CN112608977B - Gamma-glutamyl transpeptidase detection kit and detection method thereof - Google Patents

Gamma-glutamyl transpeptidase detection kit and detection method thereof Download PDF

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CN112608977B
CN112608977B CN202011436205.6A CN202011436205A CN112608977B CN 112608977 B CN112608977 B CN 112608977B CN 202011436205 A CN202011436205 A CN 202011436205A CN 112608977 B CN112608977 B CN 112608977B
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enzyme
substrate
nitroaniline
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CN112608977A (en
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冷毅斌
王长乐
敖慧龙
王振平
陈冬琴
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Elabscience Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91045—Acyltransferases (2.3)
    • G01N2333/91074—Aminoacyltransferases (general) (2.3.2)
    • G01N2333/9108—Aminoacyltransferases (general) (2.3.2) with definite EC number (2.3.2.-)
    • G01N2333/91085—Transglutaminases; Factor XIIIq (2.3.2.13)

Abstract

The invention discloses a gamma-glutamyl transpeptidase detection kit and a detection method thereof, wherein the detection kit comprises a matrix buffer solution, an enzyme extract, a substrate, a standard substance and a dissolving diluent, wherein the matrix buffer solution comprises tris (hydroxymethyl) aminomethane and diglycine, the enzyme extract is a buffer solution prepared from tris (hydroxymethyl) aminomethane and hydrochloric acid, the substrate is L-gamma-glutamyl-4-nitroaniline, the standard substance is p-nitroaniline, and the dissolving diluent is acidic DMSO. The detection kit adopts a solvent system which does not affect the reaction of catalyzing L-gamma-glutamyl-4-nitroaniline by gamma-GT and can effectively dissolve p-nitroaniline, solves the problem that the production amount of the p-nitroaniline can only be calculated by a pure theoretical mode in the prior art, can more accurately and directly determine the enzyme activity, and has low cost and small error.

Description

Gamma-glutamyl transpeptidase detection kit and detection method thereof
Technical Field
The invention relates to the technical field of biological detection, in particular to a gamma-glutamyl transpeptidase detection kit and a detection method thereof.
Background
Gamma-glutamyl transpeptidase (gamma-GT) is widely present in various organs of human body, is a key enzyme in gamma-glutamyl circulation, catalyzes GSH degradation, and has the functions of participating in adjusting glutathione level in tissues, absorbing and excreting amino acid, acylating free amino acid in peptide chain and the like. The activity of the gamma-GT in normal human serum is very low, and the activity of the gamma-GT in the serum of patients with acute hepatitis, liver cancer, obstructive yellow pox and the like is obviously improved, so the measurement of the activity of the gamma-GT has certain significance for the diagnosis of diseases of a liver and gall system and is beneficial to the diagnosis of the liver cancer.
A gamma-glutamyl transpeptidase detection kit which takes L-gamma-glutamyl-4-nitroaniline (GPNA) as a substrate is sold on the market, and a generated chromogenic substance is p-nitroaniline, and the detection wavelength is 405 nm. In commercial kits based on this principle, there are two main ways of processing the assay results:
firstly, directly taking gamma-glutamyl transpeptidase as a standard substance, measuring a standard curve, and correcting an experimental test result. The calculation method is direct, but the cost is high by taking the enzyme as the standard substance, and the enzyme standard substance is easy to partially inactivate in the processes of storage, transportation and measurement, so that the result is deviated, and the accuracy is influenced.
Second, the amount of paranitroaniline produced can be calculated by measuring the increase in OD over a period of time, thereby defining the gamma-GT enzyme activity in the sample. The method takes the p-nitroaniline as a standard substance, has stable chemical property, has characteristic absorption at 405nm, eliminates background interference by a rate method, can effectively measure the activity of the gamma-glutamyltranspeptidase in the sample, and has better stability and accuracy.
However, no p-nitroaniline standard hole is arranged in the current commercial modified test kit. And (3) calculating the result by performing pure theoretical conversion on the molar absorptivity coefficient of the p-nitroaniline or directly providing a standard curve of the p-nitroaniline for calculation. Due to such theoretical calculation, systematic errors caused by experimental conditions cannot be corrected, and the calculation result is often deviated from the true measurement value and lacks scientificity.
The reason for this is that a suitable solvent system cannot be found at present, which can not affect the reaction of L- γ -glutamyl-4-nitroaniline catalyzed by γ -GT, but can also dissolve p-nitroaniline to prepare a p-nitroaniline standard solution with a higher concentration, so that the reaction systems of the standard well and the measurement well are completely consistent, and the interference of the reagent system is eliminated.
Disclosure of Invention
In view of the above, the invention provides a gamma-glutamyltranspeptidase assay kit, and particularly provides a solvent system which does not affect the reaction of catalyzing L-gamma-glutamyl-4-nitroaniline by gamma-GT and can effectively dissolve p-nitroaniline, so that the problem that the production amount of p-nitroaniline can only be calculated by a pure theoretical mode in the prior art is solved.
In order to achieve the purpose, the invention specifically adopts the following technical scheme:
a detection kit of gamma-glutamyl transpeptidase comprises the following reagents: matrix buffer solution, enzyme extracting solution, substrate, standard substance and dissolving diluent;
the substrate buffer solution comprises tris (hydroxymethyl) aminomethane and diglycine, and the pH value is 7.8-8.4; the enzyme extract is a buffer solution prepared from tris (hydroxymethyl) aminomethane and hydrochloric acid; the substrate is L-gamma-glutamyl-4-nitroaniline, the standard substance is p-nitroaniline, and the dissolving diluent is acidic dimethyl sulfoxide (DMSO).
Preferably, in the above technical solution, the concentration of tris (hydroxymethyl) aminomethane in the matrix buffer is 0.1-0.2mol/L, and the concentration of diglycine is 0.05-0.15 mol/L.
Preferably, in the above technical means, the concentration of tris (hydroxymethyl) aminomethane in the enzyme extract is 0.08 to 0.2 mol/L.
Preferably, in the above technical scheme, the mass of the substrate is 10-20mg, and the concentration of the substrate solution is 10-20mmol/L after the substrate solution is dissolved by the dissolving diluent.
Preferably, in the above technical solution, the content of hydrochloric acid in the dilution solution is 0.2-2%.
The invention also provides a method for determining the activity of gamma-glutamyltranspeptidase, in particular to a method for detecting by using the detection kit, which comprises the following steps:
s1, treating serum, plasma, tissue or cell sample with enzyme extractive solution to obtain sample to be tested, and detecting total protein concentration Cpr；
S2, dissolving a substrate by using a dissolving diluent to obtain a substrate solution, and mixing the substrate solution with a matrix buffer solution with m times of volume to obtain a reaction working solution; dissolving the standard substance by using a dissolving diluent to obtain a series of standard substance solutions with different concentrations, and mixing the standard substance solutions with the matrix buffer solution with m times of volume to obtain a series of standard substance working solutions with different concentrations;
s3, arranging a standard hole and a measuring hole on the ELISA plate, and adding V into the standard hole2Volume of double distilled water, measuring the volume of the double distilled water, and adding V into a hole2Diluting the sample to be detected by f times in volume; adding V into the standard hole3Adding V into the measuring hole according to the volume of the standard substance working solution3Uniformly mixing the reaction working solution with the volume;
s4, incubating at the temperature T, measuring OD values of two fixed time points by an enzyme-labeling instrument at 405nm, and calculating a change value delta A;
s5, drawing a standard curve, and calculating the enzyme activity by the following formula, wherein the calculation mode of the activity of the gamma-glutamyl transpeptidase is as follows:
when the sample to be tested is a tissue or cell sample, the formula (1) is adopted
gamma-GT enzyme activity (U/gprot) ═ ([ delta ] A-b) ÷ a × V1÷V2÷Cpr÷T×f (1)
When the sample to be tested is serum or plasma, the formula (2) is adopted
gamma-GT enzyme activity (U/L) (. DELTA.A-b) ÷ a.times.V1÷V2÷T×f (2)
Wherein, a is the slope of the standard curve, b: intercept of standard curve, V1＝V3V (m +1), wherein the enzyme activity in the formula (1) is defined as: at the temperature T, the amount of enzyme catalyzing and generating 1 mu mol of paranitroaniline per gram of protein per minute is 1 activity unit; the definition of the enzyme activity in the formula (2) is as follows: the amount of enzyme catalyzing the production of 1. mu. mol of p-nitroaniline per minute per liter of liquid sample at temperature T was 1 unit of activity.
Preferably, in the above technical scheme, m in step S2 is 2-6, that is, the volume ratio of the substrate solution or the standard solution to the matrix buffer solution is 1: 2-6; more preferably, said m is equal to 4.
Preferably, in the above technical solution, when the sample to be tested is loaded in step S4, the sample is slowly loaded at the bottom of the catalase calibration plate.
Preferably, in the above technical solution, the temperature T in step S5 is 37 ℃, and the incubation process specifically includes: incubating at 37 deg.C for 1min, measuring OD at wavelength of 405nm, and recording as A1(ii) a Then placing the mixture into a 37 ℃ incubator for accurate incubation for 5min, and recording the OD value measured at the wavelength of 405nm as A2；△A＝A2-A1。
The invention has the beneficial effects that: 1) the working solution system with acid DMSO and substrate buffer solution mixed in proportion is adopted, the reaction system does not affect the reaction of catalyzing L-gamma-glutamyl-4-nitroaniline by gamma-GT, and can dissolve p-nitroaniline, so that the reaction systems of the standard hole and the measuring hole are completely consistent, the interference of the reagent system is eliminated, and the enzyme activity is determined more accurately and directly; 2) compared with a method using gamma-glutamyltranspeptidase as a standard substance, the method has the advantages that p-nitroaniline as the standard substance is lower in cost, and the standard substance is better in stability and easier to store; 3) compared with other kits taking p-nitroaniline as a standard substance on the market, the kit provided by the invention is provided with a standard curve under the same reaction system, so that the calculation result is more scientific and accurate; 4) by fully and uniformly mixing the substrate and the buffer solution before detection, the composite pore difference in the sample detection process is greatly reduced on the premise of not influencing the detection result.
Drawings
FIG. 1 Standard Curve determined in example 3.
Detailed Description
The present invention is further illustrated by the following specific examples, which should not be construed as limiting the scope of the invention.
Example 1
A gamma-glutamyl transpeptidase detection kit comprises the following reagents:
matrix solution buffer solution: weighing 1.48g of tris (hydroxymethyl) aminomethane and 1.20g of diglycine, dissolving with double distilled water, and fixing the volume to 100 mL; 30mL of the suspension was taken out and stored at 4 ℃ in 1 bottle.
Substrate powder preparation: weighing 16mg of L-gamma-glutamyl-4-nitroaniline powder, and storing at 1 count at 4 ℃ in a dark place.
Enzyme extracting solution: weighing 1.21g of tris (hydroxymethyl) aminomethane, diluting double distilled water to a constant volume of 100mL, and adjusting the pH value to 8.0 by 2mol/L hydrochloric acid; 50mL of the solution was taken and stored at 4 ℃ in 1 bottle.
Acid DMSO (dimethylsulfoxide): adding 500 mu L of concentrated hydrochloric acid with the concentration of 10-12mol/L into 30mLDMSO, uniformly mixing, and then fixing the volume to 50mL by using DMSO; 10mL of the suspension was taken out and stored at 4 ℃ in 1 bottle.
Standard powder preparation: 6.9mg of paranitroaniline powder is weighed, and 1 piece is stored at 4 ℃ in a dark place.
Example 2
The enzyme activity detection is carried out by adopting the detection kit prepared in the embodiment 1, and the specific steps are as follows:
(1) the processing of samples, the processing methods of different types of samples are different, and the following methods can be specifically referred to:
liquid samples such as serum (plasma): for such turbid samples, 1000 Xg centrifugation is required for 10min, and the supernatant is taken as a sample to be tested.
Tissue sample: accurately weighing the tissue weight according to the weight-volume ratio of 1 g: adding the enzyme extracting solution in a ratio of 9mL, homogenizing under the ice-water bath condition, centrifuging at 12000 Xg for 10min, and taking the supernatant as a sample to be detected. In addition, the total protein concentration of the sample to be tested needs to be measured, and the protein concentration can be measured by using a BCA method.
(2) Preparing a reaction working solution and a standard working solution:
preparing a reaction working solution: 16mg of substrate powder was dissolved in 3mL of acidic DMSO to obtain a substrate solution, which was then mixed with a matrix buffer in a ratio of 1: 4, and mixing the mixture fully.
Preparing a standard working solution: dissolving the standard substance powder in acidic DMSO to obtain standard substance solutions with different concentrations, and mixing with the matrix buffer solution according to the ratio of 1: 4, and mixing well.
(3) Detection of a sample to be tested
Dividing the enzyme label plate into a standard hole and a determination hole, and referring to the following table:
1 2 3 4 5 6 7 8 9 10 11 12
A A A S1 S9 S17 S25 S33 S41 S49 S57 S65 S73
B B B S2 S10 S18 S26 S34 S42 S50 S58 S66 S74
C C C S3 S11 S19 S27 S35 S43 S51 S59 S67 S75
D D D S4 S12 S20 S28 S36 S44 S52 S60 S68 S76
E E E S5 S13 S21 S29 S37 S45 S53 S61 S69 S77
F F F S6 S14 S22 S30 S38 S46 S54 S62 S70 S78
G G G S7 S15 S23 S31 S39 S47 S55 S63 S71 S79
H H H S8 S16 S24 S32 S40 S48 S56 S64 S72 S80
wherein, A-H are standard holes, and S1-S80 are measurement holes.
Detecting by using an enzyme label plate at room temperature (25-30 ℃), and specifically comprising the following steps: adding V to standard well2Volume of double distilled water, adding V to the assay well2A sample to be tested that is diluted by a factor of f in volume; adding 5 XV into the standard hole1Adding 5 XV of standard working solution with different volumes into the measuring hole1Volume of reaction working solution (V)1The actual sample loading volumes of the substrate solution and the standard solution) and a vibrating plate 10s of the microplate reader are mixed uniformly; incubating accurately for 1min at 37 deg.C in a thermostat, measuring OD at wavelength of 405nm, and recording as A1(ii) a Then placing the mixture into a 37 ℃ incubator for accurate incubation for 5min, and recording the OD value measured at the wavelength of 405nm as A2，△AAssay well＝A2-A1。
It should be noted that:
firstly, during data processing, the standard hole does not need difference values, and the OD value measured in 5min is directly taken as a standard curve; secondly, if the samples are large in batch, before formal detection, 1-2 samples with large expected differences are selected, diluted into different concentrations for preliminary experiments, and then the OD value is selected to be 0.2-0.8, and then formal batch tests are carried out.
(3) Calculation of Activity of Gamma-glutamyltranspeptidase (Gamma-GT)
When the sample to be tested is a tissue or cell sample
gamma-GT enzyme activity (U/gprot) ═ ([ delta ] A-b) ÷ a × V1÷V2÷Cpr÷T×f (1)
When the sample to be tested is serum or plasma
gamma-GT enzyme activity (U/L) (. DELTA.A-b) ÷ a.times.V1÷V2÷T×f (2)
Wherein, a: slope of the standard curve, b: the standard curve intercept, the enzyme activity in formula (1) is defined as: the amount of enzyme catalyzing the production of 1 mu mol of p-nitroaniline per gram of protein per minute is 1 activity unit under the condition of 37 ℃; the enzyme activity in formula (2) is defined as: the amount of enzyme catalyzing the production of 1. mu. mol of p-nitroaniline per minute per liter of the liquid sample at 37 ℃ is 1 unit of activity.
Example 3
The OD value of each standard well was measured by the detection method in example 2 using the kit in example 1, wherein V1Is 50 μ L, V2Is 25. mu.L, the detection results are as follows:
Figure BDA0002828858670000061
a standard curve is plotted according to the data in the table above, as shown in FIG. 1.
Human serum, horse serum, mouse serum and 10% rat liver tissue were tested in the same reaction system, with the following results:
Figure BDA0002828858670000062
from the detection results, it can be found that the complex pore difference of the measurement results is small; and in a reaction system, the OD value of the p-nitroaniline standard substance is good in linearity.
The present invention may be better understood and appreciated by those skilled in the art with reference to the following examples. However, the protection of the invention and the scope of the claims are not limited to the examples provided. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims (9)

1. The gamma-glutamyl transpeptidase detection kit is characterized by comprising the following reagents: matrix buffer solution, enzyme extracting solution, substrate, standard substance and dissolving diluent;
the substrate buffer solution comprises tris (hydroxymethyl) aminomethane and diglycine, and the pH value is 7.8-8.4; the enzyme extract is a buffer solution prepared from tris (hydroxymethyl) aminomethane and hydrochloric acid; the substrate is L-gamma-glutamyl-4-nitroaniline, the standard substance is p-nitroaniline, and the dissolving diluent is acidic DMSO.
2. The γ -glutamyl transpeptidase assay kit according to claim 1, wherein the concentration of tris (hydroxymethyl) aminomethane in said matrix buffer is 0.1-0.2mol/L, and the concentration of diglycine is 0.05-0.15 mol/L.
3. The γ -glutamyl transpeptidase assay kit according to claim 1, wherein the concentration of tris (hydroxymethyl) aminomethane in said enzyme extract is 0.08-0.2 mol/L.
4. The detection kit according to claim 1, wherein the mass of the substrate is 10 to 20mg, and the concentration of the substrate solution is 10 to 20mmol/L after dissolution by the dissolution diluent.
5. A method for determining the activity of gamma-glutamyltranspeptidase, characterized in that said method is used for the purpose of diagnosis and treatment of non-diseases, using the gamma-glutamyltranspeptidase assay kit according to claim 1, comprising the following steps:
s1, treating serum, plasma, tissue or cell sample with enzyme extract to obtain sample to be tested, and detecting total protein concentration Cpr；
S2, dissolving a substrate by using a dissolving diluent to obtain a substrate solution, and mixing the substrate solution with a matrix buffer solution with m times of volume to obtain a reaction working solution; dissolving the standard substance by using a dissolving diluent to obtain a series of standard substance solutions with different concentrations, and mixing the standard substance solutions with the matrix buffer solution with m times of volume to obtain a series of standard substance working solutions with different concentrations;
s3, arranging a standard hole and a measuring hole on the ELISA plate, and adding V into the standard hole2Volume of double distilled water, measuring the volume of the double distilled water, and adding V into a hole2Diluting the sample to be detected by f times in volume; adding V into the standard hole3Adding V into the measuring hole according to the volume of the standard substance working solution3Uniformly mixing the reaction working solution with the volume;
s4, incubating at the temperature T, measuring OD values of two fixed time points by an enzyme-labeling instrument at 405nm, and calculating a change value delta A;
s5, drawing a standard curve, and calculating the enzyme activity by the following formula, wherein the calculation mode of the activity of the gamma-glutamyl transpeptidase is as follows:
when the sample to be tested is a tissue or cell sample, the formula (1) is adopted
gamma-GT enzyme activity (U/gprot) ═ ([ delta ] A-b) ÷ a × V1 ÷V2 ÷Cpr ÷T×f(1)
When the sample to be tested is serum or plasma, the formula (2) is adopted
gamma-GT enzyme activity (U/L) (. DELTA.A-b) ÷ a.times.V1 ÷V2 ÷T×f(2)
Wherein, a is the slope of the standard curve, b: intercept of standard curve, V1 ＝V3V (m +1), wherein the enzyme activity in the formula (1) is defined as: at the temperature T, the amount of enzyme catalyzing and generating 1 mu mol of paranitroaniline per gram of protein per minute is 1 activity unit; the definition of the enzyme activity in the formula (2) is as follows: the amount of enzyme catalyzing the production of 1. mu. mol of p-nitroaniline per minute per liter of liquid sample at temperature T was 1 unit of activity.
6. The method of claim 5, wherein m is 2-6 in step S2.
7. The method of claim 5, wherein the sample to be tested is loaded at step S3 slowly at the bottom of the catalase test plate.
8. The method according to claim 5, wherein the temperature T in step S4 is 32-40 ℃, and the time interval between the two incubations is 3-10 min.
9. The method according to claim 5, wherein the temperature T in step S4 is 37 ℃, and the incubation process comprises: incubating at 37 deg.C for 1min, measuring OD at wavelength of 405nm, and recording as A1(ii) a Then placing the mixture into a 37 ℃ incubator for accurate incubation for 5min, and recording the OD value measured at the wavelength of 405nm as A2 ；△A＝A2 -A1。
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