CN110687306B - Double-reagent glycosylated hemoglobin detection kit for direct on-machine hemolysis enzyme method - Google Patents
Double-reagent glycosylated hemoglobin detection kit for direct on-machine hemolysis enzyme method Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/723—Glycosylated haemoglobin
Abstract
The invention relates to a direct on-machine hemolysis enzyme method double-reagent glycosylated hemoglobin detection kit, which comprises a sample treatment agent, a reaction reagent 1 and a reaction reagent 2. The protease for hydrolyzing the glycosylated hemoglobin in the whole blood sample is arranged in the sample treating agent with the hemolysis effect, and can be directly used for one-step hemolysis on the full-automatic biochemical analyzer and hydrolyzing the glycosylated hemoglobin in the whole blood sample, so that off-line hemolysis pretreatment is not needed in advance, and direct on-machine hemolysis is faster and more effective. Compared with the traditional glycosylated hemoglobin detection kit, the detection kit reduces one reagent, and the R1a reagent and the R1b reagent do not need to be mixed in advance like the traditional kit when in use, so that the time can be shortened and the operation can be simplified. In addition, the detection kit optimizes the components of the sample treatment agent, and the dissolution product obtained after the whole blood sample is subjected to hemolysis treatment by the sample treatment agent has good stability, and the error of the detection result within 3 hours is within 5%.
Description
Technical Field
The invention relates to the technical field of biochemical detection, in particular to a glycosylated hemoglobin detection kit, and in particular relates to a direct on-machine hemolysis enzyme method double-reagent glycosylated hemoglobin detection kit.
Background
The glycosylated hemoglobin (glycated hemoglobin, GHb) is a product of binding hemoglobin in red blood cells in blood with blood sugar, and can be classified into glycosylated glucose, glycosylated fructose, glycosylated lactose, and the like. The free glucose content in human body is far higher than that of other saccharides, and the saccharification probability of beta-chain N-terminal valine residue of hemoglobin is highest, so that most of glycosylated hemoglobin in human body is a conjugate (hemoglobin A1c, hbA1 c) of beta-chain N-terminal valine residue and glucose, accounting for 60% of glycosylated hemoglobin, and HbA1c results are used in clinical quantitative determination at present.
The production of glycosylated hemoglobin is an irreversible reaction, is proportional to the blood glucose concentration, and is maintained for about 120 days, so that the blood glucose concentration before 120 days can be detected, and is not affected by the blood drawing time, whether or not it is empty, whether or not insulin is used, and other factors that can cause the blood glucose level to fluctuate briefly. The guidelines of the american society of diabetes in 2010 have HbA1c ≡6.5% as one of the diagnostic criteria for diabetes, and the world health organization in 2011 has also proposed the use of this criterion for the diagnosis of diabetes in countries and regions where conditions are present. The glycosylated hemoglobin has strong detection specificity, good repeatability and high sensitivity, and is internationally recognized as a gold standard for monitoring diabetes.
The method for detecting glycosylated hemoglobin mainly comprises the following steps: detection methods based on charge differences, including ion exchange High Performance Liquid Chromatography (HPLC), electrophoresis methods, and the like; detection methods based on structural differences include affinity chromatography, immunochemistry, enzymatic methods, and the like. The ion exchange HPLC method has advantages of high detection throughput, high accuracy, and good reproducibility, and has been recommended as a reference method by the national glycosylated hemoglobin standardization program (NGSP), but requires a special instrument and has a long experimental time. The immunoturbidimetry is suitable for batch detection by an automatic biochemical analyzer, but is susceptible to high concentration glucose, triacetin and bilirubin, as well as hemoglobin variants.
The enzymatic reaction is based on the principle that after whole blood is subjected to a hemolysis treatment, the beta-chain N-terminus of glycosylated hemoglobin (HbA 1 c) is cleaved by neutral protease to release glycosylated dipeptide, and fructosyl amino acid oxidase (FAOD) having good specificity is used to act on the glycosylated dipeptide to produce hydrogen peroxide (H) 2 O 2 )，H 2 O 2 Is proportional to HbA1c content in blood, H 2 O 2 And reacting with chromogen under the action of peroxidase to generate chromogenic compound, and determining HbA1c content according to the change of absorbance by contrast with a calibration curve.
The enzymatic reaction is sensitive and specific, can be detected by using a full-automatic biochemical analyzer, does not need expensive instruments, and has good correlation with the conventional HPLC method and the immunoturbidimetry. The enzymatic method is divided into two types, one is to directly measure the percentage of glycosylated Hemoglobin to total Hemoglobin (referred to as one-step method) through absorbance change values in the reaction of a primary sample and a reagent, and the other is to respectively measure the absolute concentrations of glycosylated Hemoglobin and total Hemoglobin, and further calculate the percentage of glycosylated Hemoglobin to total Hemoglobin through a formula (referred to as two-step method).
The two-step method requires a measuring instrument having two channels of a measurement channel for total hemoglobin and a measurement channel for glycosylated hemoglobin, and thus requires two channel amounts of reagents, which is complicated to handle. The reagent of the one-step method is currently three reagents (R1 a, R1b and R2), because the reagent 1a contains protease, components in the R1a and the R1b cannot coexist for a long time and can only be divided into two reagents, the reagent R1a and the reagent R1b are mixed in advance when in use, and the whole blood sample needs to be subjected to off-line hemolysis pretreatment in advance for 10 to 20 minutes, so that the service life of the reagent is limited, and meanwhile, the detection turnover time is long, the operation is complex, and the artificial condition is not easy to control. Although the one-step method has double reagents, the sample is unstable after treatment, and detection must be completed within 20 minutes, which limits the use of the method.
Disclosure of Invention
Aiming at the defects of the enzymatic one-step glycosylated hemoglobin reagent in the prior art, the invention aims to provide a novel glycosylated hemoglobin detection kit which can be directly used for hemolysis of a blood sample and detecting the content of glycosylated hemoglobin on a full-automatic biochemical analyzer.
The aim of the invention is achieved by the following technical scheme:
a direct on-machine hemolysis enzyme method double-reagent glycosylated hemoglobin detection kit comprises the following reagents and components:
(1) A sample processing agent comprising:
(2) A reagent 1 comprising:
(3) Reactant 2 comprising:
in a specific embodiment of the invention, the quaternary ammonium salt in the detection kit is an alkyl trimethyl quaternary ammonium salt or an alkyl dimethylbenzyl ammonium salt. Non-limiting examples of alkyl trimethyl quaternary ammonium salts include, for example, dodecyl trimethyl ammonium chloride, tetradecyl trimethyl ammonium chloride, hexadecyl trimethyl ammonium chloride, octadecyl trimethyl ammonium chloride, dodecyl trimethyl ammonium bromide, tetradecyl trimethyl ammonium chloride, hexadecyl trimethyl ammonium bromide, octadecyl trimethyl ammonium bromide. Non-limiting examples of alkyl dimethylbenzyl ammonium salts include, for example, dodecyl dimethylbenzyl ammonium chloride, dodecyl dimethylbenzyl ammonium iodide, dodecyl dimethylbenzyl ammonium bromide.
In a specific embodiment of the invention, the polyethylene glycol in the detection kit has a molecular weight of 4000 to 250000, such as 4000, 6000, 8000, 10000, 12000, 20000, 40000, 108000, 218000. Preferably, the polyethylene glycol has a molecular weight of 10000 to 100000.
In particular embodiments of the invention, the cyclodextrin in the test kit comprises cyclodextrin and derivatives thereof known in the art, such as α -cyclodextrin, β -cyclodextrin, γ -cyclodextrin, methyl- β -cyclodextrin, 2-hydroxypropyl- α -cyclodextrin, 2-hydroxypropyl- β -cyclodextrin, 2-hydroxypropyl- γ -cyclodextrin, triacetyl- β -cyclodextrin, quaternary ammonium- β -cyclodextrin, carboxymethyl- β -cyclodextrin, 2,3, 6-tri-O-methyl- β -cyclodextrin, dimethyl β -cyclodextrin, 3-O-acetyl-2, 6-di-O-methyl- β -cyclodextrin.
It should be noted that the quaternary ammonium salt is added in the sample treatment agent in the detection kit disclosed by the invention, so that the rapid hemolysis of blood cells is facilitated, the stability of the treated sample is improved by PEG and cyclodextrin, and the cracking product is stable within 3 hours.
In a specific embodiment of the invention, the buffer in the detection kit is 2-cyclohexylaminoethanesulfonic acid (CHES), 2-morpholinoethanesulfonic acid, MES, PIPES, HEPES, TRIS-HCl, TRICINE, BICINE, MOPS, or a combination thereof.
In a preferred embodiment of the present invention, the buffer in the sample processing agent is 2-cyclohexylaminoethanesulfonic acid (CHES), the buffer in the reaction agent 1 is 2-morpholinoethanesulfonic acid, and the buffer in the reaction agent 2 is Tris-HCl.
In a specific embodiment of the invention, the surfactant in the detection kit is Triton X-100, SDS, glycerol or a combination thereof.
In a preferred embodiment of the present invention, the surfactant in the sample treatment agent is Triton X-100, SDS or a combination thereof, the surfactant in the reaction agent 1 is Triton X-100, glycerol or a combination thereof, and the surfactant in the reaction agent 2 is Triton X-100.
In a specific embodiment of the present invention, the neutral protease in the sample processing agent is a metalloprotease and the peroxidase in the reaction reagent 2 is horseradish peroxidase.
In a preferred embodiment of the present invention, the sample processing agent, the reaction reagent 1 and the reaction reagent 2 further comprise 1 to 10% by weight of a stabilizer. Further preferably, the stabilizer is trehalose or sorbitol.
In a preferred embodiment of the present invention, the reagents 1 and 2 further comprise a preservative in the range of 0.001-0.05 mg/mL. Further preferably, the preservative in the reaction reagent 1 is proclin300, sodium methyl benzoate or geneticin G418 or a combination thereof; the preservative in the reagent 2 is geneticin G418.
In a preferred embodiment of the invention, the sample processing agent further comprises 0.01-10mM of N-ethylmaleimide.
It should also be noted that the detection kit of the invention is added with metalloprotease, which can rapidly decompose beta chain of hemoglobin to release amino acid, including glycosylated valine, so as to be beneficial to the subsequent reaction. And simultaneously, ascorbic acid oxidase, catalase and N-ethylmaleimide are added, so that interference of ascorbic acid, hemoglobin variant and post-transcriptional modification in a sample can be effectively removed.
In some more specific embodiments of the invention, the detection kit comprises the following reagents and components:
(1) A sample processing agent comprising:
(2) A reagent 1 comprising:
(3) Reactant 2 comprising:
in some still more specific and preferred embodiments of the invention, the detection kit comprises the following reagents and components:
(1) A sample processing agent comprising:
(2) A reagent 1 comprising:
(3) Reactant 2 comprising:
in a more preferred embodiment of the invention, the detection kit comprises the following reagents and components:
(1) A sample processing agent comprising:
(2) A reagent 1 comprising:
(3) Reactant 2 comprising:
the sample treating agent, the reaction reagent 1 and the reaction reagent 2 in the direct on-machine hemolysis double-reagent glycosylated hemoglobin detection kit can be prepared by respectively taking distilled water as a solvent, adding all components into the distilled water and uniformly mixing, and then respectively filling all mixed liquids into a sample treating agent container, a reaction reagent 1 container and a reaction reagent 2 container, adding a kit using instruction and packaging the kit.
The invention has the beneficial effects that:
according to the kit for detecting the glycosylated hemoglobin by the direct on-machine hemolysis method, the protease for hydrolyzing the glycosylated hemoglobin in the whole blood sample is arranged in the sample treating agent with the hemolysis effect, and the quaternary ammonium salt which is helpful for accelerating the cracking of blood cells is added in the sample treating agent, so that the hemolysis is faster, and the stability of the treated sample is improved by PEG and cyclodextrin, so that the cracked product is stable within 3 hours. Therefore, through the design of the sample treating agent, the sample pretreatment can be directly finished on the full-automatic biochemical analyzer, and the glycosylated hemoglobin in the whole blood sample can be tested in real time, so that the method is more efficient, and meanwhile, the trouble of sample off-line hemolysis operation and errors possibly caused by manual hemolysis operation are avoided. The stability of the dissolved product obtained after the whole blood sample is subjected to hemolysis treatment by the sample treating agent is good, the error of the detection result within 3 hours is within 5%, the defect that the reported double-reagent method is unstable after the sample is treated and the detection must be completed within 20 minutes is overcome, and the method is applicable to wider models and is more beneficial to clinical application.
Meanwhile, the detection kit of the invention can combine the traditional R1a reagent and the R1b reagent into one reagent because the protease for hydrolyzing the glycosylated hemoglobin in the whole blood sample is arranged in the sample treatment agent for hemolysis, and compared with the traditional glycosylated hemoglobin detection kit, one reagent is reduced, and the R1a reagent and the R1b reagent do not need to be mixed in advance as the traditional kit when in use, so that the time can be shortened and the operation can be simplified.
Drawings
FIG. 1 shows that the direct on-machine hemolysis double reagent glycosylated hemoglobin detection kit of the present invention has good linearity in the range of 3.8 wt.% to 12.8 wt.% glycosylated hemoglobin HbA1 c;
FIG. 2 shows that the direct on-machine hemolysis double reagent glycosylated hemoglobin detection kit of the present invention ("Shangtai reagent results") correlates well with the commercially available one-step three reagent glycosylated hemoglobin detection kit ("control reagent results").
Detailed Description
The invention will be described in further detail by means of the following detailed description in conjunction with the accompanying drawings.
The principle of enzymatic measurement of glycosylated hemoglobin is that after whole blood is subjected to hemolysis treatment, the beta-chain N-terminus of glycosylated hemoglobin (HbA 1 c) is cleaved by neutral protease to release glycosylated dipeptide, and fructosyl amino acid oxidase (FAOD) having good specificity is used to act on glycosylated dipeptide to generate hydrogen peroxide (H) 2 O 2 )，H 2 O 2 Is proportional to HbA1c content in blood, H 2 O 2 And reacting with chromogen under the action of peroxidase to generate chromogenic compound, and determining HbA1c content according to the change of absorbance by contrast with a calibration curve.
The currently common enzymatic glycosylated hemoglobin detection kit on the market comprises a hemolysis agent, a reagent 1a, a reagent 1b and a reagent 2, and is a three-reagent detection kit. Wherein the hemolyzing agent comprises, for example, 100mmol/L of N-cyclohexyl-2-sulfamic acid (CHES) buffer, 1 wt.% of triax-100, 0.45 wt.% of sodium dodecyl sulfate; reagent 1a comprises, for example, 5mmol/L of 2-morpholinoethanesulfonic acid buffer (pH 7.0), 4KU/ml of protease, 2mmol/L of N-ethylbutenediomide; reagent 1b comprises, for example, 1 mmol/L2-morpholinoethanesulfonic acid buffer, 10. Mu. Mol/L2- (4-iodobenzene) -3- (2, 4-dinitrobenzene) -5- (2, 4-dithiobenzene) -2H-tetrazole monosodium salt; reagent 2 comprises, for example, 15mmol/L of tris buffer, 10U/ml of Fructose Valine Oxidase (FVO), 90U/ml of horseradish peroxidase, and 0.8mmol/L of DA-64, wherein Fructose Valine Oxidase (FVO) is a fructosyl amino acid oxidase (FAOD) and DA-64 is chromogen.
The reagents 1a and 1b in such a detection kit on the market must be separately contained in different containers. If the reagent 1a and the reagent 1b are directly mixed together, the component in the reagent 1b is in contact with the protease in the reagent 1a for a long period of time during the non-use of the detection kit, which affects the activity of the protease. When the detection kit is used for glycosylated hemoglobin detection, the R1a reagent and the R1b reagent are required to be mixed in advance, and the whole blood sample is required to be subjected to off-line hemolysis pretreatment 10 to 20 minutes in advance, so that the service life of the reagent is limited, and meanwhile, the detection turnover time is long, the operation is complex, and the artificial condition is not easy to control.
In order to overcome the above-mentioned drawbacks of the detection kits currently in the market, the present inventors have made intensive studies and developments and creatively proposed to dispose the protease component in the reagent 1a of the detection kit currently in the market in a sample treating agent that plays a hemolysis role, so that the remaining components of the reagent 1a and the respective components of the reagent 1b can be mixed into one reagent. In addition, the present inventors have further optimized the composition of each reagent for detection kits commonly found on the market. Finally, the inventor develops the direct on-machine hemolysis double-reagent glycosylated hemoglobin detection kit of the invention, which comprises a sample treatment agent, a reaction reagent 1 and a reaction reagent 2, belongs to the two-reagent detection kit, and reduces one reagent compared with the common glycosylated hemoglobin three-reagent detection kit on the market.
The invention is further illustrated by the following non-limiting exemplary embodiments and test examples.
Examples 1 to 3
The direct on-machine hemolysis double-reagent glycosylated hemoglobin detection kit of examples 1-3 comprises the following reagents and components:
for examples 1 to 3, the components of the sample treating agent, the reaction reagent 1 and the reaction reagent 2 were added to distilled water and mixed uniformly, respectively, using distilled water as a solvent, and then the respective mixed liquids were filled in a sample treating agent container, a reaction reagent 1 container and a reaction reagent 2 container, respectively, to prepare the detection kit of each example.
Test case
In this test example, the whole blood sample was detected by using a two-point end-point method (forward reaction) on a fully automatic biochemical analyzer (rogowski Modular P) using the direct on-machine hemolysis enzymatic double reagent glycosylated hemoglobin detection kit prepared in example 2 as a representative example, and the accuracy, stability, reproducibility, linear range and correlation with a commercially available enzymatic triple reagent glycosylated hemoglobin detection kit were examined.
The detection operation method is briefly described as follows: taking 10 mu L of whole blood sample (or calibration quality control substance) to be detected, adding the whole blood sample (or calibration quality control substance) into 125 mu L of sample treatment solution, fully and uniformly mixing, taking out 25 mu L of mixed solution, mixing with 160 mu L of R1 reagent, preserving heat for 5min at 37 ℃, and reading absorbance A1 of a first point at 700/800 nm. Then 70. Mu.L of R2 reagent was added, and after 3 minutes of reaction at 37℃the absorbance A2 at the second spot was read. And calculating an absorbance difference delta A=A2-A1 of the whole blood sample to be measured, and calculating the HbA1c content of the glycosylated hemoglobin in the whole blood sample according to a calibration curve.
1. Accuracy and stability
The glycosylated hemoglobin standards GBW09181, GBW09182 and GBW09183 in the human hemoglobin solutions with the indicated concentration percentages of 5.02%, 6.86% and 9.34%, respectively, were obtained, and were tested after 1, 20, 40, 60, 120, 180, 200min on a full-automatic biochemical analyzer using the sample treatment agent of the direct on-machine hemolysis dual reagent glycosylated hemoglobin test kit of example 2, respectively, and the test was repeated 3 times at each time point. The results are shown in table 1, and the direct on-machine hemolysis double-reagent glycosylated hemoglobin detection kit provided by the invention can be used for testing glycosylated hemoglobin in a whole blood sample within 1 minute, so that instant detection can be realized, and the error of the detection result within 3 hours is within 5%, and has good accuracy and stability.
Table 1: accuracy and stability test results
2. Repeatability of
Two samples of glycosylated hemoglobin HbA1c at a concentration percentage of 4.7% and 8.6% were obtained, and were each tested 10 times using the direct on-machine hemolysis dual reagent glycosylated hemoglobin test kit of example 2. The results are shown in Table 2 below, and it can be seen that the kit for directly detecting glycosylated hemoglobin by the on-machine hemolysis method according to the present invention has good reproducibility.
Table 2: repeatability test
3. Linear range
Whole blood samples with concentration percentages of glycosylated hemoglobin HbA1c of 3.8% and 12.8% respectively are obtained, and mixed into 6 gradients according to the proportion: 3.8%, 5.6%, 7.4%, 9.2%, 11.0% and 12.8% of the total amount of the reagent, and each concentration was measured by using the direct on-machine hemolysis double reagent glycosylated hemoglobin test kit of example 2, and each concentration was repeatedly measured 3 times. As shown in Table 3 and FIG. 1, it is seen that the direct on-machine hemolysis double reagent glycosylated hemoglobin detection kit of the present invention has good linearity in the range of the HbA1c percentage of glycosylated hemoglobin of 3.8% -12.8%.
Table 3: linear range test
4. Comparison with one-step three-reagent glycosylated hemoglobin detection kit
Whole blood samples having different concentration percentages of HbA1c of glycosylated hemoglobin were obtained, and the direct on-machine hemolysis double reagent glycosylated hemoglobin detection kit prepared in example 2 was used for detection, and meanwhile, the commercial one-step three reagent glycosylated hemoglobin detection kit (Roche Module P) was used for detection, and the results are shown in Table 4 and FIG. 2 below, which show that the direct on-machine hemolysis double reagent glycosylated hemoglobin detection kit of the present invention ("Shangtai reagent results") has good correlation with the commercial one-step three reagent glycosylated hemoglobin detection kit ("control reagent results").
Table 4: comparative test of the detection kit of the present invention with commercially available detection kit
The invention has been described with particular reference to the examples which are intended to be illustrative of the invention and not limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art according to the idea of the invention. Such deductions, modifications or alternatives fall within the scope of the claims of the present invention.
Claims (1)
1. The kit for detecting the double-reagent glycosylated hemoglobin by the direct on-machine hemolysis method is characterized by comprising the following reagents and components:
(1) A sample processing agent comprising:
(2) A reagent 1 comprising:
(3) Reactant 2 comprising:
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