Reagent and kit for detecting sugar defect type transferrin based on HPLC-FLD and application

Abstract

The invention provides a non-disease-diagnosis method for detecting carbohydrate-deficient transferrin based on HPLC-FLD (High Performance Liquid Chromatography-Flavin Like Disease). The method comprises the following steps: S1, obtaining a serum sample or a dried blood spot sample; s2, pretreating a serum sample or a dried blood spot sample, adding a terbium trivalent solution and a deferoxamine solution, incubating at 25 DEG C for 4 hours or placing in a refrigerator at 4 DEG C overnight, filtering with a 0.22 mu m water system filter membrane, and taking filtrate for detection; and S3, detecting the sugar defect type transferrin in the filtrate by adopting a high performance liquid chromatography combined fluorescence detection method. The analysis method is accurate and reliable, and has a wider linear range.

Description

Technical Field

[0001] The invention relates to a reagent and a kit for detecting sugar-deficient transferrin based on HPLC-FLD and application thereof, belonging to the technical field of sugar-deficient transferrin detection. Background Art

[0002] Alcohol dependence is a recurrent, chronic disease of central nervous system poisoning caused by habitual excessive drinking. Once contracted, it is difficult to get rid of the control of alcohol. It is characterized by psychological and physiological dependence, manifested as continuous or intermittent craving for alcohol and compulsive drinking, as well as withdrawal symptoms after stopping drinking. The harm of drinking to health and society mainly occurs through three interrelated mechanisms: first, after drinking, the person becomes physically drunk and confused for several hours, and his words and deeds can easily lead to irreversible consequences; second, after drinking, alcohol causes varying degrees of acute or chronic damage to organs and tissues in the body, such as alcoholic liver damage and complications of other organs; finally, long-term alcoholism can also cause changes in brain structure and function, leading to impaired cognitive function and other behavioral changes, such as poor self-control over drinking or mental disorders such as depression.

[0003] Transferrin (TRF) is a type of protein naturally present in the blood, which is mainly responsible for transporting iron absorbed from the digestive tract and iron released by red blood cell degradation in the human body. The naming of transferrin subtypes is related to the number of terminal sialic acid residues on its N-linked oligosaccharide chain. In normal human serum transferrin, tetrasialotransferrin (Tetrasialotransferrin, 4-TRF) is the main component, accounting for about 80%; the rest is composed of hexasialotransferrin (Hexasialotransferrin, 6-TRF), pentasialotransferrin (Pentasialotransferrin, 5-TRF), trisialotransferrin (Trisialotransferrin, 3-TRF), and disialotransferrin (Disialotransferrin, 2-TRF). Long-term alcohol abuse will lead to an increase in the level of disialotransferrin in human serum, as well as the appearance of monosialotransferrin (1-TRF) and asialotransferrin (0-TRF). These transferrin subtypes with abnormally elevated levels after alcohol abuse are called carbohydrate-deficient transferrin (CDT). Carbohydrate-deficient transferrin is currently considered to be the most suitable biomarker for the discovery of alcohol dependence and its staged detection.

[0004] At present, the main methods for CDT detection include electrophoresis, immunoassay, and high-performance liquid chromatography. The electrophoresis method uses the pI characteristics to separate different subtypes of transferrin, but this method is greatly affected by the matrix and is not suitable for analyzing actual samples. Although the immunoassay has great advantages in sensitivity, it is expensive, has complex steps, and cannot be quantified, so it is not universal in clinical applications. High-performance liquid chromatography is the preferred reference method for CDT detection. High-performance liquid chromatography technology can well separate transferrin glycoforms. At the same time, the high-performance liquid chromatogram can provide chromatographic peaks that are easy to identify, and the absolute or relative disialyl transferrin concentration can be quantified by measuring the peak area or peak height. Transferrin has specific ultraviolet absorption at 460nm. The use of high-performance liquid chromatography combined with ultraviolet method has high specificity and sensitivity, reducing the interference of other proteins in the analysis. However, considering that the chromatographic peak of CDT is easily confused with baseline fluctuations when the sample size is too small, it is not conducive to the quantification of monosialotransferrin with a small content. High performance liquid chromatography combined with ultraviolet method can usually only be used to detect the CDT content in constant samples, and it is difficult to detect CDT in trace samples. In addition, its linear range of detection is also narrow. Summary of the invention

[0005] The present invention provides a reagent and a kit for detecting sugar-deficient transferrin based on HPLC-FLD and applications thereof, which can effectively solve the above-mentioned problems.

[0006] The present invention is achieved in that:

[0007] A non-disease diagnosis method for detecting sugar-deficient transferrin based on HPLC-FLD comprises the following steps:

[0008] S1, obtain serum sample or dried blood spot sample.

[0009] S2. After pre-treating the serum sample or dried blood spot sample, add trivalent terbium solution and deferoxamine solution, incubate at 25°C for 4 hours or place in a 4°C refrigerator overnight, filter with a 0.22μm water filter membrane, and take the filtrate for testing; use deferoxamine solution to remove the coordinated iron ions contained in transferrin, and then replace them with trivalent terbium, so that transferrin has fluorescent properties for fluorescence detection.

[0010] S3, the sugar-deficient transferrin in the filtrate was detected by high performance liquid chromatography combined with fluorescence detection.

[0011] In some embodiments, the pretreatment of the serum sample is as follows: taking the upper serum, adding an equal amount of sample defatting solution, shaking and placing at 4°C for 30-60 minutes, taking out the mixed solution and centrifuging for 5 minutes to remove lipoproteins in the serum, and taking the supernatant.

[0012] In some embodiments, the pretreatment of the dried blood spot sample is: cut the dried blood spot sample into pieces, place it in a centrifuge tube, add Tris-HCl buffer with pH = 7.5-8.5, let it stand at room temperature for 4 hours or refrigerate it at 4°C overnight, take out the mixed solution and centrifuge it, and take the supernatant. The isoelectric point of transferrin is 5.4-5.7, and the pH of Tris-HCl buffer is higher than the isoelectric point. Transferrin is negatively charged, and this charged state is conducive to the effective separation of transferrin from other proteins or molecules in the subsequent high-performance liquid chromatography (HPLC) separation process. The dried blood spot sample mixed with the buffer is allowed to stand at room temperature for 4 hours, or placed in a refrigerated environment at 4°C overnight to fully dissolve and balance. Afterwards, the mixed solution is centrifuged to remove insoluble substances and cell debris, and finally the supernatant is taken for the next step of analysis and processing. This series of pretreatment steps ensures that the transferrin in the sample can be accurately detected and quantified in subsequent analysis.

[0013] In some embodiments, the sample defatting liquid is a mixed solution of dextran sulfate and calcium chloride, wherein the concentration of dextran sulfate is 0.4-0.6 g / L, and the concentration of calcium chloride is 0.4-0.6 g / L. Dextran sulfate (Dextransulfatesodium) is a polysaccharide compound with anticoagulant, cell signaling, anti-inflammatory effects and potential applications in tumor treatment. Calcium chloride (Calcium chloride) is an inorganic compound, commonly used as a dehydrating agent. In the prior art, there is no use of a mixed solution of dextran sulfate and calcium chloride directly as a sample defatting liquid. The embodiments of the present invention found that a mixed solution of dextran sulfate and calcium chloride can reduce the fat content in the blood through a specific biochemical mechanism, thereby achieving a good defatting effect.

[0014] In some embodiments, the deferoxamine solution has a concentration of 1.5-2.5×10 -2 mol / L deferoxamine methanesulfonic acid solution. This concentration is calculated based on the highest saturated concentration of iron ions in serum and blood and the optimization of the detection method. At this concentration, the iron ions in transferrin can be completely removed to ensure the sensitivity of the detection.

[0015] In some embodiments, the trivalent terbium solution has a concentration of 1.5-2.5×10 -2 mol / L TbCl3 solution. This unique coordination site of transferrin is not limited to binding with iron ions. Studies have found that transferrin can also undergo coordination reactions with other trivalent metal cations to generate a series of complexes with fluorescent labels. In particular, it can react with Tb 3+ 、Eu 3+The complex formed by the lanthanide trivalent metal cations has unique fluorescence properties. At this concentration of trivalent terbium solution, terbium ions can completely replace iron ions, making CDT saturated with terbium ions, thereby emitting strong specific fluorescence.

[0016] In some embodiments, the high performance liquid chromatography uses an anion exchange chromatography column, such as a SAX-NP 5U4.6 / 100 anion chromatography column. This chromatographic column is particularly suitable for separating and analyzing molecules with negative charges. In this embodiment, transferrin carries a negative charge under specific pH conditions. Therefore, the selection of an anion exchange chromatography column is based on its ability to effectively interact with the negative charge of transferrin, thereby achieving effective separation and detection of transferrin. The column temperature is set to 30°C. The selection of this temperature is carefully considered to provide a suitable environment for the detection of biological samples. Too high a temperature may cause denaturation or decomposition of biological molecules, while too low a temperature may affect the separation efficiency and analysis speed of the chromatographic column. Therefore, a column temperature of 30°C is a balance point that not only ensures the efficiency of chromatographic analysis, but also avoids unnecessary damage to biological samples, thereby ensuring the accuracy and reliability of the analysis results. Through such a setting, researchers can obtain high-quality chromatographic spectra, and then conduct in-depth research and analysis on the properties and behavior of transferrin.

[0017] In some embodiments, the excitation wavelength of the fluorescence detection is set to 298 nm, the emission wavelength is set to 550 nm, and the detector flow cell temperature of the fluorescence detection is 50°C.

[0018] In certain embodiments, the mobile phase of the HPLC is a Tris-HCl buffer, the pH of which is set between 7.5 and 8.5. The selection of this pH range is based on the characteristics of sialic acid proteins, and after a large number of experimental verifications, it was found that it can provide the best detection signal. Therefore, it is not recommended to use other buffer solutions, because Tris-HCl buffer is considered to be an ideal choice for processing biological samples. When the pH value is 8.0, the fluorescence intensity reaches a peak.

[0019] A reagent or kit for detecting sugar-deficient transferrin based on HPLC-FLD, comprising a sample defatting solution, a deferoxamine solution, a trivalent terbium solution and a mobile phase of a high performance liquid chromatography;

[0020] The sample defatting solution is a mixed solution of dextran sulfate and calcium chloride, wherein the concentration of dextran sulfate is 0.4-0.6 g / L, and the concentration of calcium chloride is 0.4-0.6 g / L;

[0021] The deferoxamine solution has a concentration of 1.5-2.5×10 -2 mol / L deferoxamine methanesulfonic acid solution;

[0022] The mobile phase of the high performance liquid chromatography is Tris-HCl buffer with a pH value of 7.5-8.5.

[0023] The beneficial effects of the present invention are:

[0024] In the high performance liquid chromatography-fluorescence detection and analysis method of the present invention, after the iron ions in transferrin are replaced by trivalent lanthanide ions such as terbium, the strong fluorescence emitted by the complex formed can amplify the CDT detection signal and can be used for detecting CDT in trace samples; at the same time, through improvements in detection conditions such as sample pretreatment, buffer, deferoxamine solution, trivalent terbium solution, mobile phase, etc., the improvements cooperate with each other to play a synergistic effect, so that the detection method of the present invention has high accuracy, strong specificity, high sensitivity, and a wider linear range than high performance liquid chromatography-ultraviolet method determination. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 This is a fluorescence emission spectrum of transferrin combined with terbium in Example 1.

[0027] Figure 2 is the TRF-Tb of Example 2 3+ Relationship between fluorescence intensity and pH value.

[0028] Figure 3 is the TRF-Tb of Example 3 3+ Relationship between fluorescence intensity and pH value of Tris-HCl buffer.

[0029] Figure 4 is the TRF-Tb of Example 4 3+ Plot of fluorescence intensity versus temperature.

[0030] Figure 5 This is the chromatogram of transferrin isoforms detected by high performance liquid chromatography-fluorescence detection in Example 5.

[0031] Figure 6 This is the standard curve of human transferrin in Example 5.

[0032] Figure 7 This is a picture of a dried blood spot sample collected using the blood collection filter paper of Example 6.

[0033] Figure 8These are the fluorescence detection chromatograms of normal and alcohol-dependent transferrin subtype samples in Example 6: a) healthy sample b) alcohol-dependent sample.

[0034] Fig. 9 This is a diagram of the HPLC-UV and HPLC-FLD detection of DBS samples in Example 6.

[0035] Fig.10 The stability of BDS samples under different storage conditions in Example 6 (1 and 2 are samples from alcohol-dependent persons; 3 is a sample from a healthy person).

[0036] Fig.11 This is a box plot of the CDT test results of the three methods in Example 6.

[0037] Fig.12 Receiver operating characteristic (ROC) curve for Example 6. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Example 1 Fluorescence emission spectrometry of transferrin combined with terbium

[0040] 1. Preparation of main reagents:

[0041] (1) 0.02 mol / L Tris-HCl buffer: Accurately weigh 2.4228 g of Tris into a clean beaker and dissolve in water. Adjust the pH of the solution to 8.0 with 1 mol / L HCl, make up to 1 L in a volumetric flask, and store at 4°C until use.

[0042] (2) 1.00 g / L transferrin standard stock solution: Accurately weigh 0.0100 g of TRF and place it in a clean EP tube. Use a pipette to accurately transfer 1 mL of Tris-HCl buffer to dissolve it and store it at 4°C until use.

[0043] (3)2.00×10 -2 mol / L terbium ion standard stock solution: Accurately weigh 0.0747g TbCl3·6H2O, place it in a clean beaker, dissolve it with a small amount of 1mol / L HCl, and then dilute it to a 100mL volumetric flask, and store it at 4℃ for later use.

[0044] (4) HPLC mobile phase configuration: Phase A is 0.02 mol / L Tris-HCl buffer, pH = 8.0; Phase B is a buffer of 0.02 mol / L Tris-HCl and 0.5 mol / L NaCl, pH = 8.0; Phase C is a cleaning solution, 1 mol / L NaCl.

[0045] (5) Degreasing solution: Accurately weigh 0.1000 g of dextran sulfate, dissolve it and dilute it to a 100 mL volumetric flask. Accurately weigh 0.1110 g of calcium chloride, dissolve it and dilute it to a 100 mL volumetric flask. Mix the dextran sulfate solution and the calcium chloride solution in a ratio of 1:1 (v:v=1:1) to prepare the degreasing solution, and store it at 4°C until use.

[0046] (6)2.00×10 -2 1 mol / L deferoxamine (Desferal) solution: Accurately weigh 1.3136 g of deferoxamine methanesulfonic acid and place it in a clean beaker. Dissolve it with ultrapure water, make up to 100 mL in a volumetric flask, and store at 4°C until use.

[0047] All reagents used were of analytical grade, and the experimental water was ultrapure water.

[0048] 2. Fluorescence measurement conditions:

[0049] Emission spectrum: The emission spectrum was scanned at a fixed excitation wavelength of 298 nm. The operating parameters of the fluorescence spectrophotometer were set as follows:

[0050] Excitation slit width: 5nm; emission slit width: 5nm; measurement voltage: 400V; sample pool specifications: 1cm×1cm×3cm; spectral scanning speed: 240nm / min.

[0051] 3. HPLC detection conditions

[0052] The mobile phase configuration is as follows:

[0053] Mobile phase A: 20mmol / L Tris-HCl solution;

[0054] Mobile phase B: 20 ​​mmol / L Tris-HCl solution;

[0055] Mobile phase C: 1 mol / L NaCl solution.

[0056] The injection volume was 50 μl, the flow rate was 1 mL / min, and after each sample was tested, the column was cleaned with 10-15 column volumes of mobile phase C.

[0057] The gradient elution program is shown in Table 1.

[0058] Table 1 HPLC gradient elution program

[0059]

[0060] a Before each sample injection, equilibrate the column with 10 to 15 column volumes of mobile phase A for 10 minutes, so that at time = 0, the mobile phase in the column is 100% A.

[0061] A SAX-NP 5U 4.6 / 100 anion exchange column was selected and the column temperature was set at 30°C.

[0062] 4. Pre-treatment of samples to be tested

[0063] The TRF system is 1.00 mL of 1.00 g / L transferrin standard stock solution.

[0064] Tb 3+ The system is 10 μL 2.00×10 -2 molL of TbCl3 solution.

[0065] TRF-Tb 3+ The system is to pre-treat the transferrin standard solution: take 1.00mL 1.00g / L transferrin standard stock solution into an EP tube, add 10μL deferoxamine solution, incubate at room temperature for 15min, then add 10μL 2.00×10 -2 molL TbCl3 solution, incubate at room temperature for 4h or refrigerate overnight at 4℃. Dilute the sample five times with Tris-HCl buffer, filter with 0.22μm water filter, remove bubbles by ultrasonication and set aside.

[0066] like Figure 1 As shown, the TRF-Tb was scanned under 298 nm excitation. 3+ , Tb 3+ , TRF fluorescence emission spectra. Curves 1, 2, and 3 correspond to TRF, Tb 3+ 、TRF-Tb 3+ Emission spectra of three different systems, the fluorescence intensity from high to low is TRF-Tb 3+ , Tb 3+ 、TRF. TRF-Tb 3+ The fluorescence emission intensity was greatly improved, and the fluorescence intensity increased by about 5.1 times (compared with Tb 3+ This indicates that transferrin has a significant effect on Tb 3+ The fluorescence produced a sensitization effect.

[0067] Under 298nm excitation, TRF-Tb 3+ The characteristic fluorescence emission spectra of terbium ions appear at 488nm and 550nm, corresponding to the terbium ion electrons. 5D4- 7 F6 and 5 D4- 7 F5 transition, in which the fluorescence intensity at 550nm is significantly enhanced.

[0068] The experiment was carried out at pH 9.0. Figure 1 It was found that, through protein absorption excitation, TRF-Tb 3+ The fluorescence intensity of the system was greatly improved, and this enhancement effect had a positive impact on improving the sensitivity of detection. At the same time, under this condition, it was observed that Tb 3+ The maximum emission peak of TRF-Tb red-shifted from 541nm to 550nm. 3+ There is energy transfer in the complex. In this process, TRF transfers the absorbed UV energy to Tb 3+ Subsequently, TRF-Tb was generated through the 4f orbital electron transition of terbium. 3+ characteristic fluorescence emission.

[0069] Based on this, this embodiment selects an emission wavelength of 550nm to measure the fluorescence intensity. 298nm is selected as the set excitation light of the fluorescence detector in the high performance liquid chromatography combined with fluorescence detector, and 550nm emission light is set as the detector output chromatographic signal wavelength.

[0070] Example 2 Effect of Solution pH on TRF-Tb 3+ Effect of fluorescence intensity

[0071] The effect of solution pH on TRF-Tb 3+ Effect of fluorescence intensity. The fluorescently labeled transferrin solution (TRF-Tb3+ system) was divided into 5 portions, and the pH value of each solution was accurately adjusted to 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0 using 1 mol / L NaOH and HCl. The fluorescence intensity of each solution was then measured. The test method was the same as in Example 1. The results are shown in FIG. Figure 2 As shown in the figure, the fluorescence intensity changes significantly within the pH range of 6.0 and 9.0. As the pH value gradually increases, the fluorescence intensity first increases, and then begins to weaken when the pH reaches a certain value. Figure 2 It can be observed that the fluorescence intensity reaches the maximum value when the pH value is 8.0.

[0072] Analysis of TRF-Tb 3+The reason why the fluorescence intensity is affected by pH is as follows: when the pH increases from 6.0 to 8.0, the negatively charged TRF in the solution increases with the increase of pH (the isoelectric point of TRF is 5.9), which is affected by the carbonate group in transferrin, which is the receptor for terbium ions. Under high pH conditions, the bicarbonate (HCO 3- ) loses a hydrogen ion and turns into carbonate (CO3 2- ). Therefore, increasing the pH from 7.0 to 8.0 increases the affinity of transferrin for terbium ions. Therefore, the concentration of the binary complex formed with Tb3+ in this pH range is positively correlated with the pH value, and the fluorescence intensity is observed to increase. However, when the pH continues to rise, the hydroxide ions (OH - ) concentration also increases. These hydroxide ions begin to compete with TRF for Tb 3+ , making TRF-Tb 3+ The concentration of the binary complex decreases. Therefore, as the pH value further increases, the fluorescence intensity begins to decrease.

[0073] Example 3 Effect of pH Value of Tris-HCl Buffer on TRF-Tb 3+ Effect of fluorescence intensity

[0074] Since the sample needs to be diluted with Tris-HCl solution with the same concentration as the mobile phase during the sample preparation process before injection, the effect of Tris-HCl buffer solutions with different pH values ​​on TRF-Tb 3+ The fluorescence intensity test method is the same as in Example 1. Figure 3 As shown in the experimental results, the pH value of Tris-HCl buffer has an important effect on the TRF-Tb 3+ When the pH value of Tris-HCl buffer is 8.0, TRF-Tb 3+ The fluorescence intensity is maximum.

[0075] Example 4 Effect of Temperature on TRF-Tb 3+ Effect of fluorescence intensity

[0076] Effect of temperature on TRF-Tb 3+ The effect of solution fluorescence intensity on Figure 4 The fluorescence intensity test method is the same as in Example 1. It is found in the experiment that as the temperature rises, the fluorescence intensity of the solution increases and then decreases. The mechanism of the experimental phenomenon is discussed below.

[0077] First, as the temperature rises, the luminescent fluorescence lifetime of terbium ions will become shorter. This is because high temperature accelerates the non-radiative transition process of ions, causing the excited terbium ions to return to the ground state faster, thereby shortening the fluorescence lifetime. However, when the temperature rises, the radiative fluorescence of the excited state high energy level will also increase. Therefore, in the early stage of temperature rise, the fluorescence intensity is enhanced to a certain extent.

[0078] The fluorescence intensity is also affected by both the non-radiative transition coefficient and the energy transfer efficiency. As the temperature increases further, the non-radiative transition coefficient increases, resulting in more energy being lost in the form of non-radiation rather than emitted in the form of fluorescence. High temperature may also affect the TRF and Tb 3+ The energy transfer efficiency between them increases, causing the fluorescence intensity to decrease.

[0079] Temperature rise may also cause changes in the tertiary structure of transferrin, or even denaturation. This structural change may affect the binding of TRF and Tb 3+ The fluorescence intensity will decrease significantly when the binding site is damaged or changed.

[0080] In summary, in order to obtain the best fluorescence intensity, the temperature needs to be controlled. According to the experimental results, when the temperature is 50°C, the fluorescence intensity of the TRF solution reaches the best. Therefore, when using the HPLC fluorescence detector, the temperature of the flow cell should be set to 50°C.

[0081] Example 5 Detection of Sugar-Deficient Transferrin by High Performance Liquid Chromatography-Fluorescence Method

[0082] 5.1 Transferrin isoform detection

[0083] This experiment is based on transferrin and Tb 3+ The fluorescence enhancement effect of the complex was determined by high performance liquid chromatography combined with fluorescence method to determine human serum transferrin. The test method was the same as in Example 1, and a subchromatogram of normal transferrin was obtained, such as Figure 5 shown.

[0084] The order of detection of transferrin isoforms in HPLC is disialyl transferrin (2-TRF), trisialyl transferrin (3-TRF), tetrasialyl transferrin (4-TRF), pentasialyl transferrin (5-TRF) and hexasialyl transferrin (6-TRF). The chromatogram reflects the separation of transferrin on the chromatographic column, which is related to the polarity, molecular size and isoelectric point of transferrin isoforms.

[0085] In this example, the percentage of the sum of asialotransferrin (0-TRF, when detectable), monosialotransferrin (1-TRF) and disialotransferrin (2-TRF) (0-TRF+1-TRF+2-TRF) in the total transferrin is used as Carbohydrate Deficient Transferrin. Figure 2-6 In the normal transferrin isoform samples, 0-TRF and 1-TRF were not detected, the level of 2-TRF was low, and the chromatographic peak shape of the CDT part was obvious and was not affected by the other matrices in the serum samples.

[0086] 5.2 Standard curve and detection limit

[0087] In a 1mL EP tube, add 100μL of 5.00×10 -3 mol / L TbCl3 solution, 100 μL of 1.00 g / L TRF standard solution diluted in multiple ratios (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128), and 800 μL of Tris-HCl solution with a pH of 8.0, mixed well and allowed to stand at room temperature for 15 min.

[0088] The above samples were detected by high performance liquid chromatography combined with fluorescence detector under the conditions of excitation wavelength 298nm and emission wavelength 550nm. The standard curve was obtained by plotting the CDT integral value (the integral value of the 2-TRF component of transferrin in normal samples) I in the high performance liquid chromatography fluorescence spectrum and the dilution multiple conversion value c.

[0089] like Figure 6 As shown, TRF concentration has a good linear relationship with CDT integral value in the dilution range of 1:64 to 1:1. Through linear regression analysis, the linear regression equation is I=29.92c+103.46 (the unit of c is mg / L), R2=0.9998.

[0090] To determine the detection limit of the HPLC combined with fluorescence detection method, the blank solution was measured 11 times in parallel, and the standard deviation was calculated, which was 0.49. The detection limit of the method was 0.05 mg / L by dividing the standard deviation 3 times by the slope of the standard curve. In order to evaluate the precision of the method, the TRF standard solution with a concentration of 100 mg / L was measured 11 times in parallel, and the relative standard deviation was calculated to be 1.0%.

[0091] 5.3 Determination of sugar-deficient transferrin in serum

[0092] Serum sample processing: Take 1mL of upper serum in an EP tube, add an equal amount of defatting solution, shake and place in a 4℃ refrigerator for 30-60min, take out the mixed solution and centrifuge for 5min to remove lipoprotein in the serum; add 10μL of deferoxamine solution, incubate at room temperature for 15min, add 10μL of 2.00×10-2molL TbCl3 solution and incubate at room temperature for 4h or place in a -4℃ refrigerator overnight. Dilute the sample five times with Tris-HCl buffer, filter with a 0.22μm water filter, remove bubbles by ultrasonic wave, and place in a sample injection bottle for testing.

[0093] After treatment, the serum samples were divided into 5 parts. These samples were substituted for the TRF standard solution in the preparation of the standard curve. The other operations were consistent with the preparation of the working curve in Section 2.3.5.2. According to the established standard curve I = 29.92c + 103.46 (the unit of c is mg / L), R2 = 0.9998, the content of transferrin in the serum samples was calculated.

[0094] The CDT integral values ​​of the five serum samples were obtained by high performance liquid chromatography. After conversion, the transferrin content in the serum was 3.12 g / L, as shown in Table 2. According to existing studies, the transferrin content in the serum of healthy adults is usually in the range of 2.2 to 4.0 g / L. The experimental results of 3.12 g / L are within the normal range.

[0095] Table 2 Determination results of transferrin in serum samples

[0096]

[0097] 5.4 Recovery rate experiment

[0098] To examine the accuracy of the analytical method, the serum samples from the same source in Section 5.3 were divided into 6 portions, numbered 1 to 6. 50 μL of 1.00 g / L TRF standard was added to samples 1 to 3, and 100 μL of the same concentration of TRF standard was added to samples 4 to 6 to conduct a recovery experiment.

[0099] The experimental results are shown in Table 3. The data in Table 3 show that the relative standard deviations (RSD) of the test results of samples 1 to 3 and samples 4 to 6 are 2.5% and 1.1%, respectively, and the recovery rates are 99.6% and 99.1%, respectively. This shows that the precision of this method is high and the recovery rate is also high, which fully proves the accuracy and reliability of this analytical method.

[0100] Table 3 Recovery experiment of transferrin determination in serum (n=3)

[0101] Serial number Average value / mg / L RSD / % TRF concentration in sample / mg / L Add TRF concentration / mg / L Recovery rate / % 1～3 8.10 2.5 3.12 5.00 99.6 4～6 13.03 1.1 3.12 10.00 99.1

[0102] 5.5 Comparative test between HPLC fluorescence method and HPLC-UV method

[0103] In order to further verify the accuracy of the method established in the experiment, the CDT content in the above human serum samples was determined by high performance liquid chromatography combined with ultraviolet method (HPLC-UV). High performance liquid chromatography combined with ultraviolet method is a commonly used method for detecting CDT in clinical medicine. It has been recommended by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) as a standardized method for clinical detection of CDT content in serum.

[0104] Experimental reagents: 1.7 mmol / L FeNTA solution; 0.02 mmol / L Bis-Tris buffer (pH=7.4); degreasing solution.

[0105] Experimental method: Take the prepared 100 mg / L TRF standard and make multiple dilutions (1:1, 1:2, 1:4, 1:8, 1:16, 1:32), and add a certain volume of FeNTA (vFeNTA:v=5:1) to saturate the transferrin iron. Filter with a 0.22μm filter membrane before injection. The HPLC detection conditions are the same as above. The injection volume is 100μL. The wavelength of the UV detector is set to 460nm, which is the specific absorption light of transferrin.

[0106] (1) Drawing a standard curve: Linear regression analysis was performed on the CDT integral value detected at 460 nm and the concentration of the TRF standard solution. The standard curve obtained was I=1.58c+2.27, R=0.9977 (unit of c: mg / L).

[0107] (2) Determination of TRF in serum samples: Take 1 mL of serum sample from the same source as the HPLC-FLD test, add an equal amount of defatted liquid to precipitate lipoproteins in the serum. After standing at room temperature for 15 minutes, centrifuge at 3000r / min for 5 minutes, take 1000μL of the supernatant and add 200μL of FeNTA solution. Mix the samples and store at 4°C for at least 4 hours or overnight. The integrated value of CDT of the three sample solutions at a wavelength of 460nm was determined by HPLC-UV method. According to the linear regression equation of the working curve I=1.58c+2.27, R=0.9977 (unit of c: mg / L), the concentration of TRF in the serum sample was calculated to be 3.14g / L. The results are shown in Table 4.

[0108] Table 4 Determination of transferrin in human serum by HPLC combined with UV method

[0109]

[0110]

[0111] According to the experimental results in Table 2 and Table 4, the results of the two analysis methods were tested for significant differences using SPSS26.0 software. In the test process, the homogeneity of variance was first tested to confirm whether the two sets of data were consistent in variance. The P value in the homogeneity of variance test was 0.390, which was greater than the significance level of 0.05, indicating that there was no significant difference in the variance of the two sets of data, providing a basis for the use of the F test. The F test is a commonly used statistical analysis method that judges the significance of the difference by comparing the ratio of inter-group variation to intra-group variation, and is used to evaluate whether there is a significant difference in the means of two or more groups of data.

[0112] Under the premise of meeting the homogeneity of variance, the result of the F test shows that the F value is 0.011. By consulting the F distribution table, we get the corresponding P value of 0.920, which is much larger than the significance level of 0.05. This shows that there is no statistically significant difference between the overall means obtained by the two methods.

[0113] In summary, the linear range of TRF determination by HPLC combined with UV method is 100 mg / L TRF dilution ratio 1:16 to 1:1, and the above HPLC-fluorescence method has a wider linear range. At the same time, the results of TRF determination in serum by the above HPLC-fluorescence detection analysis method are compared with the results of HPLC-UV method, and no significant difference is shown. It proves that the above analysis method is accurate and reliable.

[0114] Example 6 Detection of Sugar-Deficient Transferrin in Dried Blood Spot Samples by High Performance Liquid Chromatography-Fluorescence Method

[0115] 6.1 DBS sample testing for alcohol-dependent individuals

[0116] Twenty alcohol-dependent patients were selected and treated in the Alcohol Detoxification Department of Quanzhou Third People's Hospital from December 2022 to January 2024. All 20 alcohol-dependent people were clinically diagnosed according to the American Diagnostic and Statistical Manual of Mental Disorders, 5th edition, and the Chinese Classification and Diagnostic Criteria for Mental Disorders, 3rd edition. All alcohol-dependent patients were male (aged 46.7±13.3 years).

[0117] Exclusion criteria: ① exclude congenital glycosylation abnormalities; ② exclude recent use of antibiotics, hypoglycemic drugs and other drugs that cause liver damage; ③ exclude people with malignant tumors.

[0118] For comparative analysis, 10 healthy subjects were selected from the laboratory of the Affiliated Hospital of Ningde Normal University as the control group. They included 6 males and 4 females, whose ages matched those of the experimental group. The significance and risks of this study were fully informed to the patients and their families, who agreed and signed the informed consent.

[0119] Serum samples: The subjects were fasting and blood was collected. Then, venous blood was collected using a blood collection tube without anticoagulant. After centrifugation, the upper serum was transferred to an EP tube and placed in a 4°C refrigerator for later use.

[0120] DBS (dried blood spot) samples: Use specific blood collection filter paper, such as Figure 7 shown.

[0121] Venous blood dried blood spot sample: Take 50μL of whole blood from the previously collected but uncentrifuged venous blood sample, spot it on the designated position of the blood collection filter paper, and dry it at room temperature.

[0122] The dried DBS samples were sealed and stored to avoid moisture or other contamination to ensure the stability of the DBS samples.

[0123] The serum sample processing was the same as in Example 5.

[0124] Dried blood spot sample processing: Cut the sample on the blood collection filter paper with clean scissors and place it in a 1.5mL centrifuge tube. Add 1mL of Tris-HCl solution with pH=8.0 and let it stand at room temperature for 4h or refrigerate it in a 4℃ refrigerator overnight.

[0125] Take out the mixed solution and centrifuge it, take the supernatant, add 10 μL of terbium chloride solution and 10 μL of deferoxamine solution, incubate at room temperature for 4 hours or place in a refrigerator at 4°C overnight. Filter with a 0.22 μm water filter membrane, and then transfer to a sample bottle for testing.

[0126] The sampling, processing and storage processes of all samples strictly comply with relevant biosafety regulations and ethical standards.

[0127] The DBS samples and serum samples collected from the 30 subjects were tested using the high performance liquid chromatography combined with fluorescence (HPLC-FLD) method. The testing conditions were the same as above.

[0128] In order to investigate the advantages of HPLC-FLD method in trace detection, DBS samples from the same source were detected by HPLC-FLD method and high performance liquid chromatography combined with ultraviolet detection (HPLC-UV) method.

[0129] Typical chromatograms of DBS samples from healthy individuals and alcohol-dependent individuals are shown in Figure 2. Figure 8 shown.

[0130] Figure 8 a) is a sample from a healthy person, and b) is a sample from an alcohol-dependent person. By observing the chromatogram, it can be found that in the samples of alcohol-dependent people, 2-TRF is significantly increased, and 1-TRF appears (0-TRF is only detected in patients with heavy drinking, and it does not appear in the 20 alcohol-dependent samples collected). In this section, the ratio of the total area in the CDT and transferrin chromatogram is used for relative quantification. Figure 8 The CDT content in the normal sample was 1.23%, and the CDT content in the alcohol-dependent sample was 4.72%.

[0131] In order to compare the effects of high performance liquid chromatography-ultraviolet (HPLC-UV) and high performance liquid chromatography-fluorescence (HPLC-FLD) in detecting dried blood spot (DBS) samples, DBS samples from the same source were processed by the same method and then detected by these two methods respectively.

[0132] Test results such as Fig. 9 As shown in the figure, the small figure is the detection result using the HPLC-UV method. It can be seen from the chromatogram that no obvious chromatographic peaks appear under this method. Analysis shows that this may be because the UV method is not sensitive enough when detecting DBS samples or the concentration of the target substance in the DBS sample is too low, and the UV method cannot effectively detect it. Relatively speaking, the detection results of the HPLC-FLD method in the large figure show a better peak shape. This shows that this method has higher sensitivity and detection ability for DBS samples, and can clearly identify and quantify transferrin subtypes in DBS samples.

[0133] Therefore, by comparing the detection results of the two methods, we can conclude that the HPLC-FLD method has a better performance than the HPLC-UV method in detecting sugar-deficient transferrin in DBS samples. Therefore, the HPLC-FLD method has a trace detection advantage over the HPLC-UV method.

[0134] 6.2 Sample testing repeatability evaluation

[0135] Two samples were collected from three different periods, for a total of 6 DBS samples. Each DBS sample was measured 5 times under the same conditions (i.e., the same method, reagents, instruments, environmental conditions, and operators) to obtain inter-batch precision. Five DBS samples collected in the same period were measured 5 times under the same conditions (i.e., the same method, reagents, instruments, environmental conditions, and operators) to perform intra-batch repeatability tests to obtain intra-batch precision.

[0136] The relative standard deviation (RSD) was calculated based on the test results, and the coefficient of variation (CV) was obtained, as shown in Tables 5 and 6.

[0137] Table 5 Sample batch repeatability experiment (n = 6)

[0138]

[0139] Table 6 Sample intra-batch repeatability experiment (n=5)

[0140]

[0141] In the inter-batch (n=6) and intra-batch (n=5) repeatability tests, we obtained an inter-batch coefficient of variation CV of 5.5% and an intra-batch coefficient of variation CV of 2.5%. The inter-batch and intra-batch coefficients of variation obtained in the experiment were both less than the 10% required by the clinical inter-batch difference index.

[0142] The results of this experiment showed that the detection method used has high repeatability and precision and can meet the needs of clinical testing.

[0143] 6.3 Sample storage stability assessment

[0144] 6.3.1 Stability of DBS samples stored at different temperatures

[0145] For DBS samples, storage stability is also important for ensuring the accuracy and reproducibility of analytical results. In order to investigate the stability of dried blood spot (DBS) samples under different storage conditions, three groups of DBS samples were taken, labeled 1 to 3. Among them, No. 1 and No. 2 are samples of alcohol-dependent people, and No. 3 is a sample of healthy people. After being processed according to the method in 6.1, the three groups of samples were placed at room temperature, 4°C refrigeration, and -20°C frozen storage conditions. The CDT content in each group of samples was measured at 0, 6, 12, 24, 30, 48, 54, 60, and 72 hours, respectively.

[0146] The precision of DBS sample detection also uses the method of comparing the coefficient of variation. The mean value and relative standard deviation (RSD) of the test results are calculated, and the coefficient of variation (CV) of the ratio of the mean value to the RSD is used to evaluate the stability of the sample.

[0147] The calculation results are shown in Table 7. The CV of CDT content detection in DBS samples stored at room temperature is 6.2%, the CV of CDT content detection in DBS samples stored at 4°C is 6.8%, and the CV of CDT content detection in DBS samples stored at -20°C is 6.0%.

[0148] Table 7 Stability of DBS samples under different storage conditions

[0149]

[0150] The results showed that under different storage conditions, the coefficient of variation of CDT results in DBS samples detected by HPLC-FLD method was less than 10%, which can meet the requirements of in vitro sample detection and analysis.

[0151] In order to show the calculation results more intuitively, a box plot is made as shown in Figure 10. The CDT test results of DBS samples stored at room temperature, 4°C and -20°C are almost the same. At the same time, the CDT test results of DBS samples stored at room temperature perform well.

[0152] Compared with serum samples, DBS samples can be stored at room temperature for a week with almost no effect on the CDT test results, providing more ample conditions for the storage of samples during transportation. The storage stability and storage conditions do not have too high requirements, which means that DBS samples can replace traditional serum samples to achieve simpler detection of alcohol dependence markers, providing a scientific basis for clinical testing of changes in CDT metabolic levels. This has positive significance for the diagnosis of alcohol dependence. DBS samples also have great advantages in detecting CDT samples and even more protein biomarkers.

[0153] 6.3.2 Storage stability of DBS samples at room temperature

[0154] The stability of DBS samples stored at room temperature was further investigated and compared with serum samples stored at room temperature. DBS samples and serum samples from the same source were processed according to Section 6.1 and stored at room temperature for a long time. The CDT content in DBS samples and serum samples was tested on days 1, 2, 3, 5, 7, 14 and 28, respectively.

[0155] The experimental results are shown in Table 8. The results show that after the serum samples were stored at room temperature for 2 days, the CDT test results changed significantly. This may be because during the long-term storage at room temperature, the organic components such as proteins in the serum were affected by external factors such as temperature, light, oxygen, etc., and chemical changes such as oxidation and hydrolysis occurred, thus affecting the test results. In addition, if the serum is contaminated by microorganisms such as bacteria and mold during storage, it will also affect the test results of the protein in the serum.

[0156] In contrast, the CDT test results of DBS samples stored at room temperature remained stable within 7 days. There was no statistically significant difference between the measured data and the test results of freshly sampled samples (P>0.05). This shows that DBS samples stored at room temperature have better stability than serum samples and are suitable for clinical application scenarios that require long-term storage or transportation before testing.

[0157] Table 8 Comparison of stability of DBS samples and serum samples under different storage conditions

[0158]

[0159]

[0160] This section discusses the effect of DBS samples stored at room temperature on the repeatability of CDT content test results at different time points from 8 to 24 hours, as well as the stability of DBS samples stored at room temperature for 2 weeks. The experimental results show that compared with serum samples, the CDT content in DBS samples still maintains good stability and accuracy within 1 week of storage at room temperature.

[0161] 6.3.3 Effect of different sampling locations of DBS samples on CDT levels

[0162] First, prepare venous DBS samples of 30 samples. Take about 50μL of drops from the uncentrifuged blood sample. After drying at room temperature in the sampling area of ​​the blood collection filter paper, perform CDT testing together with the fingertip blood DBS sample from the same source. Fingerprint blood dried blood spot sample: In the fasting state, blood is collected through the fingertip and the blood is dropped directly on the designated position of the blood collection filter paper. The sampled filter paper is placed at room temperature for 1-2 hours until it is completely dry. Through comparative analysis, the consistency of the results of fingertip blood and venous blood in the DBS sample is examined.

[0163] The test results are shown in Table 9. The experimental results were verified to be normally distributed, and the data were found to be in accordance with the normal distribution. The P value of the one-way ANOVA test for homogeneity of variance was 0.818, which was greater than 0.05. The F test was then used to compare the data, and there was no statistically significant difference in variance. The F test result was F=0.070, P=0.792>0.05, indicating that there was no statistical difference in the data. The results are shown in Table 3-11. Through the analysis of the experimental results, it can be concluded that during the test, the location of blood collection will not have a significant effect on the final test results.

[0164] Table 9 CDT results of different sampling sites

[0165]

[0166] 6.3.4 HPLC-FLD detection of CDT values ​​in DBS samples and serum samples and HPLC-UV detection of CDT values ​​in serum

[0167] The carbohydrate-defective transferrin (CDT) values ​​in dried blood spot (DBS) and serum samples were detected by high performance liquid chromatography combined with fluorescence detection (HPLC-FLD) technology, and the CDT value in serum was determined by high performance liquid chromatography combined with ultraviolet detection (HPLC-UV) method.

[0168] In order to show the test results more intuitively, box plots of three sets of data are drawn, such as Fig.11 As shown, the figure clearly shows the difference in CDT levels between alcohol-dependent persons and healthy persons.

[0169] Since the overall sample size of the CDT samples tested is relatively small, the distribution of the data may be affected by individual extreme values ​​or outliers, and may deviate from the normal distribution. In order to ensure the accuracy and reliability of the data, we used SPSS26.0 software to verify the normal distribution of the experimental results. The verification results showed that all data met the characteristics of the normal distribution.

[0170] In order to analyze whether there are significant differences among the three groups of data, the homogeneity of variance of the three groups of data is first tested. The P value of the one-way ANOVA test for homogeneity of variance is 0.746, which is greater than the significance level of 0.05, indicating that there is no statistically significant difference in the variance between the groups. Therefore, the F test can be used for comparison. Furthermore, the results of the F test show that the F value is 0.069 and the P value is 0.933, which is also greater than the significance level of 0.05. This result shows that there is no statistically significant difference between the CDT results detected by the three methods. Therefore, we can assume that the CDT levels detected by the three methods are consistent and there are no significant differences. The calculation results are shown in Table 10.

[0171] Table 10 F test of CDT results detected by different methods

[0172]

[0173] Although the F test showed that the overall means of CDT level data detected by the three methods were similar, it could not reveal whether there was a statistical difference between any two groups of data. In order to further explore whether there were differences between the groups, we needed to perform multiple comparisons based on the F test.

[0174] Since the previous variance homogeneity test has confirmed the homogeneity of variance of the three groups of data, we used the least significant difference (LSD) method for multiple comparisons. LSD multiple comparisons is a statistical method used to compare whether there are significant differences between multiple groups or treatments. The F test cannot be used directly for pairwise comparisons between groups, which will increase the type I error α, that is, the false positive error.

[0175] The CDT results obtained by three different detection methods (HPLC-FLD detection of DBS samples, HPLC-FLD detection of serum samples, and HPLC-UV detection of serum samples) were compared pairwise, and the results of multiple comparisons are shown in Table 11.

[0176] As can be seen from the table, the P value of the difference between each two groups is greater than the significance level of 0.05, indicating that there is no statistically significant difference between any two groups. This result further supports the results of the F test.

[0177] Table 11 Multiple comparison LSD results

[0178]

[0179] In summary, the content of sugar-deficient transferrin (CDT) in dried blood spot (DBS) samples was determined by high performance liquid chromatography combined with fluorescence detection (HPLC-FLD) technology, and compared with the results of CDT content in serum, and it was found that there was no significant difference between the two. At the same time, the results of detecting CDT content in DBS samples by HPLC-FLD method were consistent with the results of detecting CDT content in serum by HPLC-UV method recommended by the International Federation of Clinical Chemistry and Laboratory Medicine. Therefore, it can be confirmed that DBS samples are suitable for the detection of CDT content, which provides a new sample source and detection method for CDT detection.

[0180] 6.3.5 CDT distribution levels in alcohol dependent individuals and healthy controls

[0181] The CDT content of healthy samples and samples of alcohol-dependent people was detected by high-performance liquid chromatography-ultraviolet method, and finally the receiver operating characteristic (ROC) curve was drawn, as shown in Fig.12 , thus determining that a CDT value greater than 1.92% is positive and a CDT value ≤ 1.92% is negative.

[0182] Example 7 HPLC-FLD detection of CDT in DBS, liver function index GGT and the accuracy of the combination of the two in diagnosing alcohol dependence

[0183] Among the 30 subjects who participated in the clinical diagnosis experiment of alcohol dependence, the high performance liquid chromatography combined with ultraviolet method (HPLC-UV) used 1.92% as the positive reference value. The test results showed that there were 10 healthy people and 20 alcohol-dependent people, which was consistent with the clinical diagnosis results. The diagnostic results of CDT in DBS samples detected by HPLC-FLD method showed that there were 11 healthy people and 19 diagnosed as alcohol-dependent people. By comparing with the results of HPLC-UV method, among these 19 people diagnosed as alcohol-dependent, 17 were actually real alcohol-dependent people.

[0184] According to the diagnostic results of HPLC-UV, the subjects were divided into "case group" and "control group", and the HPLC-FLD test results to be evaluated were arranged horizontally. Among them, "+" indicates a positive result and "-" indicates a negative result. The number of true positives (a), false positives (b), false negatives (c) and true negatives (d) is helpful for analyzing the sensitivity and specificity of the HPLC-FLD method for diagnosing alcohol dependence, as shown in Table 12.

[0185] Sensitivity and specificity are key indicators for evaluating the performance of diagnostic methods. Sensitivity, also known as the True Positive Rate (TPR), is a measure of the ability of the diagnostic method to be evaluated to detect sick people. Sensitivity is only related to the case group. The higher the sensitivity, the more people who are actually sick can be correctly identified as patients by the diagnostic method to be evaluated. Specificity, also known as the True Negative Rate (TNR), is a measure of the ability of the diagnostic method to be evaluated to correctly judge healthy people, and is only related to the healthy group. The higher the specificity, the more accurately the method can identify healthy people as disease-free. Both indicators need to be calculated based on the gold standard for comparison.

[0186] Table 12 HPLC-FLD detection of CDT in DBS samples

[0187]

[0188] According to Table 12, the sensitivity of HPLC-FLD for CDT in DBS samples for diagnosing alcohol dependence was calculated to be 85.0%, the specificity was 80.0%, the positive predictive value was 89.5%, the negative predictive value was 72.7%, the Youden index was 0.65, and the accuracy was 83.3%.

[0189] The Youden Index (YI) is also called the correct index. It is mainly used to evaluate the ability of the method to distinguish true patients from non-patients. It is calculated by combining sensitivity and specificity, and the formula is:

[0190] YI=TPR+TNR-1.

[0191] The higher the Youden Index, the more accurately the method can distinguish patients from non-patients.

[0192] For the aforementioned 30 subjects, their liver function index GGT data were also collected (see Appendix Table 3 for details). Taking 60UI / L as the positive reference value, the sensitivity and specificity of the diagnosis of alcohol dependence were also evaluated against the gold standard, as shown in Table 13.

[0193] Table 13 Diagnostic results of liver function index GGT

[0194]

[0195]

[0196] According to Table 13, it was calculated that the sensitivity of the liver function index GGT in diagnosing alcohol dependence was 80.0%, the specificity was 70.0%, the positive predictive value was 84.2%, and the negative predictive value was 63.6%, the Youden index was 0.50, and the accuracy was 76.7%.

[0197] Finally, the diagnostic results of DBS samples detected by HPLC-FLD were combined with the liver function index GGT for diagnosis, as shown in Table 14.

[0198] Table 14 HPLC-FLD detection of CDT and liver function index GGT in DBS samples combined diagnosis results

[0199]

[0200] According to Table 13, the sensitivity of HPLC-FLD detection of DBS samples combined with liver function index GGT for diagnosing alcohol dependence was calculated to be 89.5%, specificity was 81.9%, positive predictive value was 89.5%, negative predictive value was 81.8%, Youden index = 0.71, and accuracy was 86.7%.

[0201] The above calculation results are summarized in Table 15.

[0202] Table 15 Sensitivity and specificity of CDT, GGT and their combination

[0203]

[0204] Based on the above data, the sensitivity and specificity of the two methods of HPLC-FLD detection of CDT in DBS samples and liver function GGT values ​​were compared. Since the sample size in this study was small and the theoretical frequency T was less than 1, Fisher's exact test was suitable. SPSS IBM Data were analyzed by Software Version 26.0. Fisher's exact test gave a P value of 0.349. At a significance level of 0.05, there was no statistically significant difference in the accuracy of the diagnosis of CDT in DBS samples using the HPLC-FLD method alone and the combined use of CDT and GGT. When CDT and GGT were used together for diagnosis, the diagnostic effect was further improved. This combined application may be able to more comprehensively reflect the alcohol dependence status of the subjects, especially in the diagnosis of complex conditions involving alcohol dependence and related liver damage, showing a higher application value.

[0205] Comparison of the Youden Index showed that the Youden Index of the CDT method in DBS samples detected by HPLC-FLD was higher than that of the liver function index GGT method alone. This indicates that the CDT detected by HPLC-FLD method performs better in terms of diagnostic accuracy.

[0206] In summary, although the sensitivity of CDT in DBS samples detected by HPLC-FLD alone was not significantly different from that of the combined use of CDT and GGT, considering the comparative results of the Youden Index and the potential advantages of combined diagnosis, it is believed that the use of CDT in clinical practice will provide more accurate and comprehensive diagnostic information for alcohol dependence.

[0207] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting sugar-deficient transferrin based on HPLC-FLD for non-disease diagnosis, characterized in that: The following steps are involved: S1, serum sample or dried blood spot sample was obtained; S2, after pre-treating the serum sample or dried blood spot sample, add trivalent terbium solution and deferoxamine solution, incubate at 25°C for 4 h or place in a refrigerator at 4°C overnight, filter with a 0.22 μm water filter membrane, and take the filtrate for testing; S3, the sugar-deficient transferrin in the filtrate was detected by high performance liquid chromatography combined with fluorescence detection.

2. The method according to claim 1, characterized in that: The pretreatment of the serum sample is as follows: taking the upper serum, adding an equal amount of sample defatting solution, shaking and placing at 4°C for 30-60 minutes, taking out the mixed solution and centrifuging for 5 minutes to remove lipoprotein in the serum, and taking the supernatant.

3. The method according to claim 1, characterized in that The pretreatment of the dried blood spot sample is as follows: the dried blood spot sample is cut into pieces, placed in a centrifuge tube, a Tris-HCl buffer solution with a pH of 7.5-8.5 is added, the mixture is allowed to stand at room temperature for 4 hours or refrigerated at 4° C. overnight, the mixed solution is taken out for centrifugation, and the supernatant is taken out.

4. The method according to claim 1, characterized in that: The sample defatting solution is a mixed solution of dextran sulfate and calcium chloride, wherein the concentration of dextran sulfate is 0.4-0.6 g / L, and the concentration of calcium chloride is 0.4-0.6 g / L.

5. The method according to claim 1, characterized in that: The deferoxamine solution has a concentration of 1.5-2.5×10 - 2 mol / L deferoxamine methanesulfonic acid solution.

6. The method according to claim 1, characterized in that The trivalent terbium solution has a concentration of 1.5-2.5×10 - 2 mol / L TbCl3 solution.

7. The method according to claim 1, characterized in that The high performance liquid chromatography uses an anion exchange chromatography column, and the column temperature is set at 30°C.

8. The method according to claim 1, characterized in that: The excitation wavelength of the fluorescence detection was set to 298 nm, the emission wavelength was set to 550 nm, and the detector flow cell temperature of the fluorescence detection was 50°C.

9. The method according to claim 1, characterized in that: The mobile phase of the high performance liquid chromatography is Tris-HCl buffer with a pH value of 7.5-8.

5.

10. A reagent or kit for detecting sugar-deficient transferrin based on HPLC-FLD, characterized in that: It includes sample degreasing solution, deferoxamine solution, trivalent terbium solution and mobile phase of high performance liquid chromatography; The sample defatting solution is a mixed solution of dextran sulfate and calcium chloride, wherein the concentration of dextran sulfate is 0.4-0.6 g / L, and the concentration of calcium chloride is 0.4-0.6 g / L; The deferoxamine solution has a concentration of 1.5-2.5×10 -2 mol / L deferoxamine methanesulfonic acid solution; The mobile phase of the high performance liquid chromatography is Tris-HCl buffer with a pH value of 7.5-8.5.

Images