CN108287245B

CN108287245B - Kit for measuring glycosylated hemoglobin and preparation method thereof

Info

Publication number: CN108287245B
Application number: CN201810095525.6A
Authority: CN
Inventors: 甘萍萍; 耿英利; 黎明; 吴昌英; 龙腾镶
Original assignee: Maccura Biotechnology Co ltd
Current assignee: Maccura Biotechnology Co ltd
Priority date: 2018-01-31
Filing date: 2018-01-31
Publication date: 2021-03-16
Anticipated expiration: 2038-01-31
Also published as: CN108287245A

Abstract

The invention relates to the technical field of biology, in particular to a glycosylated hemoglobin determination kit, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises latex, double buffer solution and a surfactant; the reagent R2 comprises a glycosylated hemoglobin monoclonal antibody, a double-surfactant and a buffer solution, and the kit has the advantages of high determination accuracy, low cost and good stability.

Description

Kit for measuring glycosylated hemoglobin and preparation method thereof

Technical Field

The invention belongs to the technical field of glycosylated hemoglobin detection, and particularly relates to a glycosylated hemoglobin detection kit.

Background

Glycated hemoglobin (HbAlc) is a product of hemoglobin bound to glucose in blood, and has a value proportional to blood glucose concentration, and is irreversibly bound and disappears as red blood cells die (about 120 days in the life cycle of red blood cells). Because the glycosylated hemoglobin is related to the service life of red blood cells and the average blood sugar level, the glycosylated hemoglobin is an ideal index for evaluating the long-term blood sugar control of a diabetic patient, can reflect the average blood sugar level of the past 2-3 months, and is not influenced by the blood sugar fluctuation of each day. Closely related to the occurrence of microvascular and macrovascular complications. The glycosylated hemoglobin level is increased, and the occurrence risks of diabetic retinopathy, nephropathy, neuropathy and cardiovascular events are correspondingly increased. The glycosylated hemoglobin has better prediction capability on the occurrence of diabetes.

At present, there are many methods for clinically measuring HbA1c, and HPLC, latex immunoturbidimetry, enzymatic methods, and the like are more commonly used. The HPLC method is a gold standard for measuring HbA1c, is quick, accurate and simple, but needs special instruments, is expensive in instruments and reagents, is high in test cost, and is difficult to develop in laboratories of primary hospitals. The affinity chromatography method is troublesome in time-consuming pretreatment and long in time-consuming; the precision of enzyme method detection is poor; the latex immunoturbidimetry does not need special instruments, can be used for detection together with other biochemical projects on a biochemical instrument, has large flux in unit time and lower test cost than an HPLC method, and is suitable for batch detection.

Currently, the latex applying reagent on the market is a traditional three-reagent, wherein the reagent 1 is blank latex particles, the reagent 2 is a monoclonal antibody of glycosylated hemoglobin (HbA1C), and the reagent 3 is a corresponding anti-goat anti-mouse IgG antibody. Because the immune complex formed by the monoclonal antibody and the secondary antibody has poor long-term storage stability and gradually inactivates to cause the reduction of a measurement signal, an operator needs to mix the reagent 2 and the reagent 3 before use, the operation is complicated, and the stable period of the mixed reagents is short.

Meanwhile, as the content of HbA1c in the total Hb protein is measured as a result of latex immunoturbidimetry, the method is susceptible to detecting moderate and severe anemia samples, so that the detection result is low.

Disclosure of Invention

In order to solve the problems, the invention provides a dual-reagent glycated hemoglobin test kit and a preparation method thereof, which replace the traditional test kit containing three reagents. The invention solves the problems of complicated operation and unstable reagent and the problem of low measured value of a moderate-severe anemia sample.

Specifically, the present invention provides a kit for measuring glycated hemoglobin, comprising a reagent R1 and a reagent R2, wherein the reagent R1 comprises latex, buffer a, and surfactant a; and wherein the reagent R2 comprises glycated hemoglobin antibody, surfactant B, and buffer B.

In some embodiments, buffer a comprises any two components selected from the group consisting of: glycine, borate, Tris, phosphate.

Preferably, buffer a contains components selected from the group consisting of: glycine + borate, glycine + Tris, or glycine + phosphate; most preferably, buffer a contains the component glycine + borate. In some embodiments, the glycine concentration in reagent R1 is between 2mM and 200mM, preferably between 50mM and 150mM, most preferably 100 mM. In some embodiments, the borate concentration in reagent R1 is between 1mM and 50mM, preferably between 10 and 30mM, and most preferably 20 mM.

In some embodiments, the latex particle size in reagent R1 is between 50nm and 200 nm. In a preferred embodiment, the particle size of the latex is from 80nm to 150nm, most preferably 106 nm. In some embodiments, the concentration of the latex ranges from 0.2g/L to 10g/L, preferably the concentration of the latex ranges from 0.5g/L to 2g/L, most preferably 1 g/L.

In some embodiments, surfactant a in reagent R1 is a Tween series surfactant, preferably Tween 20 (Tween-20). Preferably, the concentration of surfactant A is from 0.01ml/L to 1ml/L, preferably from 0.01ml/L to 0.1ml/L, most preferably 0.05 ml/L.

In some embodiments, agent R1 further comprises a preservative. Preferably, the preservative in the reagent R1 may be a Proclin series preservative (e.g. PC150, PC200, PC300, PC950), CAA (2-chloroacetamide), NaN3, phenol, IZU (imidazolidinyl urea), preferably a Proclin series preservative or NaN3, most preferably PC 950. In some embodiments, the concentration of preservative in the agent R1 is between 0.01ml/L and 5ml/L, preferably between 0.2ml/L and 2ml/L, most preferably 1 ml/L.

In some embodiments, the pH of reagent R1 is 5-9, e.g., 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0. Preferably, the pH of the reagent R1 is 8-8.5, most preferably 8.1.

In some embodiments, the surfactant B in reagent R2 is a tween series surfactant + a polyoxyethylene alkyl ether type nonionic surfactant. In some embodiments, the tween series surfactant is selected from the group consisting of: tween 20(Tween-20), Tween 21(Tween-21), Tween 40(Tween-40), Tween 60(Tween-60), Tween 61(Tween-61), Tween 80(Tween-80), Tween 81(Tween-81) and Tween 85 (Tween-85). In some preferred embodiments, the Tween series surfactant is Tween-20 or Tween-80, preferably Tween-20.

In some embodiments, the polyoxyethylene alkyl ether type nonionic surfactant may be polyoxyethylene alkyl ether, polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, or distyrylphenol polyoxyethylene ether; distyrylphenol polyoxyethylene ether is preferred.

In some embodiments, the concentration of the Tween series surfactant in reagent R2 is from 0.05ml/L to 2ml/L, preferably from 0.2ml/L to 0.8ml/L, most preferably 0.5 ml/L. In some embodiments, the concentration of the polyoxyethylene alkyl ether type nonionic surfactant in the reagent R2 is 0.05ml/L to 2ml/L, preferably 0.2ml/L to 0.8 ml/L; most preferably 0.5 ml/L.

In some embodiments, the glycated hemoglobin antibody in the reagent R2 is a glycated hemoglobin monoclonal antibody (e.g., a multimeric monoclonal antibody). In some embodiments, the glycated hemoglobin monoclonal antibody can be a murine anti-human or rabbit anti-human monoclonal antibody. In some embodiments, the concentration of the glycated hemoglobin antibody in the reagent R2 is 0.01-1mg/ml, preferably 0.03-0.2mg/ml, and most preferably 0.1 mg/ml.

In some embodiments, buffer B in reagent R2 may be 2-morpholinoethanesulfonic acid (MES), 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), glycine, borate, or phosphate, among others. In a preferred embodiment, buffer B is a phosphate buffer. More preferably, the phosphate is present in a concentration of 10-200mM, preferably 20-80mM, most preferably 50 mM.

In some embodiments, the reagent R2 further comprises a protecting agent. Preferably, the protective agent is selected from the group consisting of: mannitol, sucrose, sorbitol, and trehalose. More preferably, the protective agent is mannitol. In some embodiments, the concentration of the protective agent in the reagent R2 is from 2ml/L to 50ml/L, more preferably from 5ml/L to 30ml/L, and most preferably 20 ml/L.

In some embodiments, reagent R2 further comprises a protein stabilizing agent. Preferably, the protein stabilizing agent is BSA. In some embodiments, the concentration of protein stabilizing agent in reagent R2 is from 1g/L to 50g/L, preferably from 1g/L to 10g/L, and most preferably 5 g/L.

In some embodiments, the reagent R2 further comprises an inorganic salt. Preferably, the inorganic salt is a sodium or potassium salt, most preferably NaCl or KCl. In some embodiments, the concentration of inorganic salts in reagent R2 is from 1g/L to 30g/L, preferably from 5g/L to 15g/L, and most preferably 9 g/L.

In some embodiments, the agent R2 further comprises a preservative. Preferably, the preservative in the reagent R2 may be Proclin series preservatives (e.g. PC150, PC200, PC300, PC950), CAA, NaN3, phenol, IZU, preferably IZU. In some embodiments, the concentration of preservative in the agent R2 is 0.5-5g/L, preferably 2-4g/L, and most preferably 3 g/L.

In some embodiments, the reagent R2 further comprises a chelating agent. In some embodiments, the chelating agent is selected from the group consisting of: EDTA.2Na, EDTA.2K and EDTA.4Na, preferably EDTA.2Na. In some embodiments, the concentration of the chelating agent in reagent R2 is between 0.01g/L and 4g/L, preferably between 0.1g/L and 1g/L, and most preferably 0.5 g/L.

In some embodiments, the pH of reagent R2 is 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0. Preferably, the pH of the reagent R2 is 5.5-6.5, most preferably 6.

In some preferred embodiments, the present invention provides a kit for measuring glycated hemoglobin, the kit comprising R1 and a reagent R2, which comprises the following components:

reagent R1：

Reagent R2：

reagent R1：

Reagent R2：

Advantageous effects

Compared with the prior art, the invention has the following beneficial advantages: the double buffer solution adopted in the reagent R1 can keep the pH value stable, and can eliminate the latex agglutination and the physical adsorption capacity change caused by the latex charge change in the latex storage process, so that the reagent stability is better. The double-surfactant in the reagent 2 is used as a suspending agent, so that the structural stability of the antibody is maintained, the influence of the change of latex charge on physical adsorption in the latex preservation process can be further eliminated, the antibody is not adsorbed on the latex, and the specific reaction of the antigen and the antibody is promoted. Therefore, the reagent of the present invention can maintain the capacity of latex for physical adsorption of nonspecific adsorption of hemoglobin and glycated hemoglobin at an appropriate level, and the amount of immobilized protein can be kept constant, because the hemoglobin and glycated hemoglobin in the sample are far in excess and are less affected by the total amount of hemoglobin, the influence of individual differences can be eliminated, and the reagent is more advantageous in the determination of severe anemia samples.

Detailed Description

In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described below with reference to the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. 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 application.

Example 1 detection principle and use of glycated hemoglobin assay kit according to the present invention

Principle of detection: hemoglobin and glycated hemoglobin in the sample are immobilized by the same nonspecific adsorption as in the latex of the reagent 1, and when a mouse anti-human glycated hemoglobin antibody is added to form a glycated hemoglobin antigen-antibody immune complex, the amount of the aggregate varies depending on the amount of glycated hemoglobin immobilized on the surface of the latex. The percentage of glycated hemoglobin in the sample to total hemoglobin can be determined by measuring the absorbance and comparing it to a standard curve of the percentage of glycated hemoglobin. When the amount of total hemoglobin can satisfy the latex adsorption amount, the detection result is not influenced, but when the amount of total hemoglobin decreases and is insufficient to satisfy the latex adsorption amount, the immune complex formed with the anti-glycated hemoglobin antibody in the reagent 2 decreases, resulting in a decrease in the detection result, and the antibody can be adsorbed on the latex, which is not favorable for the specific reaction of the antigen antibody. In clinical application, part of patients have different samples due to individual differenceThe total hemoglobin content is different, and when the hemoglobin concentration is reduced, the detection of the kit is influenced, such as an anemia patient sample, and the hemoglobin (Hb) concentration is clinically used for replacing the hemoglobin. According to the national standard: the Hb content of normal people is 110-160g/L, the mild anemia (90-109 g/L), the moderate anemia (60-89g/L) and the severe anemia (30-59 g/L).

An exemplary method of use of the kit of the invention is given here:

1. formulating the kit of the invention with the components and amounts according to any of the embodiments described above;

2. taking an acquired whole blood sample, diluting by 50 times by adopting purified water, combining the whole blood sample with the reagent 1 after hemolysis, and carrying out the same nonspecific adsorption and the solid phase solidification on the hemoglobin and the glycosylated hemoglobin in the sample and latex;

3. mixing with a reagent R2, and fully reacting the glycated hemoglobin monoclonal antibody in the reagent 2 with the reagent to combine to form a glycated hemoglobin antigen-antibody immune complex, wherein the amount of coagulation is different according to the amount of glycated hemoglobin immobilized on the surface of the latex;

4. measuring the absorbance difference after reaction by using a full-automatic biochemical analyzer;

5. and calculating the percentage content of HbA1c in the sample according to the absorbance change value.

It is to be understood that the manner of using the kit of the present invention is not limited to the examples provided herein, and the procedure, amount and other parameters thereof may be modified without departing from the gist of the present invention as long as the effects of the present invention are achieved.

Example 2 glycated hemoglobin assay kit of the present invention has effects on reducing the individual difference and improving stability Effect of (1)

(1)Preparation of the kit of the invention: the kits of experimental group 1 and experimental group 2 were prepared according to the components and contents described in table 1 below, respectively.

TABLE 1 preparation of test groups 1 and 2 kits

Note:^aemulgen A90, surfactant distyrylphenol polyoxyethylene ether, is a series of Emulgen products from Kao corporation.

(2)Effect verification method：

a. Individual difference verification: 10 samples having the same HbA1c concentration were measured, and the difference was that the hemoglobin (Hb) concentration was different. The HbA1c values of 10 samples are 5.2%, and the method has high specificity and the numerical value can reflect the real content of the HbA1c values measured by an internationally accepted IFCC mass spectrometry method. The Hb concentrations of 10 samples are respectively 32g/L, 45g/L, 55g/L, 65g/L, 98g/L, 110g/L, 122g/L, 125g/L, 130g/L and 155g/L, and are measured by a Hesenmeikang hemoglobometer, the Hb concentrations are in gradient distribution and can reflect different individual differences. Wherein: the 1-5# samples belong to anemia samples, the lower the Hb value indicates more significant anemia, and the 6-10# samples are routine samples. The 10 samples are respectively tested by using reagent of a commercial manufacturer of the test kit, the HbA1c test can be judged to be influenced by individual difference according to the measured value of the commercial reagent, and the test result of the test reagent is better than the measured value of the commercial reagent, so that the test reagent can have the effect of eliminating the influence of the individual difference.

b. And (3) stability verification: and (3) under the storage condition of 2-8 ℃, measuring the quality control of the item in 0 month and 3 months respectively, and observing the stability of the reagent.

(3)Results of the experiment：

The test kits of experimental groups 1 and 2 were compared with conventional test kits (i.e., commercially available reagent 1 and commercially available reagent 2), and the individual difference verification results and the long-term stability verification results are shown in the following tables 2 and 3:

TABLE 2 Individual Difference samples for test groups 1 and 2 Experimental results (#1- #10 represent pathological samples 1-10, respectively, with HbA1c values in%)

Whole blood sample	1#	2#	3#	4#	5#	6#	7#	8#	9#	10#
											Hb(g/L)	32	45	55	65	98	110	125	122	130	155
Mass Spectrometry (control)	5.2	5.2	5.2	5.2	5.2	5.2	5.2	5.2	5.2	5.2
											Commercial reagent 1	1.02	2.36	3.69	3.96	5.02	5.12	5.36	5.25	5.36	5.36
Commercial reagent 2	1.53	2.93	3.54	3.87	4.89	5.13	5.42	5.27	5.29	5.26
											Experimental group 1	4.11	4.48	4.62	4.88	5.10	5.21	5.22	5.25	5.24	5.30
Experimental group 2	4.12	4.43	4.63	4.96	5.15	5.15	5.23	5.23	5.26	5.29

The above results show that: when HbA1c is measured by the traditional kit, the measurement result is more accurate when Hb is greater than 110 g/L; when Hb is less than 110g/L, the measured result is more and more different from the real value along with the reduction of Hb value, which indicates that the Hb concentration is less than 110g/L and influences the detection of HbA1c, and the lower Hb is, the lower HbA1c value is; indicating that the individual difference sample influences the result of detecting HbA1 c.

Compared with the measurement result of a reagent sold in the market, the measurement value of the kit is closer to the true value, which shows that the kit can eliminate individual difference in the glycosylated hemoglobin; on the basis of being superior to the measurement result of the reagent sold in the market, the closer the measurement result is to the real value, the better the effect is.

The results of the HbA1c detection in the experimental groups 1 and 2 are closer to the real values than the conventional kit, which indicates that the effect of eliminating the influence of the individual difference sample on the HbA1c measurement value is better. On the basis of being superior to the measurement result of the reagent sold in the market, the closer the measurement result is to the real value, the better the effect is.

TABLE 3 Long-term stability test results for test groups 1 and 2

As can be seen from the stability data of table 3: compared with the quality control measurement data of 0 month, the measured value difference of the 3 month quality control measurement data is very small, and the deviation is within the quality control requirement range (+/-7%), which indicates that the detection kit has high stability under the storage condition of 2-8 ℃.

Example 3 Effect test of glycated hemoglobin assay kit of the present invention at various concentration values

(1)Preparation of the kit: the kits of experimental groups 3-7 were prepared according to the components and contents described in table 4 below, respectively.

TABLE 4 composition table of experimental groups 3-7

(2)Effect verification method：

The individual difference verification and stability effect verification methods were the same as in example 2.

(3)Results of the experiment：

The test kits of experimental groups 3 to 7 were compared with conventional test kits (i.e., commercially available reagent 1 and commercially available reagent 2), and the individual difference verification results and the long-term stability verification results are shown in the following tables 5 and 6:

TABLE 5 Individual Difference samples from groups 3-7 Experimental results (#1- #10 represent pathological samples 1-10, respectively, with HbA1c values in%)

The detection results of the kits of the experimental groups 3-7 on HbA1c are closer to the real values compared with the commercial reagents, especially the measured values of Hb are obviously better than those of the traditional kit when Hb is less than 110g/L, which indicates that the effect of the kits on eliminating the influence of individual difference samples on the measured value of HbA1c is remarkably improved. As can be seen from the results, the determination results of the experimental groups 3-6 are already significantly better than those of the traditional kit, and are close to the true values. The detection result of the experimental group 7 is basically not different from the true value, which shows that the influence of individual difference can be completely eliminated.

TABLE 6 Long-term stability test results for test groups 3-7

As can be seen from the stability data of table 6: compared with the quality control measurement data of 0 month, the difference of the measured values of the 3-month quality control measurement data is very small, and the deviation is within the range of +/-7 percent of the quality control requirement, so that the detection kit disclosed by the invention can be stable under the storage condition of 2-8 ℃. And the results of experimental groups 5-6 are better than those of experimental groups 3-4, and the most preferred formulation is experimental group 7.

Example 4 concentration boundary test of glycated hemoglobin assay kit of the present invention

Example 3 shows the applicable concentration range of each component of the kit of the invention, and this example tests the influence of other concentrations outside the applicable range on individual difference and stability effect.

(1)Preparation of the kit: the kits of experimental groups 8-9 were prepared according to the components and contents described in table 7 below, respectively.

TABLE 7 composition tables of Experimental groups 8-9

(2)Results of the experiment：

Experiment group 8 the calibration curve of the experiment group was poor, the sensitivity of the low end was poor, the high end had no gradient, and the calibration did not pass. Experiment group 9 the sensitivity of the experiment group is better, but the high-end signal value is out of limit, which also causes the calibration failure and the effect is worse.

Example 5 Effect test of Replacing double buffer Components in reagent R1, double surfactant Components in reagent R2

(1)Preparation of the kit: the kits of experimental groups 3-7 were prepared according to the components and contents as described in table 8 below.

TABLE 8 composition Table of experimental groups 10-12

(2)Effect verification method：

(3)Results of the experiment：

The test kits of the experimental groups 10 to 12 were compared with conventional test kits (i.e., commercially available reagent 1 and commercially available reagent 2), and the individual difference verification results and the long-term stability verification results are shown in the following tables 9 and 10:

TABLE 9 Individual Difference samples from groups 10-12 Experimental results (#1- #10 represent pathological samples 1-10, respectively, with HbA1c values in%)

Whole blood sample	1#	2#	3#	4#	5#	6#	7#	8#	9#	10#
											Hb(g/L)	32	45	55	65	98	110	125	122	130	155
Mass Spectrometry (control)	5.2	5.2	5.2	5.2	5.2	5.2	5.2	5.2	5.2	5.2
											Experimental group 10	2.52	2.92	4.12	4.55	5.12	4.89	5.11	4.98	4.78	4.96
Experimental group 11	2.53	2.92	4.13	4.50	5.25	4.78	5.33	4.85	5.06	5.06
											Experimental group 12	2.56	2.63	4.26	4.53	5.36	4.93	5.36	4.96	5.13	4.76
Commercial reagent 1	1.02	2.36	3.69	3.96	5.02	5.12	5.36	5.25	5.36	5.36
											Commercial reagent 2	1.53	2.93	3.54	3.87	4.89	5.13	5.42	5.27	5.29	5.26

The test result shows that: when HbA1c is measured by the commercial reagent 1 and the commercial reagent 2, the measurement result is more accurate when Hb is more than 110 g/L; when Hb is less than 110g/L, the measured result is more and more different from the real value along with the reduction of Hb value, which indicates that the Hb concentration is less than 110g/L and influences the detection of HbA1c, and the lower Hb is, the lower HbA1c value is; indicating that the individual difference sample influences the result of detecting HbA1 c.

After the reagent replacement as described above, the detection results of HbA1c of the three sets of test kits for samples 1# -5# (Hb < 110 g/L) were greatly different from the actual values, indicating that the measured values were not accurate enough.

TABLE 10 Long-term stability test results for test groups 10-12

The stability data show that: the deviation of the quality control measurement data of 0 month is large, and the quality control measurement data of 3 months are not in the required range (+/-7%), which indicates that the stability of the reagent at 2-8 ℃ is not good.

Example 6 Effect testing of alternative Components

This example then tested the effect of some of the alternative components in reagents R1 and R2 on the effect.

(1)Preparation of the kit: the kits of experimental groups 13-14 were prepared according to the components and contents described in table 11 below, respectively.

TABLE 11 Individual Difference samples from groups 13-14 Experimental results (#1- #10 represent pathological samples 1-10, respectively, with HbA1c values in%)

(2)Effect verification method：

(3)Results of the experiment：

The kits of experimental groups 13 to 14 were compared with conventional kits (i.e., commercially available reagent 1 and commercially available reagent 2), and the individual difference verification results and the long-term stability verification results are shown in the following tables 12 and 13:

TABLE 12 Individual Difference samples from groups 13-14 Experimental results (#1- #10 represent pathological samples 1-10, respectively, with HbA1c values in%)

Whole blood sample	1#	2#	3#	4#	5#	6#	7#	8#	9#	10#
											Hb(g/L)	32	45	55	65	98	110	125	122	130	155
Mass Spectrometry (control)	5.2	5.2	5.2	5.2	5.2	5.2	5.2	5.2	5.2	5.2
											Experimental group 13	4.02	4.45	4.72	4.99	5.11	5.21	5.22	5.23	5.27	5.25
Experimental group 14	4.10	4.36	4.69	4.98	5.14	5.23	5.25	5.25	5.31	5.26
											Commercial reagent 1	1.02	2.36	3.69	3.96	5.02	5.12	5.36	5.25	5.36	5.36
Commercial reagent 2	1.53	2.93	3.54	3.87	4.89	5.13	5.42	5.27	5.29	5.26

The test result shows that: when HbA1c is measured by the traditional kit, the measurement result is more accurate when Hb is greater than 110 g/L; when Hb is less than 110g/L, the measured result is more and more different from the real value along with the reduction of Hb value, which indicates that the Hb concentration is less than 110g/L and influences the detection of HbA1c, and the lower Hb is, the lower HbA1c value is; indicating that the individual difference sample influences the result of detecting HbA1 c.

The measured value of the kit is closer to the true value than the measured result of the reagent sold in the market, which indicates that the individual difference can be eliminated in the glycosylated hemoglobin; on the basis of being superior to the measurement result of the reagent sold in the market, the closer the measurement result is to the real value, the better the effect is.

As can be seen from the above results, the above replacement (e.g., replacing Tween-20 with Tween-80 and replacing A-90 with A-60) can still achieve the effect of reducing the influence of individual differences.

TABLE 13 Long-term stability test results for test groups 13-14

The stability data show that: compared with the quality control measurement data of 0 month, the difference of the measured values of the 3-month quality control measurement data is very small, and the deviation is within the range of +/-7 percent of the quality control requirement, so that the detection kit disclosed by the invention can be stable under the storage condition of 2-8 ℃.

As can be seen from the above results, the stability effect of the present invention can still be achieved by using the above replacement (e.g., Tween-20 replaced by Tween-80 and A-90 replaced by A-60).

It is to be understood that this invention is not limited to the particular methodology, protocols, and materials described, as these may vary without departing from the spirit of the invention. It is also to be understood that the examples described herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention, which is defined only by the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A kit for assaying glycated hemoglobin, comprising a reagent R1 and a reagent R2, wherein the reagent R1 comprises latex, buffer a, and surfactant a; and wherein the reagent R2 comprises glycated hemoglobin antibody, surfactant B, and buffer B; wherein the buffer A is a combination of glycine and borate; the surfactant B is a Tween series surfactant and a distyrylphenol polyoxyethylene ether type nonionic surfactant;

wherein the pH value of the reagent R1 is 5-9;

wherein the pH value of the reagent R2 is 4-9;

the concentration of the latex in the reagent R1 is 0.2g/L-10 g/L; the concentration of glycine in the reagent R1 is 2mM-200mM, and the concentration of borate is 1mM-50 mM; the concentration of the surfactant A in the reagent R1 is 0.01ml/L-1 ml/L; the concentration of the glycosylated hemoglobin antibody in the reagent R2 is 0.01mg/ml-1 mg/ml; the concentration of the Tween series surfactant in the reagent R2 is 0.05ml/L-2ml/L, and the concentration of the distyrylphenol polyoxyethylene ether type nonionic surfactant is 0.05ml/L-2 ml/L; the concentration of the buffer B in the reagent R2 is 10mM-200 mM.

2. The kit of claim 1, wherein the tween series surfactant is selected from the group consisting of: tween 20, tween 21, tween 40, tween 60, tween 61, tween 80, tween 81, and tween 85.

3. The kit according to any one of claims 1 to 2, wherein the latex particle size in the reagent R1 is between 50nm and 200 nm.

4. The kit of any one of claims 1-2, wherein the reagent R1 further comprises a preservative.

5. The kit of any one of claims 1-2, wherein the reagent R2 further comprises one or more of: protective agent, protein stabilizer, inorganic salt, preservative and chelating agent.

6. A kit for assaying glycated hemoglobin, the kit comprising a reagent R1 and a reagent R2, each of which comprises the following components:

reagent R1：

Reagent R2：

7. A kit for assaying glycated hemoglobin, the kit comprising a reagent R1 and a reagent R2, each of which comprises the following components:

reagent R1：

Reagent R2：