CN115032188A - A kind of detection method of serum glycated albumin - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a method for detecting serum albumin. The detection method effectively reduces the interference of reagent blank background, simultaneously reduces the interference of endogenous glycated amino acid in the sample, greatly improves the accuracy of glycated albumin test, and eliminates the test result deviation caused by the reagent blank background change possibly occurring in the long-term storage process.

Description

A kind of detection method of serum glycated albumin

technical field

The invention relates to the technical field of medicine, in particular to a detection method for serum glycated albumin.

Background technique

For the current clinical biochemical testing, most of the laboratory equipment in medical institutions above the second grade are imported brands, and routine testing is the mainstay. POCT belongs to the IVD market segment. For primary hospitals below the second grade, the laboratory department has a small sample size, relatively low technical level, and poor practicability of large-scale equipment. Therefore, the laboratory department usually uses POCT products instead of traditional inspection methods for diagnosis. Among conventional biochemical testing methods, liquid biochemical testing is often more precise and accurate than dry biochemical testing. Therefore, liquid biochemical testing still has a large application market for such POCT testing products.

Due to the relatively low market threshold for POCT products, the competition is particularly fierce. For quantitative products, it is often necessary to ensure that a calibration before leaving the factory can ensure the accuracy and reliability of the test results throughout the life cycle of the product. For liquid biochemical reagents, the stability and accuracy of the reagents are required to be further improved. For a class of reagents that use end-point colorimetric determination, it is often found in the development of reagents that once the two reagents are mixed, a large change in the light absorption signal will occur, and with the prolongation of the reagent storage time, the blank background signal of this reagent will be reduced. Changes may be more pronounced than initially. The change of this signal may have the following two effects: ① The blank background signal is too high, which affects the detection sensitivity. ②During long-term storage, due to the increase or decrease of the blank background signal of the reagent, the calculated result will appear falsely increased or falsely decreased, resulting in the deviation of the detection result.

In the enzymatic assay kits for glycated albumin, most of them firstly use the protease in the R1 reagent to digest and hydrolyze the glycated albumin into glycated amino acids or glycated polypeptides, and measure the first absorbance; then add the fructose amino acid oxidase in the R2 reagent. /Fructose ground state oxidase, peroxidase coupling reaction, color development through Trinder reaction, measure the second absorbance at the equilibrium end point, and calculate the difference between the two absorbances. The concentration of glycated albumin in the sample was determined via an absorbance-concentration calibration curve. The reaction process is as follows:

In this assay method, the reagent blank has a high degree of reactivity, and it mainly occurs at the moment when the two reagents are mixed. To analyze the reasons, there are mainly the following two points: First, in order to make the decomposition reaction of glycated albumin better to reach the end point, the amount of protease in the general reagent is high enough, and the enzyme activity of the protease is reduced during the incubation reaction of the first period of time. It is fully activated or has reached a high level. When the second reagent is added, it is likely that other protein components contained in the reagent will be digested by enzyme digestion to produce insoluble factors or light absorption at specific wavelengths; second, the active components of Trinder reaction (such as 4-Aminoantipyrine produces rapid color development when mixed with phenol/amines, peroxidase, resulting in increased background.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a detection method for serum glycated albumin. The kit and detection method effectively reduce the determination interference caused by the high blank background of the reagent, and at the same time reduce the interference of endogenous glycated amino acids in the sample, greatly improve the test accuracy of the glycated albumin detection kit, and eliminate the Deviation of test results caused by changes in reagent blank background that may occur during long-term storage.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

A detection method for serum glycated albumin, comprising:

Take reagent 1, add the sample to be tested or calibrator, and incubate for the first time; then add reagent 2, mix well, and measure the first absorbance value A1 at a predetermined time; incubate for a second time, measure the second absorbance value A2; calculate and deduct The absorbance change value after blank background signal ΔA=A2-A1, the serum glycated albumin concentration was calculated by the absorbance change value ΔA;

Wherein, the reagent 1 includes fructose amino acid oxidase and peroxidase; the reagent 2 includes protease.

In the present invention, the serum glycated albumin concentration is calculated by the absorbance change value ΔA, specifically:

The concentration of serum glycated albumin was calculated by substituting the absorbance change value ΔA into the standard calculation function of serum glycated albumin.

In the present invention, the serum glycated albumin standard calculation function is determined by the following method:

Establish the functional relationship between the concentration C of different calibrators and the corresponding absorbance change value ΔA, and obtain the standard calculation function.

In the present invention, the reagent 1 is taken, the sample to be tested or the calibrator is added, and incubated for the first time; the reagent 2 is added, mixed well, and the first absorbance value A1 is measured at a predetermined time. Wherein, the predetermined time is specifically: 0-20s, preferably 0-18s, start timing after the reagent 2 is added and mixed. That is, the measurement of the first absorbance value A1 is performed within 20 s after adding the reagent 2 and mixing. It can be measured immediately after mixing (ie, 0 s), or it can be measured at any time point between 0 and 20 s.

In the present invention, the first duration and the second duration are both selected from any duration of 3 to 5 minutes, specifically 3 min, 4 min or 5 min.

In the present invention, the wavelengths for detecting the first absorbance value and the second absorbance value are both 500-600 nm, specifically 500 nm, 550 nm or 600 nm.

In order to solve the problem of excessive interference of the blank background signal caused by the mixing of the two reagents, the applicant tried different methods, such as: changing the measurement of the first absorbance from the end point of the first incubation period before adding the R2 reagent to after adding the R2 reagent The rapid determination, and then through the second incubation reaction to measure the second absorbance. However, the study found that after the first step of incubation, the protease has converted most of the glycated albumin into glycated amino acids or glycated polypeptide intermediates. Once the R2 reagent containing fructose amino acid oxidase or fructose ground state oxidase and peroxidase is added After the reaction, the reaction occurs very quickly (that is, the degree of reactivity rises sharply), and the equilibrium end point is quickly reached. At this time, no matter how close the time of collecting the first absorbance is to the time point of adding the R2 reagent, the absorbance change is not a simple reagent blank absorbance. The signal changes of the samples participating in the reaction are superimposed, and the reaction signal test deviation of samples with different concentrations is more likely to occur at this time.

After long-term research and exploration, the applicant finally recombined the components of the traditional glycosylated hemoglobin detection kit, and detected the blank background signal at a specific time point, effectively reducing the detection interference due to the high blank background of the reagent. In this method, the positions of protease, fructose amino acid oxidase (FAOD) and peroxidase (POD) are exchanged, and fructose amino acid oxidase (FAOD) and peroxidase (POD) are placed in reagent 1, and protease is placed in reagent 1. In Reagent 2, on the one hand, the kit of the present invention after adjusting the components utilizes fructose amino acid oxidase or fructose ground state oxidase to effectively eliminate the endogenous interference of glycated amino acids in the sample. On the other hand, the initial catalytic reaction starts slowly, and the conversion of glycated albumin into glycated amino acids or glycated polypeptide intermediates is very low, so this delayed reaction leaves enough time for the collection of the first absorbance (A1). It can more accurately reflect the real-time reagent blank background signal, and then collect the second absorbance (A2) at the end point of the second incubation equilibrium, and the calculated absorbance change ΔA (A2-A1) at the corresponding sample concentration is accurately subtracted. Real-time measurement signal of reagent blank background absorbance. Therefore, using the kit of the present invention for detection not only reduces the detection interference due to the high blank background of the reagent, but also reduces the interference of endogenous glycated amino acids in the sample, greatly improves the testing accuracy of the glycated albumin detection kit, and eliminates the need for The deviation of test results caused by the change of reagent blank background that may occur during long-term storage is avoided.

In the present invention, the types and amounts of other components of reagent 1 and reagent 2 in the kit, such as anti-interference agents, buffers, preservatives, stabilizers, etc., are not specifically limited. Normal selection is fine. In the present invention, the reagent 1 includes buffer, fructose amino acid oxidase, peroxidase, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS ), anti-interference agents, preservatives and stabilizers; Reagent 2 includes buffer, proteinase K, 4-aminoantipyrine, preservatives and stabilizers.

Further, in the present invention, the reagent 1 includes:

The reagent 2 includes:

In some embodiments, the buffer is tris-hydroxymethylaminomethane hydrochloride (Tris-HCl), 3-(N-morpholine)propanesulfonic acid (MOPS), 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), at least one of piperazine-1,4-dihydroxypropanesulfonic acid (POPSO), and 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO).

In some embodiments, the preservative is sodium azide and/or Proclin 30.

In the present invention, the purpose of the anti-interference agent is to remove interfering substances in the sample, such as ascorbic acid or bilirubin. In some embodiments, the anti-interfering agent is ascorbate oxidase or bilirubin oxidase.

In some embodiments, the stabilizer is one or more of water-soluble calcium salts, glycerol, trehalose, sucrose, bovine serum albumin, and disodium EDTA.

In some specific embodiments, in the kit, the reagent 1 is composed as follows:

Reagent 2 is composed as follows:

In some embodiments, the time of incubation and re-incubation in step 1 is 3-5 minutes.

In some embodiments, the determination of the first absorbance value in step 1 is performed immediately (within 18s) after adding reagent 2 and mixing.

The invention recombines and combines the components of the traditional kit, detects the blank background signal at a specific time node, effectively reduces the determination interference of the high blank background of the reagent, and reduces the interference of endogenous glycosylated amino acids in the sample , which greatly improves the test accuracy of the glycated albumin detection kit, and eliminates the test result deviation caused by the blank background change of the reagent that may occur during the long-term storage process.

Description of drawings

Fig. 1 shows the determination curve of the blank background absorbance of the reagent of Comparative Example 1;

Fig. 2 shows the sample absorbance measurement curve of the linear lower limit concentration of Comparative Example 1;

Fig. 3 shows the sample absorbance measurement curve of the linear upper limit concentration of Comparative Example 1;

Figure 4 shows the reagent blank background absorbance measurement curve of Example 1;

Fig. 5 shows the sample absorbance measurement curve of the linear lower limit concentration of Example 1;

6 shows the sample absorbance measurement curve of the linear upper limit concentration of Example 1.

Detailed ways

The invention provides a detection method for serum glycated albumin. Those skilled in the art can learn from the content of this document and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the present invention. Invention technology.

Unless otherwise specified, the test materials used in the present invention are all common commercial products and can be purchased in the market.

Below in conjunction with embodiment, the present invention is further elaborated:

Example 1

1. Prepare Reagent 1 and Reagent 2 with the following compositions.

Reagent 1 composition:

Reagent 2 composition:

2. Detection parameters: take 150 μL of reagent 1, add 10 μL of sample/calibrator, and incubate for 3-5 minutes; then add 50 μL of reagent 2, mix well, and immediately (within 18s) measure the first absorbance A1 at a wavelength of 546 nm, incubate for 3-5 minutes Then record the second absorbance value A2.

Comparative Example 1

1. Prepare Reagent 1 and Reagent 2 with the following compositions.

Reagent 1 composition:

Reagent 2 composition:

2. Detection parameters: take 150 μL of reagent 1, add 10 μL of sample/calibrator, incubate for 3 to 5 minutes, and measure the first absorbance A1 at 546 nm wavelength; then add 50 μL of reagent 2, mix well, and incubate for 3 to 5 minutes to measure the second absorbance value A2.

Comparative Example 2

Prepare Reagent 1 and Reagent 2 with the following compositions.

Reagent 1 composition:

Reagent 2 composition:

2. Detection parameters: take 150 μL of reagent 1, add 10 μL of sample/calibrator, and incubate for 3-5 minutes; then add 50 μL of reagent 2, mix well, and immediately (within 18s) measure the first absorbance A1 at a wavelength of 546 nm, incubate for 3-5 minutes Then measure the second absorbance value A2.

test case

Detected according to the kits and detection methods of Comparative Example 1, Comparative Example 2, and Example 1, and determined the reagent blank absorbance, signal gradient, and clinical comparison correlation of glycated albumin detection reagents under different shelf life by Trinder reaction color development, The specific results are shown in Table 1 and Figures 1-6.

Table 1

The results show that the comparative example 1 has a larger reagent blank background, and the reagent blank signal increases significantly with the longer the reagent is placed. For the POCT-like glycated albumin detection kit with only one calibration, the accuracy of the low-value samples easily affected. Comparative Example 2 had a smaller reagent blank background than Comparative Example 1, but the linear gradient was significantly smaller, and the clinical correlation was poor. Compared with Comparative Example 1 and Comparative Example 2, Example 1 has lower reagent blank background (close to zero level), better clinical correlation, and its linear gradient also meets general detection requirements.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be regarded as It is the protection scope of the present invention.

Claims (10)

1. a detection method of serum glycated albumin, is characterized in that, comprises: Take reagent 1, add the sample to be tested or calibrator, and incubate for the first time; then add reagent 2, mix well, and measure the first absorbance value A1 at a predetermined time; incubate for a second time, measure the second absorbance value A2; calculate and deduct The absorbance change value after blank background signal ΔA=A2-A1, the serum glycated albumin concentration was calculated by the absorbance change value ΔA; Wherein, the reagent 1 includes fructose amino acid oxidase and peroxidase; the reagent 2 includes protease. 2. detection method according to claim 1, is characterized in that, calculates serum glycated albumin concentration with absorbance change value ΔA, is specially: The concentration of serum glycated albumin was calculated by substituting the absorbance change value ΔA into the standard calculation function of serum glycated albumin. 3. detection method according to claim 2 is characterized in that, described serum glycated albumin standard calculation function is determined by the following method: Establish the functional relationship between the concentration C of different calibrators and the corresponding absorbance change value ΔA, and obtain the standard calculation function. 4 . The detection method according to claim 1 , wherein the predetermined time is specifically: 0-20 s after the reagent 2 is added and mixed evenly. 5 . 5 . The detection method according to claim 1 , wherein the first duration and the second duration are both selected from any duration from 3 to 5 minutes. 6 . 6 . The detection method according to claim 1 , wherein the detection wavelengths of the first absorbance value and the second absorbance value are both 500-600 nm. 7 . 7. detection method according to claim 1, is characterized in that, The reagent 1 includes buffer, fructose amino acid oxidase, peroxidase, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt, anti-interference agent, preservative agents and stabilizers; The reagent 2 includes buffer, proteinase K, 4-aminoantipyrine, preservatives and stabilizers. 8. detection method according to claim 7 is characterized in that, described buffer solution is tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 3-(N-morpholine) propanesulfonic acid (MOPS) , 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), piperazine-1,4-dihydroxypropanesulfonic acid (POPSO), 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO) at least one of them. 9 . The detection method according to claim 7 , wherein the anti-interference agent is ascorbate oxidase or bilirubin oxidase. 10 . 10. detection method according to claim 7 is characterized in that, described stabilizer is a kind of in water-soluble calcium salt, glycerol, trehalose, sucrose, bovine serum albumin, disodium EDTA or several.

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