Kit for determining sugar deficiency transferrin and application thereof

Abstract

The invention provides a kit for determining sugar deficiency transferrin and application thereof, the kit for determining sugar deficiency transferrin comprises a first reagent, a second reagent and a separation tube, the first reagent comprises a buffer solution, a coagulant, a preservative, a protective agent and a surfactant, and the second reagent comprises a buffer solution, a coagulant, a preservative, a protective agent and a surfactant; the second reagent comprises a buffer solution, a transferrin antibody and microsphere conjugate, a preservative, a surfactant and a stabilizer; the separation pipe comprises an upper half part separation pipe and a lower half part collection pipe, the separation pipe comprises a first separation chamber and a second separation chamber, the second separation chamber is a hollow cylinder and is arranged in the center of the first separation chamber, and the top of the second separation chamber is communicated with the first separation chamber. The whole kit is simple to operate, realizes rapid separation of a target object, does not need elution, and has a good test effect.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection, and in particular to a kit for determining sugar-deficient transferrin and an application thereof. Background Art

[0002] Alcoholism has become a global problem that can lead to irreversible psychological and physiological damage, as well as family and social problems. For example, pregnant women who are addicted to alcohol may cause their children to suffer from fetal alcohol syndrome. Therefore, correctly judging the drinking status plays an important role in different fields such as clinical medicine, forensic medicine and justice. Traditional detection methods include physical examination, investigation and questioning, and some clinical biochemical markers. Although the latter is relatively objective, it has poor sensitivity and specificity for judging drinking status. In recent years, scholars have also paid more and more attention to some new drinking markers, such as acetaldehyde protein adducts, fatty acid ethyl esters, 5-hydroxytryptol, etc. Among them, carbohydrate-deficient transferrin (CDT) is undoubtedly the most popular new marker at present.

[0003] CDT is an isomer of transferrin (TRF), which is mainly caused by the loss of TRF sialic acid (SA) residues. The proportion of different transferrin subtypes in the total amount of transferrin in normal human serum is also different. The most abundant is the tetrasialic acid molecule subtype. After drinking, the sialic acid molecules contained in transferrin in the body decrease, which increases the levels of trisialic acid, disialic acid, monosialic acid and asialic acid transferrin subtypes. Studies have shown that asialo transferrin, which accounts for the main part of CDT, is caused by the loss of two sugar chains. In clinical practice, asialo transferrin, monosialic acid transferrin and disialic acid transferrin subtypes are collectively referred to as CDT. Its increased content is often seen in alcoholics and disappears after a period of abstinence, with a half-life of 14 days. Studies have shown that compared with the absolute quantitative level of CDT, the ratio of CDT to the total amount of transferrin has a higher diagnostic specificity for drinking status.

[0004] At present, the main detection methods of CDT include isoelectric focusing electrophoresis, chromatography and immunoassay. Isoelectric focusing electrophoresis is to separate different subtypes according to their different isoelectric points on a gel with a pH gradient. After the immunofixation and staining steps, the bands of various transferrin subtypes can be seen, and finally the density measurement is performed to obtain the CDT results. The absolute concentration of CDT can be obtained according to the calibration curve. The commonly used chromatography method is anion exchange chromatography, but its sensitivity is low; high performance liquid chromatography can also detect hereditary TRF in blood samples of healthy people, but column exchange is time-consuming and expensive, which limits its application in large-scale CDT detection. In recent years, CDT can be separated from plasma by plant lectin affinity chromatography, and then anti-human transferrin serum is added for quantitative detection. This detection method combining chromatography and immunoassay has a good detection effect. In this regard, a kit for determining sugar-deficient transferrin is proposed to improve the effect of separating CDT from plasma, which is of great significance for improving the detection accuracy. Summary of the invention

[0005] The present invention aims to provide a kit for determining sugar-deficient transferrin and its application. The kit does not require an elution step, has high CDT separation efficiency and high test accuracy.

[0006] To achieve the above object, the present invention provides a kit for measuring sugar-deficient transferrin, the kit for measuring sugar-deficient transferrin includes a first reagent, a second reagent and a separation tube, the first reagent includes a buffer, a coagulant, a preservative, a protective agent and a surfactant, and the second reagent includes a buffer, a transferrin antibody and a microsphere conjugate, a preservative, a surfactant and a stabilizer. The separation tube includes a separation portion in the upper half and a collection portion in the lower half, the separation portion includes a first separation chamber and a second separation chamber, the second separation chamber is a hollow cylinder and is arranged in the center of the first separation chamber and the top of the second separation chamber is connected to the first separation chamber, and a filter membrane is provided at the top of the second separation chamber. The first separation chamber is filled with agarose magnetic beads, the agarose magnetic beads are coupled with rhinoceros beetle lectin AlloA, and two-thirds of the height of the second separation chamber is filled with agarose gel, the agarose gel is coupled with pea lectin; the bottom of the second separation chamber is connected to the collection portion, and a one-way filter membrane is provided at the bottom of the second separation chamber.

[0007] Preferably, the first reagent comprises 20-80mmol / L buffer, 5.0-30g / L coagulant, 0.5-2g / L preservative, 0.1-1g / L protective agent and 10-30mmol / L surfactant. The second reagent comprises 100-300mmol / L buffer, 50-200mg / L transferrin antibody and microsphere conjugate, 0.5-2.0g / L preservative, 10-30mmo / L surfactant and 10-30mmo / L stabilizer.

[0008] Preferably, the buffer in the first reagent is phosphate buffer, the coagulant is dextran 20000, the preservative is sodium azide, the protective agent is gelatin and the surfactant is Tween 20; the buffer in the second reagent is phosphate buffer, the preservative is sodium azide, the surfactant is Tween 20 and the stabilizer is glycerol.

[0009] Preferably, the preparation steps of the conjugate of transferrin antibody and microspheres are:

[0010] S1. Washing: First, take carboxyl polystyrene microspheres of a predetermined particle size and add them to a washing buffer; then centrifuge, remove the supernatant, and resuspend the precipitate with MES buffer by ultrasonication;

[0011] S2, activation: carbodiimide and N-hydroxysulfosuccinimide are added to the resuspended carboxyl polystyrene microsphere suspension for activation. After activation, centrifugation is performed again, and the precipitate is ultrasonically resuspended with a coupling buffer to obtain a carboxyl polystyrene microsphere suspension. The coupling buffer is a phosphate buffer with a concentration of 100 mmol / L and a pH of 7.0;

[0012] S3, coupling: adding the transferrin antibody solution to the activated carboxyl polystyrene microsphere suspension, reacting at room temperature for 2 hours to obtain coupled carboxyl polystyrene microspheres;

[0013] S4, blocking: adding bovine serum albumin (BSA) to the above-mentioned coupled carboxyl polystyrene microsphere suspension for blocking. After the blocking is completed, centrifugation is performed and the precipitate is stored in a 100 mmol / L pH 7.0 phosphate buffer to obtain the transferrin antibody and microsphere conjugate.

[0014] Preferably, the preparation steps of the agarose magnetic beads are:

[0015] S21, washing: adding agarose magnetic beads of a predetermined diameter range into a container, adding anhydrous ethanol, mixing evenly, magnetically separating and removing the supernatant, repeating 2-3 times to obtain washed agarose magnetic beads;

[0016] S22, coupling: add the agarose magnetic beads obtained in S21 to the coupling buffer, then add the AlloA lectin, mix well, shake at room temperature for 2-3 hours, and remove the supernatant by magnetic separation; the coupling buffer is 0.1 mol / L NaHCO3, 0.8 mol / L NaCl, pH 8.3;

[0017] S23. Blocking: Add bovine serum albumin buffer without exosomes for blocking treatment, shake at room temperature for 2-3 hours, and remove the supernatant by magnetic separation; add TRIS buffer, shake at room temperature to mix, and remove the supernatant by magnetic separation. Repeat the operation 2-3 times to obtain agarose beads coupled with Rhinoceros Beetle lectin AlloA.

[0018] Preferably, 6-15 mg of Rhizoma Dioscoreae lectin AlloA is coupled to each 1 ml of agarose magnetic beads.

[0019] Preferably, the preparation steps of the agarose gel are:

[0020] S31, transfer the activated agarose gel 6B into the core funnel, and wash it with 1-100 mM HCl solution for standby use;

[0021] S32, dissolving pea lectin in a coupling buffer, and coupling it with the washed agarose gel 6B in S31, reacting at room temperature for 5 hours, and then washing the coupled agarose gel 6B with a coupling buffer to wash away the uncoupled pea lectin;

[0022] S33, using 0.5 mol / L glycine to block the remaining activated groups of Sepharose 6B, and then washing the blocked Sepharose 6B 3 times with TRIS buffer to obtain Sepharose coupled with pea lectin.

[0023] Preferably, the coupling buffer contains 1 mmol / L CaCl2, 1 mmol / L MgCl2, 1 mmol / L NaCl2 and 20 mmol / L Tris-HCL; the pH value of the coupling buffer is 7.0-7.3.

[0024] Preferably, 1 g of agarose gel is coupled with no less than 4.5 g of pea lectin.

[0025] The present invention also provides an application of a kit, wherein the kit is the above-mentioned kit for determining sugar-deficient transferrin, and the kit is used to detect the content of sugar-deficient transferrin in a body fluid sample, and the body fluid sample is at least one of blood, serum, and plasma.

[0026] The beneficial effects of the present invention are as follows: the present invention proposes a kit for measuring sugar-deficient transferrin, wherein a separation tube is provided in the kit, wherein the separation portion of the separation tube is provided with a second separation chamber located in the middle of the first separation chamber by arranging a first separation chamber, wherein the first separation chamber is filled with agarose magnetic beads coupled with rhinoceros beetle lectin AlloA, wherein the rhinoceros beetle lectin AlloA can specifically bind to sialic acid (Sia) α2→6 galactose (Gal) β1→4 N-acetylglucosamine (GlcNAc) residues in oligosaccharide chains, thereby achieving the purpose of separating transferrin and sugar-deficient transferrin and extracting sugar-deficient transferrin, wherein the second separation chamber is filled with agarose gel coupled with pea lectin, wherein the pea lectin cooperates with the rhinoceros beetle lectin AlloA to achieve the purpose of secondary separation of transferrin, thereby improving the separation effect of sugar-deficient transferrin, wherein the sample processed by the separation tube contains almost only sugar-deficient transferrin, thereby laying a foundation for the subsequent detection of the content of sugar-deficient transferrin. The present invention is to improve the separation effect of the classification tube, and the first separation chamber adopts agarose magnetic beads, and the magnetic microspheres are coupled with lectins. The surface area of ​​the ball is large, and more lectins are cross-linked, and more proteins can be combined. The second separation chamber is set in a hollow cylindrical form, and the agarose gel coupled with pea lectin is filled inside. When the sample liquid to be tested undergoes the first separation chamber, a preliminary pre-separation is achieved, and most of the transferrin has been adsorbed. The pre-separated sample passes through the second separation chamber again, and a secondary separation is achieved, and the residual transferrin can be captured by affinity again. At the same time, the first separation chamber and the second separation chamber are set to change the flow route of the liquid, first move upward and then move downward from the internal hollow cylindrical cavity, greatly increasing the contact surface of the sample to be tested and the adsorption medium. And the agarose gel in the second separation chamber is not filled completely, and when the liquid enters the second separation, a similar "siphon" effect will be produced, which can speed up the separation efficiency, and the separation tube does not need centrifugal assistance and can also quickly and accurately separate the sugar-deficient transferrin. The sample separated by the separation tube is used in conjunction with the first reagent and the second reagent, and the content of sugar-deficient transferrin in the sample can be detected and calculated. The entire kit is easy to operate, achieves rapid separation of the target, does not require elution, and has good test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of the structure of the separation tube of the present invention.

[0028] Figure 2 It is a linear analysis diagram of the test values ​​obtained by the test kit of the present invention and the Siemens test method in Table 1.

[0029] Figure 3 It is a linear analysis diagram of the test values ​​obtained by the test kit of the present invention and the Siemens test method in Table 2.

[0030] Among them, 1-separation part, 2-collection part, 3-agarose magnetic beads, 4-agarose gel, 5-one-way filter membrane. DETAILED DESCRIPTION

[0031] The embodiments described below are only some embodiments of the present invention, not all 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.

[0032] The present invention provides a kit for measuring sugar-deficient transferrin, the kit for measuring sugar-deficient transferrin includes a first reagent, a second reagent and a separation tube, the first reagent includes a buffer, a coagulant, a preservative, a protective agent and a surfactant, the second reagent includes a buffer, a transferrin antibody and a microsphere conjugate, a preservative, a surfactant and a stabilizer. The separation tube includes a separation portion 1 in the upper half and a collection portion 2 in the lower half, the separation portion 1 includes a first separation chamber and a second separation chamber, the second separation chamber is a hollow cylinder and is arranged in the center of the first separation chamber and the top of the second separation chamber is connected to the first separation chamber, and a filter membrane is provided on the top of the second separation chamber. The first separation chamber is filled with agarose magnetic beads 3, the agarose magnetic beads 3 are coupled with rhinoceros beetle lectin AlloA, and two-thirds of the height of the second separation chamber is filled with agarose gel 4, the agarose gel 4 is coupled with pea lectin; the bottom of the second separation chamber is connected with the collection portion 2, and a one-way filter membrane 5 is provided at the bottom of the second separation chamber. The first separation chamber is provided with a liquid inlet near the bottom side wall, and a liquid outlet is provided near the bottom side wall of the collecting part 2. The liquid inlet is arranged on the bottom side wall of the first separation chamber, and the liquid to be tested gradually reaches the top of the second separation chamber from bottom to top, and then moves downward from the top of the second separation chamber to the one-way filter membrane 5, which can extend the distance of the liquid to be tested, improve the contact effect of the liquid to be tested with the medium, and is conducive to completely capturing the transferrin in the liquid to be tested, and only sugar-deficient transferrin is included in the liquid to be tested flowing out through the liquid outlet. The filter membrane at the top of the second separation chamber can effectively block agarose magnetic beads, preventing agarose magnetic beads from mistakenly entering the second separation chamber. The one-way filter membrane at the bottom of the second separation chamber can block the affinity medium (i.e., agarose gel) coupled with lectin from passing through without blocking liquid and protein from passing through, and plays the effect of auxiliary filtration.

[0033] The first reagent includes 20-80mmol / L buffer, 5.0-30g / L coagulant, 0.5-2g / L preservative, 0.1-1g / L protective agent and 10-30mmol / L surfactant. The second reagent includes 100-300mmol / L buffer, 50-200mg / L transferrin antibody and microsphere conjugate, 0.5-2.0g / L preservative, 10-30mmo / L surfactant and 10-30mmo / L stabilizer.

[0034] The buffer in the first reagent is phosphate buffer, the coagulant is dextran 20000, the preservative is sodium azide, the protective agent is gelatin and the surfactant is Tween 20; the buffer in the second reagent is phosphate buffer, the preservative is sodium azide, the surfactant is Tween 20 and the stabilizer is glycerol.

[0035] The preparation steps of the conjugate of transferrin antibody and microspheres are as follows:

[0036] S1. Washing: First, carboxyl polystyrene microspheres of a predetermined particle size are taken and added to a washing buffer (50 mM MES buffer, pH 6.0); then centrifuged, the supernatant is removed, and the precipitate is resuspended by ultrasound in a MES buffer (100 mM MES buffer, pH 7.0);

[0037] S2, activation: carbodiimide and N-hydroxysulfosuccinimide are added to the resuspended carboxyl polystyrene microsphere suspension for activation. After activation, centrifugation is performed again, and the precipitate is ultrasonically resuspended with a coupling buffer to obtain a carboxyl polystyrene microsphere suspension. The coupling buffer is a phosphate buffer with a concentration of 100 mmol / L and a pH of 7.0;

[0038] S3, coupling: adding the transferrin antibody solution to the activated carboxyl polystyrene microsphere suspension, reacting at room temperature for 2 hours to obtain coupled carboxyl polystyrene microspheres;

[0039] S4, blocking: adding bovine serum albumin (BSA) to the above-mentioned coupled carboxyl polystyrene microsphere suspension for blocking. After the blocking is completed, centrifugation is performed and the precipitate is stored in a 100 mmol / L pH 7.0 phosphate buffer to obtain the transferrin antibody and microsphere conjugate.

[0040] The transferrin antibody refers to an antibody that can specifically bind to transferrin. The antibody can be purchased from the market and can be a separated natural antibody or a recombinant antibody. The transferrin antibody can bind to various subtypes of transferrin, mainly including asialo transferrin, monosialo transferrin, disialo transferrin, trisialo transferrin, and tetrasialo transferrin.

[0041] The particle size distribution range of the carboxyl polystyrene microspheres is 150-300 nm. The particle sizes of the carboxyl polystyrene microspheres include but are not limited to 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, and 300 nm.

[0042] The preparation steps of the agarose magnetic beads are:

[0043] S21, washing: adding agarose magnetic beads of a predetermined diameter range into a container, adding anhydrous ethanol, mixing evenly, magnetically separating and removing the supernatant, repeating 2-3 times to obtain washed agarose magnetic beads;

[0044] S22, coupling: add the agarose magnetic beads obtained in S21 to the coupling buffer, then add the AlloA lectin, mix well, shake at room temperature for 2-3 hours, and remove the supernatant by magnetic separation; the coupling buffer is 0.1 mol / L NaHCO3, 0.8 mol / L NaCl, pH 8.3;

[0045] S23. Blocking: Add bovine serum albumin buffer without exosomes for blocking treatment, shake at room temperature for 2-3 hours, and remove the supernatant by magnetic separation; add TRIS buffer, shake at room temperature to mix, and remove the supernatant by magnetic separation. Repeat the operation 2-3 times to obtain agarose beads coupled with Rhinoceros Beetle lectin AlloA.

[0046] 6-15 mg of AlloA, a lectin from Rhinoceros Beetle, is coupled to each 1 ml of agarose magnetic beads.

[0047] The particle size distribution range of the agarose magnetic beads is 100μm-200μm, including but not limited to 100μm, 110μm, 120μm, 130μm, 140μm, 150μm, 160μm, 170μm, 180μm, 190μm, and 200μm.

[0048] The preparation steps of the agarose gel are:

[0049] S31, transfer the activated agarose gel 6B into the core funnel, and wash it with 1-100 mM HCl solution for standby use;

[0050] S32, dissolving pea lectin in a coupling buffer, and coupling it with the washed agarose gel 6B in S31, reacting at room temperature for 5 hours, and then washing the coupled agarose gel 6B with a coupling buffer to wash away the uncoupled pea lectin;

[0051] S33, using 0.5 mol / L glycine to block the remaining activated groups of Sepharose 6B, and then washing the blocked Sepharose 6B 3 times with TRIS buffer to obtain Sepharose coupled with pea lectin.

[0052] The coupling buffer contains 1mmol / L CaCl2, 1mmol / L MgCl2, 1mmol / L NaCl2 and 20mmol / L Tris-HCL; the pH value of the coupling buffer is 7.0-7.3.

[0053] 1g of agarose gel is coupled with no less than 4.5g of pea lectin.

[0054] The Sepharose 6B was activated using NHS.

[0055] The present invention also provides an application of a kit, wherein the kit is the above-mentioned kit for determining sugar-deficient transferrin, and the kit is used to detect the content of sugar-deficient transferrin in a body fluid sample, wherein the body fluid sample is at least one of blood, serum, and plasma. For blood, serum, and plasma samples, the samples are centrifuged at a speed of less than 3000g for 10-15 minutes to remove cell debris and precipitate impurities in the samples, and the supernatant after centrifugation is taken for standby use.

[0056] The method for determining the content of sugar-deficient transferrin may include the following steps: taking an appropriate amount of serum or plasma sample supernatant 50-300 μL and adding it to a separation tube, placing it at room temperature for 10-20 minutes, collecting the flow-through and mixing it with the first reagent, incubating it for 2-5 minutes, adding the second reagent, recording the instrument reading point as A1, and detecting the transmission or scattered light signal at a specific wavelength of 340 to 800 nm. After continuing to incubate for 1 to 5 minutes, the instrument reading point is recorded as A2, and the transmission or scattered light signal is detected at a specific wavelength of 340 to 800 nm. The difference in light signal between the reaction end point A2 after adding the second reagent and the reaction starting point A1 after adding the second reagent is recorded, that is, the amplitude of the transmission or scattered light signal change between the reaction end point A2 and the reaction starting point A1, and the standard curve is brought in to calculate the content of sugar-deficient transferrin in the sample. The detection principle can be turbidimetry or turbidimetry, and the inspection instrument can be applicable to but not limited to biochemical analysis and specific protein analyzers.

[0057] Example 1

[0058] This embodiment provides a kit for measuring sugar-deficient transferrin, and the kit for measuring sugar-deficient transferrin includes:

[0059] The first reagent: 50mmol / L buffer, 15g / L coagulant, 1.4g / L preservative, 0.6g / L protective agent and 20mmol / L surfactant; according to the above formula, accurately weigh each component in the same container, mix evenly, adjust the volume, filter, and prepare the first reagent.

[0060] The second reagent includes 200mmol / L buffer, 100mg / L transferrin antibody and microsphere conjugate, 1.3g / L preservative, 20mmo / L surfactant and 15mmo / L stabilizer. According to the above formula, each component is accurately weighed in the same container, mixed evenly, and fixed to volume to obtain the second reagent.

[0061] Separation tube: The first separation chamber of the separation part of the separation tube is filled with agarose magnetic beads coupled with Rhinoceros beetle lectin AlloA, with 13 mg of Rhinoceros beetle lectin AlloA coupled to each 1 ml of agarose magnetic beads; the second separation part is filled with two-thirds of the height of agarose gel coupled with pea lectin, with 1 g of agarose gel coupled with 4.5 g of pea lectin.

[0062] Example 2

[0063] This embodiment provides a kit for measuring sugar-deficient transferrin, and the kit for measuring sugar-deficient transferrin includes:

[0064] The first reagent: 60mmol / L buffer, 25g / L coagulant, 1.0g / L preservative, 0.8g / L protective agent and 25mmol / L surfactant; according to the above formula, accurately weigh each component in the same container, mix evenly, adjust the volume, filter, and prepare the first reagent.

[0065] The second reagent includes 250mmol / L buffer, 130mg / L transferrin antibody and microsphere conjugate, 1.5g / L preservative, 25mmo / L surfactant and 20mmo / L stabilizer. According to the above formula, each component is accurately weighed in the same container, mixed evenly, and fixed to volume to prepare the second reagent.

[0066] Separation tube: The first separation chamber of the separation part of the separation tube is filled with agarose magnetic beads coupled with rhinoceros beetle lectin AlloA, with 14 mg of rhinoceros beetle lectin AlloA coupled to each 1 ml of agarose magnetic beads; the second separation part is filled with two-thirds of the height of agarose gel coupled with pea lectin, with 1 g of agarose gel coupled with 5.0 g of pea lectin.

[0067] Example 3

[0068] This embodiment provides a kit for measuring sugar-deficient transferrin, and the kit for measuring sugar-deficient transferrin includes:

[0069] The first reagent: 70mmol / L buffer, 25g / L coagulant, 1.8g / L preservative, 0.5g / L protective agent and 22mmol / L surfactant; according to the above formula, accurately weigh each component in the same container, mix evenly, adjust the volume, filter, and prepare the first reagent.

[0070] The second reagent: includes 260mmol / L buffer, 160mg / L transferrin antibody and microsphere conjugate, 1.7g / L preservative, 22mmo / L surfactant and 19mmo / L stabilizer; according to the above formula, accurately weigh each component in the same container, mix evenly, and adjust the volume to obtain the second reagent.

[0071] Separation tube: The first separation chamber of the separation part of the separation tube is filled with agarose magnetic beads coupled with Rhinoceros beetle lectin AlloA, with 10 mg of Rhinoceros beetle lectin AlloA coupled to each 1 ml of agarose magnetic beads; the second separation part is filled with two-thirds of the height of agarose gel coupled with pea lectin, with 1 g of agarose gel coupled with 5.3 g of pea lectin.

[0072] In the above embodiment, the preparation steps of the conjugate of transferrin antibody and microspheres are as follows:

[0073] S1. Cleaning: First, carboxyl polystyrene microspheres with a particle size distribution range of 270 nm were taken and added to 50 mM MES buffer, pH 6.0; then centrifuged, the supernatant was removed, and the precipitate was precipitated with 100 mM MES buffer, pH 7.0, and ultrasonically resuspended to a concentration of 1% (w / v) of carboxyl polystyrene microspheres;

[0074] S2, activation: add carbodiimide and N-hydroxysulfosuccinimide in excess of the carboxyl density to the resuspended carboxyl polystyrene microsphere suspension for activation, stir at room temperature for 20 minutes, centrifuge again after activation, and resuspend the precipitate with coupling buffer by ultrasonication to obtain a carboxyl polystyrene microsphere suspension, the coupling buffer is a phosphate buffer with a concentration of 100 mmol / L and a pH of 7.0, and the concentration of the carboxyl polystyrene microsphere suspension is 1% (w / v);

[0075] S3, coupling: take an equal volume of transferrin antibody solution (concentration of 0.6 mg / mL) and add it to the activated carboxyl polystyrene microsphere suspension, react at room temperature for 2 hours to obtain coupled carboxyl polystyrene microspheres;

[0076] S4, blocking: adding bovine serum albumin (BSA) to the above-mentioned coupled carboxyl polystyrene microsphere suspension for blocking. After the blocking is completed, centrifugation is performed and the precipitate is stored in a 100 mmol / L pH 7.0 phosphate buffer to obtain the transferrin antibody and microsphere conjugate.

[0077] The preparation steps of agarose magnetic beads coupled with AlloA are as follows:

[0078] S21, washing: adding agarose magnetic beads with a direct range of 200 μm into a container, adding anhydrous ethanol, mixing evenly, performing magnetic separation to remove the supernatant, and repeating 2-3 times to obtain washed agarose magnetic beads;

[0079] S22, coupling: the agarose beads obtained in S21 were added to the coupling buffer, and then the Hercules beetle lectin AlloA was added, mixed evenly, and oscillated at room temperature for 2-3 hours, and the supernatant was removed by magnetic separation; the coupling buffer was 0.1 mol / L NaHCO3, 0.8 mol / L NaCl, pH 8.3, and the ratio of the volume of agarose beads (mL) to the Hercules beetle lectin AlloA (mg) was set to 1:13, 1:14 and 1:10, respectively;

[0080] S23, blocking: add bovine serum albumin buffer (0.5M, pH 6-7) without exosomes for blocking treatment, shake the reaction at room temperature for 2h, and remove the supernatant by magnetic separation; add TRIS buffer (200mM), shake and mix at room temperature, remove the supernatant by magnetic separation, repeat the operation 2-3 times to obtain agarose beads coupled with Rhinoceros Beetle lectin AlloA.

[0081] The steps for preparing agarose gel coupled with pea lectin are as follows:

[0082] S31, transfer the NHS-activated agarose gel 6B into the core funnel, and wash it with 100 mM HCl solution for standby use;

[0083] S32, dissolving the pea lectin in a coupling buffer, and coupling it with the washed agarose gel 6B in S31, reacting at room temperature for 5 hours, then washing the coupled agarose gel 6B with a coupling buffer to wash away the uncoupled pea lectin, and setting the ratio of the mass (g) of agarose gel 6B to the mass (g) of pea lectin to 1:4.5, 1:5, and 1:5.3, respectively;

[0084] S33, using 0.5 mol / L glycine to block the remaining activated groups of Sepharose 6B, and then washing the blocked Sepharose 6B three times with 0.02 mol / L, pH 7.4 TRIS buffer to obtain agarose gel coupled with pea lectin.

[0085] The coupling buffer contains 1mmol / L CaCl2, 1mmol / L MgCl2, 1mmol / L NaCl2 and 20mmol / L Tris-HCL; the pH value of the coupling buffer is 7.0.

[0086] The agarose beads coupled with the rhinoceros beetle lectin AlloA were filled into the first separation chamber of the separation tube, and the agarose gel coupled with pea lectin was filled to two-thirds of the height of the second separation chamber, and stored at 4°C for later use.

[0087] Performance evaluation of the kit for measuring sugar-deficient transferrin.

[0088] Test instrument: Hitachi 7180 biochemical analyzer.

[0089] Sugar-deficient transferrin test method:

[0090] Take 100 μL of serum sample and add it to the separation tube, incubate at room temperature for 10 minutes, collect the separation solution after 10 minutes, take 2 μL of separation and mix with 200 μL of the first reagent, incubate for 5 minutes, add 50 μL of the second reagent, continue incubation for 5 minutes, detect the transmitted light signal at a specific wavelength of 600 nm (the signal value is calculated by Hitachi 7180 biochemical analyzer based on the turbidity of the reaction solution), record the difference between the amplitude of the reaction end point and the reaction starting point after adding the second reagent, and substitute it into the standard curve to calculate the content of sugar-deficient transferrin in the sample.

[0091] The standard curve is a calibration curve obtained by fitting the concentration of the calibration product with the measured reactivity, the ordinate is the reactivity, specifically the transmitted light signal value calculated by the Hitachi 7180 biochemical analyzer, and the abscissa is the concentration of each calibration product. The transferrin antigen used to prepare the calibration product and the quality control product was purchased from Sigma-Aldrich, item number: T3309. The calibration product and the quality control product are prepared by dissolving the transferrin antigen in a buffer solution, and the composition of the buffer solution is as follows: 20mM PBS buffer solution, 1% BSA, pH 7.4. The drawing of the standard curve is a common analysis operation for those skilled in the art, which will not be described in detail here.

[0092] Transferrin content determination method:

[0093] Take 2 μL of serum sample and 200 μL of the first reagent and mix them. After incubation for 5 minutes, add 50 μL of the second reagent, the instrument reading point is recorded as A3, and the transmission or scattered light signal is detected at a specific wavelength of 600nm (the signal value is calculated by Hitachi 7180 biochemical analyzer according to the turbidity automatic detection of the reaction solution), continue to incubate for 5 minutes, the instrument reading point is recorded as A4, and the transmission or scattered light signal is detected at a specific wavelength of 600nm, and the light signal difference between the reaction end point A4 and the reaction starting point A3 after the second reagent is added is recorded, i.e., the transmission or scattered light signal variation amplitude of the reaction end point A4 and the reaction starting point A3 is brought into the standard curve of transferrin (pre-established, and the establishment method is a common technology in this area, which will not be repeated here), and the content of transferrin in the sample can be calculated. Transferrin herein includes various types of transferrin such as asialo transferrin, monosialo transferrin, disialo transferrin, trisialo transferrin, and tetrasialo transferrin.

[0094] The buffer composition of the quality control product is the same as that of the calibrator.

[0095] The transferrin antigen used in preparing the calibration and quality control products was purchased from Sigma-Aldrich, catalog number: T3309. The calibration and quality control products were prepared by dissolving the transferrin antigen in a buffer solution, the composition of which was as follows: 20 mM PBS buffer, 1% BSA, pH 7.4.

[0096] The kit for measuring sugar-deficient transferrin in Example 1 was selected for performance evaluation. The evaluation method is as follows:

[0097] 40 fresh sera were taken and tested with a sugar-deficient transferrin assay kit (latex enhanced nephelometry), which was purchased from Siemens Medical Diagnostics Co., Ltd. (referred to as Siemens), Germany, and the kit name is NLatexCDT Kit. The test was performed with reference to its product manual. At the same time, the kit of Example 1 of the present invention was used in combination with the above test method, and the correlation coefficient of the two groups of test values ​​was calculated by linear regression method.

[0098] The Siemens test kit directly tests CDT in serum. The kit contains specific CDT antibodies. The test method of the Siemens test kit is to test CDT and TRF separately, use immunoturbidimetry to obtain CDT and TRF test values, and then calculate the ratio.

[0099] Table 1

[0100]

[0101] Table 2

[0102]

[0103] Note: “%CDT” indicates the ratio of sugar-deficient transferrin to transferrin in serum samples, which is expressed as a mass percentage.

[0104] Figure 2 and Figure 3 The linear regression analysis results of the Siemens test value and the test value obtained by the kit of Example 1 of the present invention combined with the above test method are shown, wherein the correlation R 2 =0.9973, R =0.9986; %CDT correlation R 2 =0.9938, R=0.9969. The above results show that the sugar-deficient transferrin assay kit of Example 1 of the present invention has high measurement accuracy.

[0105] The present invention is described in detail above in conjunction with the embodiments. It should be noted that the various specific technical features described in the above specific embodiments can be combined and changed in any suitable manner without contradiction, and the present invention will not further describe various possible combinations. In addition, other variations and combinations of the various technical features of the present invention should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A kit for measuring sugar-deficient transferrin, characterized in that: The kit for determining sugar-deficient transferrin includes a first reagent, a second reagent and a separation tube, wherein the first reagent includes a buffer, a coagulant, a preservative, a protective agent and a surfactant, and the second reagent includes a buffer, a conjugate of transferrin antibody and microspheres, a preservative, a surfactant and a stabilizer; the separation tube includes a separation portion in the upper half and a collection portion in the lower half, the separation portion includes a first separation chamber and a second separation chamber, the second separation chamber is a hollow cylinder and is arranged at the center of the first separation chamber, and the top of the second separation chamber is connected to the first separation chamber, and a filter membrane is provided on the top of the second separation chamber; the first separation chamber is filled with agarose magnetic beads, and the agarose magnetic beads are coupled with the rhinoceros beetle agglutinin AlloA, and two-thirds of the height of the second separation chamber is filled with agarose gel, and the agarose gel is coupled with pea agglutinin; the bottom of the second separation chamber is connected to the collection portion, and a one-way filter membrane is provided at the bottom of the second separation chamber.

2. The kit for measuring sugar-deficient transferrin according to claim 1, characterized in that: The first reagent includes 20-80mmol / L buffer, 5.0-30g / L coagulant, 0.5-2g / L preservative, 0.1-1g / L protective agent and 10-30mmol / L surfactant; the second reagent includes 100-300mmol / L buffer, 50-200mg / L transferrin antibody and microsphere conjugate, 0.5-2.0g / L preservative, 10-30mmo / L surfactant and 10-30mmo / L stabilizer.

3. The kit for measuring sugar-deficient transferrin according to claim 2, characterized in that: The buffer in the first reagent is phosphate buffer, the coagulant is dextran 20000, the preservative is sodium azide, the protective agent is gelatin and the surfactant is Tween 20; the buffer in the second reagent is phosphate buffer, the preservative is sodium azide, the surfactant is Tween 20 and the stabilizer is glycerol.

4. The kit for measuring sugar-deficient transferrin according to claim 3, characterized in that: The preparation steps of the conjugate of transferrin antibody and microspheres are as follows: S1. Washing: First, take carboxyl polystyrene microspheres of a predetermined particle size and add them to a washing buffer; then centrifuge, remove the supernatant, and resuspend the precipitate with MES buffer by ultrasonication; S2, activation: carbodiimide and N-hydroxysulfosuccinimide are added to the resuspended carboxyl polystyrene microsphere suspension for activation. After activation, centrifugation is performed again, and the precipitate is ultrasonically resuspended with a coupling buffer to obtain a carboxyl polystyrene microsphere suspension. The coupling buffer is a phosphate buffer with a concentration of 100 mmol / L and a pH of 7.0; S3, coupling: adding the transferrin antibody solution to the activated carboxyl polystyrene microsphere suspension, reacting at room temperature for 2 hours to obtain coupled carboxyl polystyrene microspheres; S4, blocking: adding bovine serum albumin (BSA) to the above-mentioned coupled carboxyl polystyrene microsphere suspension for blocking. After the blocking is completed, centrifugation is performed and the precipitate is stored in a 100 mmol / L pH 7.0 phosphate buffer to obtain the transferrin antibody and microsphere conjugate.

5. The kit for measuring sugar-deficient transferrin according to claim 1, characterized in that: The preparation steps of the agarose magnetic beads are: S21, washing: adding agarose magnetic beads of a predetermined diameter range into a container, adding anhydrous ethanol, mixing evenly, magnetically separating and removing the supernatant, repeating 2-3 times to obtain washed agarose magnetic beads; S22, coupling: add the agarose magnetic beads obtained in S21 to the coupling buffer, then add the AlloA lectin, mix well, shake at room temperature for 2-3 hours, and remove the supernatant by magnetic separation; the coupling buffer is 0.1 mol / L NaHCO3, 0.8 mol / L NaCl, pH 8.3; S23. Blocking: Add bovine serum albumin buffer without exosomes for blocking treatment, shake at room temperature for 2-3 hours, and remove the supernatant by magnetic separation; add TRIS buffer, shake at room temperature to mix, and remove the supernatant by magnetic separation. Repeat the operation 2-3 times to obtain agarose beads coupled with Rhinoceros Beetle lectin AlloA.

6. The kit for measuring sugar-deficient transferrin according to claim 5, characterized in that: 6-15 mg of AlloA, a lectin from Rhinoceros Beetle, is coupled to each 1 ml of agarose magnetic beads.

7. The kit for measuring sugar-deficient transferrin according to claim 1, characterized in that: The preparation steps of the agarose gel are: S31, transfer the activated agarose gel 6B into the core funnel, and wash it with 1-100 mM HCl solution for standby use; S32, dissolving pea lectin in a coupling buffer, and coupling it with the washed agarose gel 6B in S31, reacting at room temperature for 5 hours, and then washing the coupled agarose gel 6B with a coupling buffer to wash away the uncoupled pea lectin; S33, using 0.5 mol / L glycine to block the remaining activated groups of Sepharose 6B, and then washing the blocked Sepharose 6B 3 times with TRIS buffer to obtain Sepharose coupled with pea lectin.

8. The kit for measuring sugar-deficient transferrin according to claim 7, characterized in that: The coupling buffer contains 1mmol / L CaCl2, 1mmol / L MgCl2, 1mmol / L NaCl2 and 20mmol / L Tris-HCL; the pH value of the coupling buffer is 7.0-7.

3.

9. The kit for measuring sugar-deficient transferrin as claimed in claim 7, characterized in that , each 1g of agarose gel is coupled with no less than 4.5g of pea lectin.

10. An application of a kit, characterized in that: The kit is a kit for determining sugar-deficient transferrin as claimed in any one of claims 1 to 9, and the kit is used to detect the content of sugar-deficient transferrin in a body fluid sample, wherein the body fluid sample is at least one of blood, serum and plasma.

Images