A method for detecting serum glycated albumin

Abstract

This invention relates to the field of biotechnology, and more particularly to a method for detecting serum albumin. The detection method of this invention effectively reduces interference from reagent blank background and also reduces interference from endogenous glycated amino acids in the sample, greatly improving the accuracy of glycated albumin testing and eliminating test result deviations caused by changes in reagent blank background that may occur during long-term storage.

Description

Technical Field

[0001] This invention relates to the field of medical technology, and in particular to a method for detecting serum glycated albumin. Background Technology

[0002] Currently, in clinical biochemical testing, most laboratory equipment in Grade II and above medical institutions is imported, primarily used for routine testing. Point-of-care testing (POCT) is a sub-market of in-vitro diagnostics (IVD). For primary care hospitals below Grade II, the sample volume is small, the technical level is relatively low, and large equipment is less practical. Therefore, these hospitals often use POCT products to replace traditional testing methods for diagnosis. Among routine biochemical testing methods, liquid biochemistry tests often have better precision and accuracy than dry biochemistry tests. Therefore, liquid biochemistry testing still has a significant market application for this type of POCT product.

[0003] Because the market for POCT products has relatively low barriers to entry, competition is particularly fierce. For quantitative products, it is often necessary to ensure the accuracy and reliability of test results throughout the product's lifecycle through a single calibration before shipment. For liquid biochemical reagents, the requirements for reagent stability and accuracy are even higher. For reagents using endpoint colorimetric determination, it is often found during reagent development that significant changes in light absorption signal occur when two reagents are mixed, and this change in reagent blank background signal may become more significant as the reagent storage time increases. This signal change may have the following two effects: ① An excessively high blank background signal affects detection sensitivity. ② During long-term storage, the increase or decrease in reagent blank background signal can lead to false increases or decreases in calculated results, causing deviations in detection results.

[0004] In most enzymatic assay kits for glycated albumin, the protease in reagent R1 is used to enzymatically hydrolyze glycated albumin into glycated amino acids or glycated peptides, and the first absorbance is measured. Then, the fructose amino acid oxidase / fructose ground-state oxidase and peroxidase in reagent R2 are added for a coupled reaction, followed by Trinder reaction for color development. The second absorbance is measured at the equilibrium endpoint, and the difference between the two absorbances is calculated. The concentration of glycated albumin in the sample is determined using an absorbance-concentration correction curve. The reaction process is as follows:

[0005]

[0006]

[0007]

[0008] In this assay, a high reagent blank reactivity was observed, primarily occurring at the instant of mixing the two reagents. The reasons for this are mainly twofold: First, to ensure the glycated albumin decomposition reaction reaches its endpoint more effectively, the amount of protease in the reagent is generally high. Furthermore, during the initial incubation period, the protease activity is fully activated or reaches a high level. Adding the second reagent may cause other protein components in the reagent to be enzymatically digested, producing insoluble factors or light absorption at specific wavelengths. Second, the rapid color development when the active components of the Trinder reaction (such as 4-aminoantipyrine, phenol / amine substances, and peroxidase) are mixed can lead to an increased background. Summary of the Invention

[0009] In view of this, the present invention provides a method for detecting serum glycated albumin. This kit and detection method effectively reduce interference from high reagent blank background, while also reducing interference from endogenous glycated amino acids in the sample, greatly improving the accuracy of the glycated albumin detection kit and eliminating test result deviations caused by changes in reagent blank background that may occur during long-term storage.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0011] A method for detecting serum glycated albumin, comprising:

[0012] Take reagent 1, add the sample to be tested or calibrator, and incubate for the first duration; then add reagent 2, mix well and measure the first absorbance value A1 at the predetermined time; then incubate for the second duration and measure the second absorbance value A2; calculate the absorbance change value ΔA after subtracting the blank background signal ΔA = A2 - A1, and calculate the serum glycated albumin concentration based on the absorbance change value ΔA.

[0013] Wherein, reagent 1 includes fructose amino acid oxidase and peroxidase; reagent 2 includes protease.

[0014] In this invention, the serum glycated albumin concentration is calculated using the absorbance change value ΔA, specifically as follows:

[0015] The serum glycated albumin concentration was calculated by substituting the absorbance change value ΔA into the standard calculation function for serum glycated albumin.

[0016] In this invention, the standard calculation function for serum glycated albumin is determined by the following method:

[0017] Establish the functional relationship between different calibrator concentrations C and the corresponding absorbance change value ΔA to obtain the standard calculation function.

[0018] In this invention, reagent 1 is taken, and the sample to be tested or calibrator is added and incubated for a first duration; reagent 2 is added, mixed well, and the first absorbance value A1 is measured at a predetermined time. Specifically, the predetermined time is 0-20 seconds, preferably 0-18 seconds, starting from the time after adding reagent 2 and mixing. That is, the measurement of the first absorbance value A1 is performed within 20 seconds after adding reagent 2 and mixing, which can be performed immediately after mixing (i.e., 0 seconds) or at any time point between 0 and 20 seconds.

[0019] In this 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.

[0020] In this invention, the wavelengths for detecting the first absorbance value and the second absorbance value are both 500-600nm, specifically 500nm, 550nm or 600nm.

[0021] To address the interference caused by excessive changes in the blank background signal due to the mixing of two reagents, the applicant attempted different methods, such as changing the measurement of the first absorbance from the endpoint of the first incubation before the addition of reagent R2 to a rapid measurement after the addition of reagent R2, followed by a second incubation reaction to measure the second absorbance. However, the study found that after the first incubation, the protease had already converted most of the glycated albumin into glycated amino acids or glycated polypeptide intermediates. Once reagent R2 containing fructose amino acid oxidase or fructose ground-state oxidase / peroxidase was added, the reaction occurred extremely rapidly (i.e., the reactivity increased sharply) and quickly reached the equilibrium endpoint. At this point, no matter how close the time of collecting the first absorbance was to the time of adding reagent R2, the absorbance change was not simply the reagent blank absorbance, but actually superimposed with the signal changes of the sample participating in the reaction. This made it easier for the reaction signal test deviation of samples with different concentrations to occur.

[0022] After long-term research and exploration, the applicant finally achieved a breakthrough by reconfiguring the components of a traditional glycated hemoglobin assay kit and detecting blank background signals at specific time points, effectively reducing the interference from high reagent blank background. This method involves swapping the positions of the protease and fructose amino acid oxidase (FAOD) and peroxidase (POD). FAOD and POD are placed in reagent 1, while the protease is placed in reagent 2. On one hand, the kit with the adjusted components effectively eliminates endogenous interference from glycated amino acids in the sample using fructose amino acid oxidase or fructose ground-state oxidase. On the other hand, the initial catalytic reaction starts slowly, and the degree to which glycated albumin is converted into glycated amino acids or glycated polypeptide intermediates is very low. Therefore, this delayed reaction allows sufficient time for the acquisition of the first absorbance (A1), which can accurately reflect the real-time reagent blank background signal. Subsequently, the second absorbance (A2) is acquired at the end of the second incubation equilibrium. The calculated absorbance change ΔA(A2-A1) at the corresponding sample concentration is the measurement signal that accurately subtracts the real-time reagent blank background absorbance. Therefore, using the kit of this invention for detection reduces the measurement interference of high reagent blank background and reduces the interference of endogenous glycated amino acids in the sample, greatly improving the test accuracy of the glycated albumin detection kit and eliminating the test result deviation caused by reagent blank background changes that may occur during long-term storage.

[0023] In this invention, the types and amounts of other components in reagents 1 and 2 of the kit, such as anti-interference agents, buffer solutions, preservatives, and stabilizers, are not specifically limited. Those skilled in the art can select them conventionally according to their needs and actual circumstances. In this invention, reagent 1 includes buffer solution, fructose amino acid oxidase, peroxidase, sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), anti-interference agents, preservatives, and stabilizers; reagent 2 includes buffer solution, proteinase K, 4-aminoantipyrine, preservatives, and stabilizers.

[0024] Furthermore, in this invention, reagent 1 comprises:

[0025]

[0026] The reagent 2 includes:

[0027]

[0028] In some embodiments, the buffer solution is at least one of tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), piperazine-1,4-dihydroxypropanesulfonic acid (POPSO), and 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO).

[0029] In some embodiments, the preservative is sodium azide and / or Proclin 30.

[0030] In this 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-interference agent is ascorbic acid oxidase or bilirubin oxidase.

[0031] In some embodiments, the stabilizer is one or more of water-soluble calcium salts, glycerol, trehalose, sucrose, bovine serum albumin, and disodium EDTA.

[0032] In some specific embodiments, reagent 1 in the kit comprises the following components:

[0033]

[0034] Reagent 2 has the following composition:

[0035]

[0036] In some implementations, the incubation and re-incubation time in step 1 is 3 to 5 minutes.

[0037] In some implementations, the determination of the first absorbance value in step 1 is performed immediately (within 18 seconds) after the addition of reagent 2 and mixing.

[0038] This invention reconfigures and combines the components of traditional reagent kits, detects blank background signals at specific time points, effectively reduces the interference of high reagent blank background, and reduces the interference of endogenous glycated amino acids in the sample, greatly improving the test accuracy of the glycated albumin detection kit, and eliminating test result deviations caused by changes in reagent blank background that may occur during long-term storage. Attached Figure Description

[0039] Figure 1 The absorbance curve of reagent blank background in Comparative Example 1 is shown.

[0040] Figure 2 The absorbance curve of the sample with the linear lower limit concentration is shown in Comparative Example 1.

[0041] Figure 3The absorbance measurement curve of the sample at the linear upper limit concentration of Comparative Example 1 is shown.

[0042] Figure 4 Example 1 shows the absorbance measurement curve against a blank background.

[0043] Figure 5 This shows the absorbance measurement curve of the sample at the linear lower limit concentration in Example 1;

[0044] Figure 6 The absorbance curve of the sample at the linear upper limit concentration is shown in Example 1. Detailed Implementation

[0045] This invention provides a method for detecting serum glycated albumin. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method and application of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the method and application described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0046] Unless otherwise specified, the test materials used in this invention are all commercially available products that can be purchased on the market.

[0047] The present invention will be further illustrated below with reference to the embodiments:

[0048] Example 1

[0049] 1. Prepare reagent 1 and reagent 2 with the following compositions.

[0050] Reagent 1 consists of:

[0051]

[0052] Reagent 2 consists of:

[0053]

[0054]

[0055] 2. Detection parameters: Take 150 μL of reagent 1, add 10 μL of sample / calibrator, and incubate for 3-5 min; then add 50 μL of reagent 2, mix well, and immediately (within 18 s) measure the first absorbance A1 at a wavelength of 546 nm. After incubating for 3-5 min, record the second absorbance value A2.

[0056] Comparative Example 1

[0057] 1. Prepare reagent 1 and reagent 2 with the following compositions.

[0058] Reagent 1 consists of:

[0059]

[0060] Reagent 2 consists of:

[0061]

[0062] 2. Detection parameters: Take 150 μL of reagent 1, add 10 μL of sample / calibrator, incubate for 3-5 min, and measure the first absorbance A1 at a wavelength of 546 nm; then add 50 μL of reagent 2, mix well, incubate for 3-5 min, and measure the second absorbance value A2.

[0063] Comparative Example 2

[0064] Prepare reagent 1 and reagent 2 with the following compositions.

[0065] Reagent 1 consists of:

[0066]

[0067]

[0068] Reagent 2 consists of:

[0069]

[0070] 2. Detection parameters: Take 150 μL of reagent 1, add 10 μL of sample / calibrator, and incubate for 3-5 min; then add 50 μL of reagent 2, mix well, and immediately (within 18 s) measure the first absorbance A1 at a wavelength of 546 nm. After incubating for 3-5 min, measure the second absorbance value A2.

[0071] Test case

[0072] The assays were performed according to the kits and detection methods of Comparative Examples 1, 2, and 1. The absorbance of the reagent blank, signal gradient, and clinical correlation of the glycated albumin assay reagents under different shelf-life conditions were determined using the Trinder reaction. Specific results are shown in Table 1 and... Figures 1-6 .

[0073] Table 1

[0074]

[0075] The results showed that Comparative Example 1 had a large reagent blank background, and the reagent blank signal increased significantly with the longer the reagent was stored. For a POCT-type glycated albumin detection kit that only requires one calibration, the accuracy of low-value samples is 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 relevance was poor. Compared with Comparative Examples 1 and 2, Example 1 had a lower reagent blank background (close to zero), better clinical relevance, and its linear gradient also met general detection requirements.

[0076] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting serum glycated albumin, characterized in that, include: Take reagent 1, add the sample to be tested or calibrator, and incubate for the first duration; Add reagent 2, mix well, and measure the first absorbance value A1 at the predetermined time; After incubation for a second time, the second absorbance value A2 was measured; the absorbance change value ΔA = A2-A1 after subtracting the blank background signal was calculated, and the serum glycated albumin concentration was calculated based on the absorbance change value ΔA. Wherein, reagent 1 includes fructose amino acid oxidase and peroxidase; reagent 2 includes protease; The predetermined time is specifically 0-20 seconds after adding reagent 2 and mixing; The first duration and the second duration are both selected from any duration between 3 and 5 minutes.

2. The detection method according to claim 1, characterized in that, Serum glycated albumin concentration is calculated using the absorbance change value ΔA, specifically as follows: The serum glycated albumin concentration was calculated by substituting the absorbance change value ΔA into the standard calculation function for serum glycated albumin.

3. The detection method according to claim 2, characterized in that, The standard calculation function for serum glycated albumin was determined by the following method: Establish the functional relationship between different calibrator concentrations C and the corresponding absorbance change value ΔA to obtain the standard calculation function.

4. The detection method according to claim 1, characterized in that, The wavelengths for detecting the first and second absorbance values ​​are both 500-600 nm.

5. The detection method according to claim 1, characterized in that, The reagent 1 includes a buffer solution, fructose amino acid oxidase, peroxidase, sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, an anti-interference agent, a preservative, and a stabilizer; Reagent 2 includes buffer, proteinase K, 4-aminoantipyrine, preservative and stabilizer.

6. The detection method according to claim 5, characterized in that, The buffer solution is at least one of tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 3-(N-morpholino)propanesulfonic acid (MOPS), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), piperazine-1,4-dihydroxypropanesulfonic acid (POPSO), and 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO).

7. The detection method according to claim 5, characterized in that, The anti-interference agent is ascorbic acid oxidase or bilirubin oxidase.

8. The detection method according to claim 5, characterized in that, The stabilizer is one or more of the following: water-soluble calcium salt, glycerol, trehalose, sucrose, bovine serum albumin, and disodium EDTA.

Images