CN114878839A - Glycated hemoglobin dissociation solution - Google Patents

Glycated hemoglobin dissociation solution Download PDF

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CN114878839A
CN114878839A CN202210484542.5A CN202210484542A CN114878839A CN 114878839 A CN114878839 A CN 114878839A CN 202210484542 A CN202210484542 A CN 202210484542A CN 114878839 A CN114878839 A CN 114878839A
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glycated hemoglobin
concentration
reaction
dissociation solution
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周翔
丁超
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Jiangsu Aikangrunsi Biotechnology Co ltd
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Abstract

The invention discloses a glycosylated hemoglobin dissociation solution, which comprises a phosphate buffer solution, ethylphenyl polyethylene glycol, sodium dodecyl sulfate and sodium caseinate. The dissociation liquid can efficiently dissociate the glycosylated hemoglobin in the whole blood sample, the reaction is carried out under the condition that the pH value is 7.0 +/-0.5, the reaction time is short, the influence on the subsequent reaction is small, and the release is thorough. The invention also discloses a kit comprising the glycosylated hemoglobin dissociation solution.

Description

Glycated hemoglobin dissociation solution
Technical Field
The invention relates to the field of biological detection, and particularly relates to a glycosylated hemoglobin dissociation solution.
Background
Hemoglobin (Hb) is generally composed of HbA (97%), HbA2 (2.5%) and HbF (0.5%). Chromatographic analysis of HbA revealed minor haemoglobins HbA1a, HbA1b, HbA1c and HbA 0. HbA1a, HbA1b, and HbA1c are collectively referred to as glycated hemoglobin (HbA1), and their glycosylation site is a.beta.chain N-terminal valine residue. HbA0 refers to hemoglobin without a carbohydrate attached.
Glycated hemoglobin is a stable compound in which glucose is covalently bound to the N-terminal valine residue of the beta chain of hemoglobin in human blood, and is collectively referred to as the beta chain of hemoglobin (blood) -N- (1-deoxyfructo-1-yl) hemoglobin. HbA1c is a main component of glycated hemoglobin, accounts for 60% of total glycated hemoglobin (GHb), and is the result of HbA1c in clinical quantitative determination and application. HbA1c undergoes a non-enzymatic conjugation reaction from the free aldehyde group of glucose to the amino group of the N-terminal valine of the beta chain of HbA, forming first the labile Schiff base (aldimine), followed by Amadori (glucosamine) rearrangement, and finally the stable ketoamine compound.
The prior glycated hemoglobin dissociation solution has the following several types, but all have corresponding technical defects: 1) organic solvent extraction method: for example, the extraction using acetonitrile is performed by dissolving glycated hemoglobin in an organic phase by the principle of similar phase dissolution, and centrifuging the solution using a centrifuge to obtain an extract containing glycated hemoglobin. In the method, the glycated hemoglobin needs to be centrifuged after dissociation, so that the automatic operation is not convenient to realize; 2) acid-base method: depending on the higher or lower pH, the glycated hemoglobin is denatured or the conformational change is dissociated for release. The vitamin D solution obtained by the method is a solution with higher pH or lower pH, which is not beneficial to the next immunoreaction; 3) a combination method. The currently used method is to dissociate glycosylated hemoglobin by combining 1% dodecyl trimethyl ammonium bromide, 0.1% Tween-80, 0.1% Tween-20, 0.1% Triton X-100 and 0.8% ammonium chloride. The method contains dodecyl trimethyl ammonium bromide and ammonium chloride which are highly toxic substances and have biological safety risk. Excessive use of Triton X-100 causes environmental pollution.
Therefore, the hemoglobin dissociation solution which is safe and efficient and has small influence on subsequent reaction is obtained, and the method has important significance.
Disclosure of Invention
In order to solve the above problems, the present invention provides, in a first aspect, a glycated hemoglobin dissociation solution, including a phosphate buffer, ethylphenylpolyethylene glycol, sodium dodecyl sulfate, and sodium caseinate.
Further, the concentration of the phosphate buffer solution is 10-50mM, the concentration of the ethyl phenyl polyethylene glycol is 0.3% -1%, the concentration of the lauryl sodium sulfate is 0.1% -0.5%, and the concentration of the casein sodium is 0.3% -1%.
Further, the concentration of the phosphate buffer solution is 20-40 mM.
Further, the concentration of the ethyl phenyl polyethylene glycol is 0.5% -1%.
Further, the concentration of the ethyl phenyl polyethylene glycol is 0.8% -1%.
Further, the concentration range of the casein sodium is 0.5% -1%.
In a second aspect, the present invention provides a use of the glycated hemoglobin dissociation solution according to the first aspect of the present invention in the preparation of a kit.
Further, the kit can be used for separating and detecting the glycosylated hemoglobin in the blood sample.
In a third aspect, the present invention provides a kit comprising the glycated hemoglobin dissociation solution according to the first aspect of the present invention.
Compared with the prior art, the invention has the beneficial effects that:
according to the technical scheme, phosphate, ethyl phenyl polyethylene glycol, sodium dodecyl sulfate and sodium caseinate are combined according to a certain proportion to form a new dissociation solution, glycosylated hemoglobin in a whole blood sample can be efficiently dissociated, the reaction is carried out under the condition that the pH is 7.0 +/-0.5, the reaction time is short, the influence on subsequent reaction is small, and the release is thorough. Wherein, the phosphate buffer solution is mainly used for providing a buffer environment, so that the reaction is carried out under the condition that the pH is 7.0 +/-0.5, and the glycated hemoglobin after the most suitable dissociation in the buffer zone carries out double-antibody sandwich reaction with the hemoglobin monoclonal antibody and the glycated hemoglobin monoclonal antibody. The ethyl phenyl polyethylene glycol is a surfactant, and can change the structure of the hemoglobin to release the glycosylated hemoglobin. Sodium dodecyl sulfate is a surfactant that disrupts the phospholipid bilayer in a whole blood sample, facilitating repeated mixing of hemoglobin with the reaction mass. The sodium caseinate is a thickening agent and an emulsifying agent, can be combined with other components in a whole blood sample, and enables the dissociated glycosylated hemoglobin to have specificity when being subjected to double-antibody sandwich reaction with the hemoglobin monoclonal antibody and the glycosylated hemoglobin monoclonal antibody.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a line graph showing the correlation between the concentration of a whole blood sample measured by a reaction and the data of an ichroma assay in example 1 of the glycated hemoglobin dissociation solution of the present invention.
FIG. 2 is a line graph showing the correlation between the concentration of a whole blood sample measured by a reaction in example 2 of the glycated hemoglobin dissociation solution of the present invention and the data of an ichroma assay.
FIG. 3 is a line graph showing the correlation between the concentration of a whole blood sample measured by a reaction and the data of an ichroma assay in example 3 of the glycated hemoglobin dissociation solution of the present invention.
FIG. 4 is a line graph showing the correlation between the concentration of a whole blood sample measured by a reaction in example 4 of the glycated hemoglobin dissociation solution of the present invention and the data of an ichroma assay.
FIG. 5 is a line graph showing the correlation between the concentration of a whole blood sample measured by a reaction in example 5 of the glycated hemoglobin dissociation solution of the present invention and the data of the ichroma assay.
FIG. 6 is a line graph showing the correlation between the concentration of a whole blood sample measured by a reaction in example 6 of the glycated hemoglobin dissociation solution of the present invention and the data of the ichroma assay.
FIG. 7 is a line graph showing the correlation between the concentration of a whole blood sample measured by a reaction in example 7 of the glycated hemoglobin dissociation solution of the present invention and the data of the ichroma assay.
FIG. 8 is a line graph showing the correlation between the concentration of a whole blood sample measured by a reaction in example 8 of the glycated hemoglobin dissociation solution of the present invention and the data of an ichroma assay.
FIG. 9 is a line graph showing the correlation between the concentration of a whole blood sample measured by a reaction in example 9 of the glycated hemoglobin dissociation solution of the present invention and the data of the ichroma assay.
FIG. 10 is a line graph showing the correlation between the concentration of a whole blood sample measured by a reaction with a glycated hemoglobin dissociation solution according to example 10 of the present invention and the data of an ichroma assay.
FIG. 11 is a line graph showing the correlation between the concentration of the glycated hemoglobin dissociation solution of the present invention measured in the whole blood sample reaction in comparative example 1 and the data of the ichroma assay.
FIG. 12 is a line graph showing the correlation between the concentration of the glycated hemoglobin dissociation solution of the present invention measured in the whole blood sample reaction in comparative example 2 and the data of the ichroma assay.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings of the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.
Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. The use of "first," "second," and similar terms in the description and claims of the present application do not denote any order, quantity, or importance, but rather the terms are used to distinguish one element from another. Also, the use of the terms "a" or "an" and the like do not denote a limitation of quantity, but rather denote the presence of at least one.
As used herein, the term "phosphate buffer solution", also known as PBS solution, is the buffer solution most widely used in biochemical research, the main component of which is Na 2 HPO 4 、KH 2 PO 4 NaCl and KCl, generally act as solvents to solubilize the protective agent.
As used herein, the term "ethylphenylpolyethylene glycol", Nonidet P-40, is a nonionic detergent and surfactant intended for use in lysis, thawing, protein stabilization, electrophoresis, and the like. It is often used in cell lysis, protein treatment, histochemical treatment, hybridization washing, etc.
As used herein, the term "sodium dodecyl sulfate", also known as SDS, is an organic compound of formula C 12 H 25 SO 4 Na, white or light yellow powder, is easily soluble in water and is not sensitive to alkali and hard water. Has detergency, emulsification and excellent foaming power, is an anionic surfactant slightly toxic to human body, and is used for biodegradationDegree of solution>90％。
As used herein, the term "sodium caseinate", also known as sodium caseinate, sodium caseinate, is a water soluble emulsifier with the functions of stabilizing, strengthening protein, thickening, foaming, etc.
In order to verify the effect of the reaction of the present invention on glycated hemoglobin, phosphate buffer (Na) was used 2 HPO 4 The manufacturer is Shanghai Aladdin Biotechnology Co., Ltd, product number S112446; KH (Perkin Elmer) 2 PO 4 The manufacturer is Shanghai Aladdin Biotechnology Ltd, product number P104075; the NaCl manufacturer is Shanghai Aladdin Biotechnology Co., Ltd, product number C111533; the KCl manufacturer is Shanghai Allan Biotechnology Co., Ltd., product No. P112134), ethylphenylpolyethylene glycol (Shanghai Allan Biotechnology Co., Ltd., product No. N274337), sodium lauryl sulfate (Shanghai Merlin Biotechnology Co., Ltd., product No. S817788) and sodium caseinate (Sigma Aldrich trade Co., product No. C8654) in the following ratios.
Specifically, the concentration of phosphate in the glycated hemoglobin dissociation solution is 10-50mM, 0.3-1% of ethyl phenyl polyethylene glycol, 0.1-0.5% of sodium dodecyl sulfate and 0.3-1% of sodium caseinate.
Specifically, 10 examples and 2 groups are shown in tables 1-3 for comparative ratios:
TABLE 1 formulation of dissociation liquids of examples 1 to 5
Figure BDA0003629175830000061
TABLE 2 formulations of dissociation liquids of examples 6-10
Figure BDA0003629175830000062
TABLE 3 recipe of dissociation liquid for comparative examples 1-2
Figure BDA0003629175830000063
Firstly, verification of linearity:
using 80 whole blood samples, the 80 whole blood samples were dissociated using the above 10 examples and 2 set proportions, and the glycated hemoglobin concentration (%) was measured by fluorescence immunochromatography; ichroma measurements (%) were taken simultaneously for 80 whole blood samples. The correlation was then obtained by linear fitting of the measurement values of each example to the measurement values of ichroma (tables 4-15, FIGS. 1-12).
Table 4 measurement of example 1 and ichroma
Figure BDA0003629175830000071
Table 5 example 2 and ichroma measurements
Figure BDA0003629175830000081
Table 6 example 3 and ichroma measurements
Figure BDA0003629175830000091
Table 7 example 4 and ichroma measurements
Figure BDA0003629175830000101
TABLE 8 measurement of example 5 and ichroma
Figure BDA0003629175830000111
TABLE 9 measurement of example 6 and ichroma
Figure BDA0003629175830000121
TABLE 10 measurement of example 7 and ichroma
Figure BDA0003629175830000131
TABLE 11 measurement of example 8 and ichroma
Figure BDA0003629175830000141
TABLE 12 measurement of example 9 and ichroma
Figure BDA0003629175830000151
TABLE 13 measurement of example 10 and ichroma
Figure BDA0003629175830000161
TABLE 14 measurement of comparative example 1 and ichroma
Figure BDA0003629175830000171
TABLE 15 measurement of comparative example 2 and ichroma
Figure BDA0003629175830000181
From fig. 1 to 12, it can be seen that the linear correlations of examples 1 to 10 are better, and the R2 values are all above 0.975, indicating better correlations.
As can be seen from fig. 11 and 12, the R2 values were 0.9401 and 0.9426, respectively, and the correlation was low, and in comparative example 1, the phosphate buffer, ethylphenylpolyethylene glycol, and sodium dodecylsulfate in the glycated hemoglobin dissociation solution were out of the predetermined ranges, so that the effects on the reaction were general, resulting in a large deviation of the final measurement values. In comparative example 2, the phosphate buffer, sodium lauryl sulfate and sodium caseinate were out of the predetermined ranges, so that they were generally effective in the reaction, resulting in a large deviation of the final measured values.
In addition, as can be seen from examples 1 to 12, in terms of linear correlation, example 3> example 2> example 1> example 6> example 5> example 7> example 4> example 8> example 9> example 10. However, in 10 of the examples, the R2 values for examples 3, 2, 1, 6, 5 and 7 were better than for the other examples, whereas in examples 3, 2, 1, 6, 5 and 7 the phosphate concentration was 20mM to 40mM, so that relatively speaking phosphate concentrations in the range of 20mM to 40mM had a relatively positive effect on the R2 values.
In addition, from a linear ranking, examples 9 and 10 are lower, while examples 9 and 10 correspond to lower concentrations of ethylphenylpolyethylene glycol (0.3% and 0.4% in that order). In other embodiments, the concentration of ethylphenylpolyethylene glycol is 0.5% to 1%, and thus, the concentration of ethylphenylpolyethylene glycol is not likely to be too low, preferably in the range of 0.5% to 1%.
Second, determination of reaction time
20 whole blood samples of glycated hemoglobin were collected, dissociated with a product A (component of dissociation solution of reagent of ichroma) on the market (product A, Baditai Biotech Co., Ltd., product No. CFPC-38) and then measured for concentration (%) by fluorescence immunochromatography, and the results are shown in tables 16 to 18.
TABLE 16 FIELD OF FIGHTNESS IMMUNOCHROMATOGRAPHIC DETERMINATION OF FULL BLOOD SAMPLES AFTER TREATMENT OF PRODUCT A AND DISSOLUTION SOLUTIONS FROM EXAMPLES 1-3
Figure BDA0003629175830000191
Figure BDA0003629175830000201
TABLE 17 results of fluorescence immunochromatography assay of whole blood samples treated with dissociation solutions of examples 4 to 9
Figure BDA0003629175830000202
Figure BDA0003629175830000211
TABLE 18 results of FIFIFICATION IMMUNOCHROMATOGRAPHY MEASUREMENT OF FULL BLOOD SAMPLES AFTER TREATMENT WITH DISCONNECTING SOLUTION OF EXAMPLE 10 AND COMPARATIVE EXAMPLES 1-2
Figure BDA0003629175830000212
Figure BDA0003629175830000221
From the reaction time, the reaction of the A product to 20 glycated hemoglobin stabilized after 10 minutes. However, it can be seen from this example that most of examples 1 to 10 can achieve the effect of product A within 10 minutes in 5 minutes. Therefore, the reaction time of the glycated hemoglobin dissociation solution of the present invention is greatly shortened, and the reaction time of the dissociation solution of the present invention is shortened 1/2 compared to products on the market.
Influence on subsequent reactions
After the dissociation solution dissociated the glycated hemoglobin, in order to verify whether the residual solution after dissociation has an influence on the subsequent measurement of the glycated hemoglobin, the following experiment was performed:
20 whole blood samples of glycated hemoglobin were collected, dissociated with a product a on the market, and then a signal value T/C of fluorescence immunochromatography was measured. The higher the signal value, the more accurate the measurement, and the lower the signal value, indicating that the measurement error is larger, and the subsequent reaction is large after the release of the glycated hemoglobin is also reflected. The results are shown in tables 19 to 21.
TABLE 19 values of signals measured by fluorescence immunochromatography after treatment of whole blood with product A and dissociation solutions of examples 1 to 4
Figure BDA0003629175830000222
Figure BDA0003629175830000231
TABLE 20 values of signals measured by fluorescence immunochromatography after treatment of whole blood with dissociation solutions of examples 5 to 9
Figure BDA0003629175830000232
Figure BDA0003629175830000241
TABLE 21 Signal values of the whole blood after treatment with dissociation liquid of example 10 and comparative examples 1 to 2 measured by fluorescence immunochromatography
Figure BDA0003629175830000242
Figure BDA0003629175830000251
From the effect on the subsequent reaction, the signal values T/C of examples 1 to 10 were about 50% higher than that of product a, and it can be seen that the dissociation liquid of the present invention has much less effect on the subsequent reaction than that of product a.
In addition, sodium caseinate is a thickener and an emulsifier, and can be combined with other components in the whole blood sample, so that the double-antibody sandwich reaction of the dissociated glycosylated hemoglobin, the hemoglobin monoclonal antibody and the glycosylated hemoglobin monoclonal antibody is more specific. Therefore, the concentration of sodium caseinate has an influence on the signal intensity. Several whole blood samples with lower concentrations of whole blood samples were selected: the percentage increase in signal for the whole blood samples 5, 8, 12 and 14 of the 4 whole blood samples, respectively, of example 1 was 46.64%, 54.16%, 52.42% and 48.34% in this order, averaging 50.39%, and similarly, the percentage increase in signal for example 2 relative to the whole blood samples 5, 8, 12 and 14 averaged 50.35%, 50.27% for example 3, 50.54% for example 4, 40.22% for example 5, 47.41% for example 6, 49.00% for example 7, 51.31% for example 8, 41.43% for example 9 and 48.87%. Wherein examples 1, 2, 3, 4, 6, 7, 8 and 10 all correspond to an average value higher than 45% and in the following order: 50.39%, 50.35%, 50.27%, 50.54%, 47.41%, 49.00%, 51.31%, and 48.87%. And examples 1, 2, 3, 4, 6, 7, 8 and 10 correspond to sodium caseinate concentration ranges: from 0.5% to 1%, it can be seen that the concentration of sodium caseinate in this range has a large contribution to the sufficient dissociation of glycated hemoglobin.
Fourth, release thorough verification
Taking 20 glycosylated hemoglobin whole blood samples, and respectively measuring the concentration of the glycosylated hemoglobin by using a product A on the market through reactions at two time points of 10min and 12 min; the glycated hemoglobin concentration was measured using 10 groups of examples, 2 groups of ratios, and two time point reactions of 5min and 10min, respectively. The results are shown in tables 22 to 25.
TABLE 22 concentration of glycated hemoglobin for the products of product A and the reactions of examples 1-3 at various time points
Figure BDA0003629175830000261
TABLE 23 glycated hemoglobin concentrations of the reactions of examples 4-6 at different time points
Figure BDA0003629175830000262
Figure BDA0003629175830000271
TABLE 24 glycated hemoglobin concentrations of the reactions of examples 7-9 at different time points
Figure BDA0003629175830000272
Figure BDA0003629175830000281
TABLE 25 glycated hemoglobin concentrations of example 10, comparative examples 1-2, reacted at different time points
Figure BDA0003629175830000282
Figure BDA0003629175830000291
The product A in the market is used for processing the glycosylated hemoglobin whole blood sample, the reaction time is increased after the reaction is carried out for 10 minutes, the amount of the glycosylated hemoglobin is not increased any more, and the maximum reaction effect is achieved.
By using the invention, the maximum reaction effect can be achieved after 5 minutes of reaction, but the reaction result is 10-25% more than that of the product A, which shows that the dissociation liquid of the invention is more thorough to the reaction. The ethyl phenyl polyethylene glycol is a surfactant, and can change the structure of the hemoglobin to release the glycosylated hemoglobin. Therefore, the amount of ethylphenylpolyethylene glycol used may be such that the effect of the dissociation liquid on the reaction is exhibited to some extent.
The concentrations of the whole blood samples 2, 10 and 16 were high, and after dissociation in 10 examples, the average reaction results of examples 3, 4 and 7 relative to the product A were 21.15%, 20.85% and 20.69%, respectively. While the concentration of ethylphenylpolyethylene glycol for examples 3, 4 and 7 is 0.8% -1%.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and the description is given here only for clarity, and those skilled in the art should integrate the description, and the embodiments may be combined appropriately to form other embodiments understood by those skilled in the art.

Claims (9)

1. The glycosylated hemoglobin dissociation solution is characterized by comprising phosphate buffer solution, ethyl phenyl polyethylene glycol, sodium dodecyl sulfate and sodium caseinate.
2. The glycated hemoglobin dissociation solution of claim 1, wherein the phosphate buffer solution has a concentration of 10 to 50mM, the ethylphenylpolyethylene glycol has a concentration of 0.3 to 1%, the sodium dodecyl sulfate has a concentration of 0.1 to 0.5%, and the sodium caseinate has a concentration of 0.3 to 1%.
3. The glycated hemoglobin dissociation solution according to claim 2, wherein the phosphate buffer solution has a concentration of 20 to 40 mM.
4. The glycated hemoglobin dissociation solution as set forth in claim 2 or 3, wherein the ethylphenylpolyethylene glycol is present at a concentration of 0.5% to 1%.
5. The glycated hemoglobin dissociation solution as set forth in claim 4, wherein the ethylphenylpolyethylene glycol is present in a concentration of 0.8% to 1%.
6. The glycated hemoglobin dissociation solution as set forth in claim 2, wherein the concentration of the sodium caseinate ranges from 0.5% to 1%.
7. Use of the glycated hemoglobin dissociation solution according to any one of claims 1 to 6 for the preparation of a kit.
8. The use according to claim 7, wherein the kit is used for separation of glycated hemoglobin in a blood sample.
9. A kit comprising the glycated hemoglobin dissociation solution according to any one of claims 1 to 6.
CN202210484542.5A 2022-05-06 2022-05-06 Glycated hemoglobin dissociation solution Pending CN114878839A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115951072A (en) * 2022-12-06 2023-04-11 北京鸿宇泰生物科技有限公司 Glycosylated hemoglobin-C peptide combined detection kit

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115951072A (en) * 2022-12-06 2023-04-11 北京鸿宇泰生物科技有限公司 Glycosylated hemoglobin-C peptide combined detection kit
CN115951072B (en) * 2022-12-06 2024-05-14 北京鸿宇泰生物科技有限公司 Glycosylated hemoglobin-C peptide joint detection kit

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