the polypeptide for having proline (Pro) or alanine (Ala) at the 2nd
The hydrolysis of the N-terminal dipeptides of N-terminal.In T2In D, GLP-1 once secretion release after, just rapidly by blood and wide expression in endothelium
It is decomposed with the DPP-4 enzyme of surface epithelial cell.DPP-4 enzyme plays a crucial role insulin synthesis as a result, DPP-4
Main characteristic parameters one of of the activity of enzyme as current diabetes.
Detection kit is depended on to the detection of DPP-4 enzymatic activity at this stage, has there is sale phase on the market at present
The detection kit for the DPP-4 enzymatic activity answered, and have shown that increasingly increased for clinical diagnosis, particularly Rezulin
The hope of object exploitation.However, actual detecting step is more at present when using kit detection dipeptidyl peptidase IV activity
It is cumbersome, and detection time process tedious, read plate time point are more, and the insufficient problem of detection accuracy.Therefore, it is badly in need of developing one kind
It is easy to operate, detection cycle is short, the method for high sensitivity is to realize the measurement of DPP-4 enzymatic activity.
Summary of the invention
In view of the deficiencies of the prior art, the present invention intends to provide DPP- in a kind of quantitative detection human plasma
The method of 4 enzymatic activitys has the characteristics that easy to operate, detection cycle is short, high sensitivity.
To realize above-mentioned first purpose, the present invention provides the following technical scheme that
A kind of method of DPP-4 enzymatic activity in quantitative detection human plasma, comprising the following steps:
1., start experiment before each reagent and sample to be tested be placed on equilibrium at room temperature；
2., plus 50 holes μ L/ quality-control sample and sample to be tested to detection orifice plate in, 4 holes of each sample to be tested；
3., 2 holes of each sample to be tested as control wells, the inhibitor working solution in 10 holes μ L/ is added；Other two hole conduct
10 1 × PBS buffer solution of the hole μ L/ are added, with sealing plate film sealing plate, 37 DEG C of 10 ± 2min of incubation in instrument connection；
4. plus 100 hole μ L/ of standard sample of prepared 7- amino -4- methylcoumarin is detected in orifice plate to blank, multiple holes 2
A operation；
5. plus the Gly-Pro -7- amino -4- methylcoumarin hydrobromate in 40 holes μ L/ is to quality-control sample and to test sample
In sample wells；
6., with sealing plate film sealing plate, with microplate reader in Ex/Em=355/460nm read plate after 37 DEG C of 10 ± 1min of incubation；
7., use sealing plate film sealing plate after read plate, in Ex/Em=355/460nm read plate again after 37 DEG C of 30 ± 2min of incubation；
The calculation of the DPP-4 enzymatic activity of sample to be tested and quality-control sample is as follows:
Enzymatic activity (μ U/mL)=B/ ((T2-T1) × V) × sample extension rate
Wherein, T2-T1For the incubation time at 37 DEG C before second of read plate, unit min；
V is the sample volume being added in detection orifice plate, unit mL；
Amount of the B for the substance of the 7- amino -4- methylcoumarin generated between first time read plate for the second time, unit pmol,
Calculation method is the concentration that uses Δ RFU as signal value and be back-calculated out through 7- amino -4- methylcoumarin standard curve multiplied by every
The volume in hole, concentration unit nM, volume unit mL；
Δ RFU=(RT2-RB2)-(RT1-RB1)
Wherein, RT2And RB2The multiple holes signal value of 1 × PBS buffer solution and inhibitor working solution is added when respectively second of read plate
Average value；RT1And RB1Respectively first time read plate when be added 1 × PBS buffer solution and inhibitor working solution multiple holes signal value
Average value.
Further, the quality-control sample is mankind K2Edta plasma, or be mankind K2Edta plasma and 1 × PBS buffer solution
The mixture prepared by volume.
Further, the quality-control sample includes upper limit of quantification quality-control sample, high concentration quality-control sample, intermediate concentration Quality Control
Sample, low concentration quality-control sample and lower limit of quantitation quality-control sample；The quality-control sample includes upper limit of quantification quality-control sample, high concentration
Quality-control sample, intermediate concentration quality-control sample, low concentration quality-control sample and lower limit of quantitation quality-control sample；Wherein,
Upper limit of quantification quality-control sample is mankind K2Edta plasma, activity level are 170.66 μ U/mL；
High concentration quality-control sample is mankind K2The mixture of edta plasma and 1 × PBS, activity level are 129.12 μ U/mL；
Intermediate concentration quality-control sample is mankind K2The mixture of edta plasma and 1 × PBS, activity level are 66.28 μ U/mL；
Low concentration quality-control sample is mankind K2The mixture of edta plasma and 1 × PBS, activity level are 44.35 μ U/mL；
Lower limit of quantitation quality-control sample is mankind K2The mixture of edta plasma and 1 × PBS, activity level are 21.23 μ U/mL.
Further, the standard sample is 1 × PBS buffer solution, or fragrant for 1 × PBS buffer solution and 7- amino -4- methyl
The mixture that legumin intermediate solution is prepared by volume.
Further, the 7- ammonia that the 7- amino -4- methylcoumarin intermediate solution is 1mg/mL by 175.18 μ L concentration
Base -4- methylcoumarin standard solution and 1 × PBS buffer solution mixed preparing of 824.82 μ L form.
Further, the 7- amino -4- methylcoumarin standard solution is by 0.01g 7- amino -4- methylcoumarin powder
End is added 10mL dimethyl sulfoxide (DMSO) dissolution and is formulated.
By using above-mentioned technical proposal, the application uses Gly-Pro -7- amino -4- methylcoumarin hydrogen bromine
Substrate specificity of the hydrochlorate (Gly-Pro-7-Amino-4-methylcoumarin hydrobromide) as DPP-4 enzyme utilizes
DPP-4 enzyme releases fluorescent material 7- amino -4- methylcoumarin (7-Amino-4- in sample after being decomposed
Methylcoumarin) this principle is measured.Wherein, in detection process the addition of inhibitor working solution be able to suppress it is to be measured
The activity of DPP-4 enzyme in sample can obtain the signal value generated by DPP-4 enzyme effect after obtaining background value.This signal
The increase of value within a certain period of time is directly proportional to DPP-4 enzymatic activity in sample to be tested, therefore the application uses 1 × PBS buffer solution
The standard curve of a 7- amino -4- methylcoumarin is made, then passes through four parameter fittings (weight factor 1/Y2), with this
Assess the DPP-4 enzymatic activity in sample to be tested.At the same time, 1 × PBS buffer solution being capable of DPP-4 enzyme preferably in maintenance system
Activity, the application using 1 × PBS buffer solution as blank control, use mankind K2The quality-control sample that edta plasma is prepared, by one
It rises and is analyzed, as reference.In addition, 1 × PBS buffer solution can assess background signal value with the presence or absence of different as blank control
Often, quality-control sample is then used to ensure experiment process itself that there is no problem according to certain standard that receives, and guarantees the accuracy of detection.
From loading to obtain as a result, the application detection total duration control within 1h, by read plate twice, can detect
DPP-4 enzymatic activity can down to 1.1 μ U/L, in addition, the orientation range of the application detection method can achieve 21.23~
170.66 μ U/mL can be maximum by 8 times of dilutions when concentration, which occurs, in sample to be tested is higher than detection upper limit of quantification, and guarantees dilute
After releasing in the detection range and sample to be tested still be able to accurately be detected, there is good sensitivity and accuracy.Relatively
, common kit needs by adopting more than data twice the measurement total time-consuming about 4-6h of DPP-4 enzymatic activity on the market
Collect (read plate) operation, therefore the detection efficiency of the application significantly improves, and effectively shortens the detection cycle of DPP-4 enzymatic activity, tool
Have the characteristics that simple and convenient.
Wherein, quality-control sample is according to the actually detected situation of DPP-4 enzyme, design corresponding upper limit of quantification quality-control sample,
High concentration quality-control sample, intermediate concentration quality-control sample, low concentration quality-control sample and lower limit of quantitation quality-control sample, and limit every germplasm
The component for controlling sample, the detection accuracy of DPP-4 enzymatic activity is helped to improve with this.
In standard sample, the 7- amino -4- methyl of isoconcentration gradient is prepared by the adjustment to 1 × PBS buffer solution dosage
Cumarin enables standard sample preferably to provide accurate standard curve for sample to be tested, improves the accurate of detection with this
Degree.
Further, the inhibitor in the inhibitor working solution is Xi Gelieting.
Further, the concentration of inhibitor working solution Chinese and Western Ge Lieting is 20 μ g/mL.
By using above-mentioned technical proposal, Xi Gelieting is by the level of incretin, improvement pancreas islet B in lifting body
Cell function adjusts a kind new medicine of blood glucose.In this application using Xi Gelieting as inhibitor, have it is highly selective, can
It is preferable to inhibit DPP-4 enzymatic activity, DPP-4 enzyme is reduced to Gly-Pro -7- amino -4- methylcoumarin hydrobromate
The amount being decomposed finally measures DPP-4 enzymatic activity using the fluorescent characteristic of 7- amino -4- methylcoumarin.Wherein, believe
Number value is weaker, and the inhibitory effect of DPP-4 enzyme is better, and when the concentration of Xi Gelieting reaches 20mg/mL, the inhibitor is to DPP-4
Inhibitory effect reach best.
Further, the detection orifice plate is 96 orifice plates.
By using above-mentioned technical proposal, 96 orifice plates are also known as 96 porocyte culture plates, select the pure polystyrene of optical clear
It is fabricated, cell can be made to reach optimal adsorption state, with this convenient for thin in the blood plasma of sample to be tested and quality-control sample
Born of the same parents, which stablize, to be adsorbed on detection orifice plate, to improve the accuracy of detection.
Further, the microplate reader is EnVision microplate reader.
By using above-mentioned technical proposal, EnVision microplate reader is the goldstandard of multi-function microplate reader in the market, is used
Optical filter and grating mixed optical path have brilliant sensitivity and stability, and detect speed block, can reduce detection process
In light path loss, improve the detectability of weak signal, and prevent periphery hole to the signal cross-talk etc. of detection hole.It is applied
In the detection method of the application, guarantee the high sensitivity and high accuracy of detection.
In conclusion the invention has the following advantages:
1, the application is from loading to out as a result, detection total duration control effectively shortens detection DPP-4 enzymatic activity within 1h
Detection cycle is short；
2, only data acquire (read plate) process twice in the entire experimental procedure of the application, far less than reagent common on the market
Box, therefore its detection method is more convenient；
3, in the application, the orientation range of detection method is 21.23~170.66 μ U/mL, at the same can at least carry out 8 times it is dilute
It releases, so that the detection of DPP-4 enzymatic activity is sensitiveer and accurate；
4, the characteristics of the application combination DPP-4 enzyme, sets matched quality-control sample, standard sample, inhibitor, detection hole
Plate and microplate reader guarantee the high sensitivity and high accuracy of detection with this.
Detailed description of the invention
Fig. 1 is the operating procedure figure of DPP-4 enzymatic activity in quantitative detection human plasma；
Fig. 2 is the standard curve illustrated example of 7- amino -4- methylcoumarin.
Specific embodiment
Below in conjunction with attached drawing, invention is further described in detail.
1, reagent prepares
1.1, quality-control sample
Including upper limit of quantification quality-control sample, high concentration quality-control sample, intermediate concentration quality-control sample, low concentration quality-control sample and quantify
Lower limit quality-control sample.
It is mankind K that quality-control product, which prepares matrix used,2Edta plasma (anti-coagulants K2EDTA purchases online shopping by import reagent
), by background value screening, matrix 001, matrix 002, the matrix 003 of different activities level are chosen, is matched according to following table one
The quality-control sample of different activities level processed.3 groups of every special quality control sample preparation, 3 repetition activity level measurements of every group of carry out.9
After numerical value is averaged, measure quality-control product activity level be respectively as follows: 170.66 μ U/mL, 129.12 μ U/mL, 66.28 μ U/mL,
44.35μU/mL、21.23μU/mL。
The preparation of one quality-control sample of table
1.2,7- amino -4- methylcoumarin (AMC) standard solution
10mL dimethyl sulfoxide (DMSO) is added by 0.01g 7- amino -4- methylcoumarin powder (being purchased from Mike woods) to dissolve afterwards
It is formulated, concentration 1mg/mL.
1.3,7- amino -4- methylcoumarin (AMC) intermediate solution
1 × PBS buffer solution mixed preparing of the AMC standard solution as made from the 1.2 of 175.18 μ L and 824.82 μ L form.
The standard curve according to the form below two of 7- amino -4- methylcoumarin (AMC) and the proportion of table three are prepared, and standard is bent
Line exemplary diagram is referring to fig. 2.The concentration of the bent point of mark be respectively 100.00nM, 80.00nM, 60.00nM, 40.00nM, 25.00nM,
In addition 10.00nM, 8.00nM, 6.00nM, 4.00nM take appropriate 1 × PBS buffer solution as blank control to assess background signal
Value is not involved in standard curve fit with the presence or absence of exception, blank control.
The preparation steps one of the standard curve of two 7- amino -4- methylcoumarin of table
The preparation steps two of the standard curve of three 7- amino -4- methylcoumarin of table
2, embodiment
2.1 embodiments 1
A kind of method of DPP-4 enzymatic activity in quantitative detection human plasma, referring to Fig. 1, comprising the following steps:
1., start experiment before each reagent and sample to be tested be placed on equilibrium at room temperature；
2., the minimum extension rate of sample to be tested and quality-control sample be 64 times, i.e., before contact plate, at least to carry out 64 times of dilutions,
It is mixed to get by 10 μ L quality-control samples/sample to be tested and 630 μ 11 × PBS buffer solution of L.
3., respectively plus 50 holes μ L/ diluted quality-control sample and sample to be tested into 96 orifice plates, each sample to be tested 4
Hole；
4., 2 holes of each sample to be tested the Xi Gelieting in 10 holes μ L/ is added as control wells, and the concentration of Xi Gelieting is
20mg/mL；10 1 × PBS buffer solution of the hole μ L/ are added as instrument connection in other two hole, with sealing plate film sealing plate, 37 DEG C of incubations
10min；
5., plus prepared 7- amino -4- methylcoumarin 100 hole μ L/ of standard sample into 96 orifice plate of blank, multiple holes 2
Operation；
6. plus the Gly-Pro -7- amino -4- methylcoumarin hydrobromate in 40 holes μ L/ is to quality-control sample and to test sample
In sample wells；
7., with sealing plate film sealing plate, with EnVision microplate reader in Ex/Em=355/460nm read plate after 37 DEG C of incubation 10min；
8., use sealing plate film sealing plate after read plate, in Ex/Em=355/460nm read plate again after 37 DEG C of incubation 30min.
The calculation of the DPP-4 enzymatic activity of sample to be tested and quality-control sample is as follows:
Enzymatic activity (μ U/mL)=B/ ((T2-T1) × V) × sample extension rate
Wherein, T2-T1For the incubation time at 37 DEG C before second of read plate, unit min；
V is that the sample volume detected in orifice plate is added, unit mL, is 0.05 in the present embodiment；
Amount of the B for the substance of the 7- amino -4- methylcoumarin generated between first time read plate for the second time, unit pmol,
Calculation method is the concentration that uses Δ RFU as signal value and be back-calculated out through 7- amino -4- methylcoumarin standard curve multiplied by every
The volume in hole, concentration unit nM, volume unit mL are 0.1 in the present embodiment.
Δ RFU=(RT2-RB2)-(RT1-RB1)
Wherein, RT2And RB2The multiple holes signal value of 1 × PBS buffer solution and inhibitor working solution is added when respectively second of read plate
Average value；RT1And RB1Respectively first time read plate when be added 1 × PBS buffer solution and inhibitor working solution multiple holes signal value
Average value.
The application determines the accuracy of detection method by the detection of quality-control sample.In the present embodiment, according to above-mentioned step
Rapid and method, analyzes quality-control sample three times, and analysis includes 1 group of standard curve point, 3 groups of quality-control samples every time, same
It is carried out on 96 orifice plates, analysis detection result is referring to following table four three times.
The testing result of four quality-control sample of table
In conjunction with the detecting step of the present embodiment and the testing result of table four, available, the detection method of the present embodiment
From loading to as a result, detection total duration is within 1h, relative to the kit sold on the market, the detection cycle of the application is obtained out
To effectively shortening, to improve the efficiency of detection DPP-4 enzymatic activity.Moreover, only having 2 times in the entire detecting step of the present embodiment
Data acquire (read plate) process, far less than kit common on the market, therefore it is more convenient.In addition, through this embodiment,
It can illustrate that the detection method can accurately measure concentration in the sample of 21.23-170.66 μ U/mL, the rate of recovery of quality-control sample exists
Within the scope of 90.4-103.2%, it is strict in the rate of recovery requirement of industry 80-120%, there is good detection accuracy and accuracy.
2.2, embodiment 2-3
Additive amount, inhibition of the embodiment 2-3 on the basis of method of embodiment 1, to incubation time, quality-control sample and sample to be tested
The concentration of agent is adjusted, and the specific situation that adjusts is referring to following table five, and corresponding testing result is referring to following table six.
The parameter of five embodiment 2-3 of table adjusts table
| Embodiment 2 | Embodiment 3 | |
| 1st incubation time (min) | 12 | 8 |
| 2nd incubation time (min) | 11 | 9 |
| 3rd incubation time (min) | 32 | 28 |
The testing result of six embodiment 2-3 of table
| Embodiment 3 | Embodiment 4 | |
| The rate of recovery (%) | 84.4-113.0 | 85.0-111.7 |
| The coefficient of variation (%) | ≤14.2 | ≤8.7 |
In conjunction with table five and table six, embodiment 2-3 is mainly adjusted the time of the incubation of quality-control sample, corresponds to Quality Control
Though the testing result of sample and sample to be tested is close with embodiment 1, the testing result of embodiment 1 is more excellent, i.e. the inspection of embodiment 1
Survey method is more accurate sensitive, therefore as a preferred embodiment by embodiment 1.
3, the depth of parallelism
Choose above-mentioned mankind K2Matrix 001, matrix 002 and the matrix 003 of edta plasma, each matrix are dilute according to such as following table seven
It releases step to be handled, obtains the depth of parallelism sample to be tested of three different activities levels.
The preparation of seven depth of parallelism sample of table
Quality-control sample and standard curve point sample are prepared respectively according to upper table one, table two and table three, in the method for embodiment 1
On the basis of, the concentration mensuration of 3 groups of samples, every group of 3 dilution is carried out, and calculate the inspection that same sample to be tested measures after diluting
The rate of recovery of result is surveyed, testing result verifies the depth of parallelism of the sample to be tested under this detection method with this referring to following table eight.
The testing result of eight depth of parallelism sample of table
Available in conjunction with the detecting step of the present embodiment and the testing result of table eight, unknown sample is with 1 under the application
× PBS buffer solution carries out the dilution of 64-256 (64 × 4) again, and activity level dilutes line with standard curve with preferable parallel
Degree, the middle horizontal rate of recovery of sample activity reaches 98.2-109.5%, that is, provable this point through this embodiment.
4, stability is detected
According to the detection method of embodiment 1, the detection stability of sample to be tested under this method is assessed.Sample to be tested derives from the mankind
Blood plasma (anti-coagulants K2EDTA), subsequent survey is carried out using the sample of a greater activity level and a lower activity level
It is fixed.The sample of different activities level is dispensed in equal volume, using packing same day first time minute as the benchmark time, is placed in
A period of time is stored under varying environment, and 3 groups of measurements are carried out to different condition of storage samples.Testing conditions and corresponding testing result
Referring to following table nine.
The testing conditions and testing result of nine sample to be tested of table
By nine result of table it is found that under this detection method, sample to be tested of the activity level in 50.64-139.6 μ U/mL exists
It is above it is several under the conditions of store a period of time after, average recovery rate still can reach 102.8-109.1%, have good detection
Stability.
To sum up, the method for DPP-4 enzymatic activity is short with detection cycle in the application quantitative detection human plasma, operates just
Prompt, the accurate sensitive feature of detection.
This specific embodiment is only explanation of the invention, is not limitation of the present invention, those skilled in the art
Member can according to need the modification that not creative contribution is made to the present embodiment after reading this specification, but as long as at this
All by the protection of Patent Law in the scope of the claims of invention.
Claims (10)
1. a kind of method of DPP-4 enzymatic activity in quantitative detection human plasma, which comprises the following steps:
1., start experiment before each reagent and sample to be tested be placed on equilibrium at room temperature；
2., plus 50 holes μ L/ quality-control sample and sample to be tested to detection orifice plate in, 4 holes of each sample to be tested；
3., 2 holes of each sample to be tested as control wells, the inhibitor working solution in 10 holes μ L/ is added；Other two hole conduct
10 1 × PBS buffer solution of the hole μ L/ are added, with sealing plate film sealing plate, 37 DEG C of 10 ± 2min of incubation in instrument connection；
4. plus 100 hole μ L/ of standard sample of prepared 7- amino -4- methylcoumarin is detected in orifice plate to blank, multiple holes 2
A operation；
5. plus the Gly-Pro -7- amino -4- methylcoumarin hydrobromate in 40 holes μ L/ is to quality-control sample and to test sample
In sample wells；
6., with sealing plate film sealing plate, with microplate reader in Ex/Em=355/460nm read plate after 37 DEG C of 10 ± 1min of incubation；
7., use sealing plate film sealing plate after read plate, in Ex/Em=355/460nm read plate again after 37 DEG C of 30 ± 2min of incubation；
The calculation of the DPP-4 enzymatic activity of sample to be tested and quality-control sample is as follows:
Enzymatic activity (μ U/mL)=B/ ((T2-T1) × V) × sample extension rate
Wherein, T2-T1For the incubation time at 37 DEG C before second of read plate, unit min；
V is the sample volume being added in detection orifice plate, unit mL；
Amount of the B for the substance of the 7- amino -4- methylcoumarin generated between first time read plate for the second time, unit pmol,
Calculation method is the concentration that uses Δ RFU as signal value and be back-calculated out through 7- amino -4- methylcoumarin standard curve multiplied by every
The volume in hole, concentration unit nM, volume unit mL；
Δ RFU=(RT2-RB2)-(RT1-RB1)
Wherein, RT2And RB2The multiple holes signal value of 1 × PBS buffer solution and inhibitor working solution is added when respectively second of read plate
Average value；RT1And RB1Respectively first time read plate when the multiple holes signal value of 1 × PBS buffer solution and inhibitor working solution is added
Average value.
2. the method for DPP-4 enzymatic activity in a kind of quantitative detection human plasma according to claim 1, which is characterized in that
The quality-control sample is mankind K2Edta plasma, or be mankind K2Edta plasma and 1 × PBS buffer solution are prepared mixed by volume
Close object.
3. the method for DPP-4 enzymatic activity in a kind of quantitative detection human plasma according to claim 2, which is characterized in that
The quality-control sample includes upper limit of quantification quality-control sample, high concentration quality-control sample, intermediate concentration quality-control sample, low concentration Quality Control sample
Product and lower limit of quantitation quality-control sample；Wherein,
Upper limit of quantification quality-control sample is mankind K2Edta plasma, activity level are 170.66 μ U/mL；
High concentration quality-control sample is mankind K2The mixture of edta plasma and 1 × PBS, activity level are 129.12 μ U/mL；
Intermediate concentration quality-control sample is mankind K2The mixture of edta plasma and 1 × PBS, activity level are 66.28 μ U/mL；
Low concentration quality-control sample is mankind K2The mixture of edta plasma and 1 × PBS, activity level are 44.35 μ U/mL；
Lower limit of quantitation quality-control sample is mankind K2The mixture of edta plasma and 1 × PBS, activity level are 21.23 μ U/mL.
4. the method for DPP-4 enzymatic activity in a kind of quantitative detection human plasma according to claim 1, which is characterized in that
The standard sample is 1 × PBS buffer solution, or presses body for 1 × PBS buffer solution and 7- amino -4- methylcoumarin intermediate solution
Mixture of the product than preparation.
5. the method for DPP-4 enzymatic activity in a kind of quantitative detection human plasma according to claim 4, which is characterized in that
7- amino -4- methylcoumarin the mark that the 7- amino -4- methylcoumarin intermediate solution is 1mg/mL by 175.18 μ L concentration
1 × PBS buffer solution mixed preparing of quasi- solution and 824.82 μ L forms.
6. a kind of method of quantitative detection human plasma enzymatic activity according to claim 5, which is characterized in that the 7- ammonia
Base -4- methylcoumarin standard solution is added 10mL dmso solution by 0.01g7- amino -4- methylcoumarin powder and matches
It makes.
7. the method for DPP-4 enzymatic activity in a kind of quantitative detection human plasma according to claim 1, which is characterized in that
Inhibitor in the inhibitor working solution is Xi Gelieting.
8. the method for DPP-4 enzymatic activity in a kind of quantitative detection human plasma according to claim 7, which is characterized in that
The concentration of inhibitor working solution Chinese and Western Ge Lieting is 20 μ g/mL.
9. the method for DPP-4 enzymatic activity in a kind of quantitative detection human plasma according to claim 1, which is characterized in that
The detection orifice plate is 96 orifice plates.
10. the method for DPP-4 enzymatic activity in a kind of quantitative detection human plasma according to claim 1, which is characterized in that
The microplate reader is EnVision microplate reader.
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