Kit for detecting sialylated sugar chain antigen kl-6, and method for using the same and application thereof

Abstract

The application provides a kit for detecting sialylated carbohydrate antigen KL-6 and a use method and application thereof, and relates to the field of in vitro detection. The kit for detecting sialylated carbohydrate antigen KL-6 provided by the application comprises magnetic beads, KL-6 antibodies, acridinium ester, a magnetic bead storage solution, an acridinium ester storage solution, a sample processing solution, a pre-priming solution and a priming solution. The sample processing solution comprises, in terms of mass fraction, 0.03-0.3% polyoxyethylene fatty acid ester, 0.3-2.0% bovine serum albumin, 0.3-2.0% raffinose, 0.3%-2.0% sodium hydroxide, 0.01-1.5% NaN3 and 0.3-1.5% sodium chloride, the rest is purified water, and the pH is 8-10. The kit can be used for determining sialylated carbohydrate antigen KL-6 in serum, and has high accuracy and strong anti-interference ability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of in vitro detection, and relates to a sialylated sugar chain antigen KL-6, in particular to a kit for detecting the sialylated sugar chain antigen KL-6 and a method and application thereof. BACKGROUND

[0002] The sialylated sugar chain antigen KL-6 (Krebs Von den Lungen-6) was discovered by Professor Kohno of Japan in 1985. A plurality of monoclonal antibodies were prepared by immunizing mice with a human lung adenocarcinoma cell line (VMRC-LCR), and the sialylated sugar chain antigen recognized by the sixth antibody was named KL-6. KL-6 belongs to epithelial mucin 1 (MUC1) classified as Cluster9, and is mainly expressed on the surface of type II alveolar epithelial cells. In normal lung tissue and terminal bronchial epithelial cells, the expression amount of KL-6 is small, but the expression is enhanced in degenerated type II alveolar epithelial cells. The domestic KL-6 detection technology mainly includes latex enhanced immunoturbidimetry and chemiluminescent immunoassay.

[0003] Chemiluminescence is a luminescence phenomenon occurring in living organisms. In 1888, Wiedemann of Germany first described the basic principle of the chemiluminescence phenomenon, that is, chemiluminescence is the result of a chemical reaction process. Chemiluminescence can be divided into two categories of gas phase and liquid phase according to the state of its reaction medium, and liquid phase chemiluminescence is the most widely used. Common luminescent reagents include luminol, acrid ester, 1,2-dioxetane and peroxyoxalic acid ester chemiluminescence systems.

[0004] Acridines are a kind of chemical luminescent reagent with very high quantum yield, and its molecular structure is composed of at least two parts: a luminescent group and a leaving group. The most widely used are acrid ester and acrid sulfonamide. The conventional reaction mechanism is that under alkaline conditions, hydrogen peroxide adds to the 9-position carbon atom of acridine, and the addition product forms a peroxide anion under alkaline conditions, which further attacks the carbonyl carbon, the leaving group is removed and further forms an unstable four-membered ring intermediate, and after ring opening, an excited state acridone is formed, which releases photons in the process of returning to the ground state.

[0005] Chemiluminescent immunoassay as a quantitative detection technology for trace substances has been developed to be advanced and mature, and plays a very important role in human disease screening and health detection. According to the different luminescent systems, chemiluminescence can be divided into: direct chemiluminescence immunoassay, luminescent oxygen channel immunoassay, enzyme-catalyzed chemiluminescence immunoassay, electrochemiluminescence immunoassay, and among them, magnetic particle chemiluminescence technology has gradually become the main character in the field of domestic clinical detection.

[0006] In chemiluminescence immunoassay for serum or plasma sample testing, sample pretreatment is crucial. Samples may contain impurities such as lipids, hemolysin products, and fibrinogen, which can interfere with the KL-6 detection signal or cause false results. Pretreatment with a sample processing solution effectively removes these interfering substances, reduces background noise, and improves detection specificity. Pretreatment standardizes sample conditions (e.g., pH, ionic strength), reducing the impact of inter-sample differences on test results and improving batch-to-batch consistency and comparability. Simultaneously, particulate matter in serum / plasma (such as cell debris and fibrin clots) can clog the microfluidic channels or sensors of the instrument; pretreatment removes these particles, reducing the risk of instrument malfunction.

[0007] The existing technology CN119322180A discloses a chemiluminescence detection kit containing the following components: KL-6 calibrator, amino-magnetic bead-conjugated KL-6 monoclonal antibody, alkaline phosphatase-labeled KL-6 monoclonal antibody, AMPPD rapid chemiluminescence substrate solution, and washing buffer. This technology requires the additional addition of AMPPD rapid chemiluminescence substrate and has a long detection time.

[0008] Existing technology CN112946255A discloses a reagent for the chemiluminescent detection of salivary glycan antigens using magnetic microparticles. The reagent comprises a magnetic microparticle reagent consisting of 2-8 μg / ml magnetic microparticle-labeled KL-6 antibody 1, 20%-30% newborn bovine serum, 0.01%-0.1% preservative, 20%-30% glycerol, 0.01%-0.1% Tween 20, 0.1%-1% BSA, and 50%-70% PBS buffer; and an acridine ester reagent consisting of 6-10 μg / ml acridine ester-labeled KL-6 antibody 2, 10%-40% glycerol, 0.5%-5% BSA, and 60%-90% PBS buffer. This technology does not utilize sample processing solutions, and its anti-interference capability is generally poor. Summary of the Invention

[0009] This invention addresses the problems existing in the prior art by providing a kit for detecting salivary glycan antigen KL-6, along with its usage and application. The kit for detecting salivary glycan antigen KL-6 provided by this invention comprises magnetic beads, KL-6 antibody, acridine ester, magnetic bead preservation solution, acridine ester preservation solution, sample processing solution, pre-activation solution, and activation solution. The sample processing solution contains, by mass fraction, 0.03%-0.3% polyoxyethylene fatty acid ester, 0.3%-2.0% bovine serum albumin, 0.3%-2.0% raffinose, 0.3%-2.0% sodium hydroxide, 0.01%-1.5% NaN3, and 0.3%-1.5% sodium chloride, with the balance being purified water at pH 8-10. This kit can be used to determine salivary glycan antigen KL-6 in serum with high accuracy and strong anti-interference ability.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] On one hand, the present invention provides a kit for detecting the salivary glycoprotein antigen KL-6, the kit comprising...

[0012] The sample contains magnetic beads, KL-6 antibody, acridine ester, magnetic bead preservation solution, acridine ester preservation solution, sample processing solution, pre-activation solution, and activation solution. The sample processing solution contains, by mass fraction, 0.03%-0.3% polyoxyethylene fatty acid ester, 0.3%-2.0% bovine serum albumin, 0.3%-2.0% raffinose, 0.3%-2.0% sodium hydroxide, 0.01%-1.5% NaN3, and 0.3%-1.5% sodium chloride, with the balance being purified water at pH 8-10.

[0013] Preferably, the sample processing solution contains, by mass fraction, 0.03%-0.3% polyoxyethylene fatty acid ester, 0.5%-1.5% bovine serum albumin, 0.3%-2.0% raffinose, 0.5%-1.5% sodium hydroxide, 0.03%-1.0% NaN3 and 0.5%-1.0% sodium chloride, with the balance being purified water at pH 9-10.

[0014] Preferably, the sample processing solution contains, by mass fraction, 0.03%-0.3% polyoxyethylene fatty acid ester, 1% bovine serum albumin, 0.3%-2.0% raffinose, 1% sodium hydroxide, 0.05% NaN3 and 0.9% sodium chloride, with the balance being purified water at pH 9.5.

[0015] Preferably, the magnetic bead preservation solution contains, by volume fraction, 70%-80% phosphate buffer, 5%-15% fetal bovine serum, 5%-15% glycerol, 0.05%-0.15% Proclin 300 and 0.05%-0.15% Tween 20.

[0016] Specifically, the magnetic bead preservation solution contains, by volume fraction, 79.8% phosphate buffer, 10% fetal bovine serum, 10% glycerol, 0.1% Proclin 300 and 0.1% Tween 20.

[0017] Preferably, the acridinium ester preservation solution comprises 60%-80% phosphate buffer, 20%-40% glycerol and 0.05%-0.15% Proclin 300 by volume, and then BSA is added so that the mass fraction of BSA in the solution is 0.5%-1.5%.

[0018] Specifically, the acridinium ester preservation solution contains 69.9% phosphate buffer, 30% glycerol and 0.1% Proclin 300 by volume, and then BSA is added so that the mass fraction of BSA in the solution is 1.0%.

[0019] Preferably, the concentration of the phosphate buffer solution is 0.005-0.1M.

[0020] Preferably, the concentration of the phosphate buffer solution is 0.01M.

[0021] Preferably, the pH of the phosphate buffer solution is 7.0-8.0.

[0022] Specifically, the pH of the phosphate buffer solution is 7.4.

[0023] Preferably, the pre-activation solution contains 4-8 g / L hydrogen peroxide and 0.05-0.10 M nitric acid.

[0024] Specifically, the pre-activation solution contains 6 g / L hydrogen peroxide and 0.07 M nitric acid.

[0025] Preferably, the activating solution contains 0.5-1.5 mol / L sodium hydroxide and 0.3-3 g / L Triton X-100.

[0026] Specifically, the activation solution contains 1.0 mol / L sodium hydroxide and 1.0 g / L Triton X-100.

[0027] On the other hand, the present invention provides the application of the above-mentioned kit in in vitro sample detection, and the application is not a disease diagnosis or treatment application.

[0028] Preferably, the in vitro sample includes a body fluid sample.

[0029] Preferably, the body fluid sample includes serum or plasma.

[0030] On the other hand, the present invention provides a method for using the above-mentioned reagent kit.

[0031] Preferably, the method of use includes sample processing and detection using the magnetic beads, KL-6 antibody, acridine ester, magnetic bead preservation solution, acridine ester preservation solution, sample processing solution, pre-activation solution, or activation solution.

[0032] Preferably, the method of use includes the following steps:

[0033] Antibody-coated magnetic beads, acridine ester-labeled antibody, sample pretreatment, and detection of KL-6 content in the sample.

[0034] Preferably, the antibody-coated magnetic beads include the following steps:

[0035] (1) Add magnetic beads to a centrifuge tube, separate magnetically and remove the supernatant; add magnetic bead cleaning solution to the centrifuge tube, mix well, separate magnetically and remove the supernatant, and wash.

[0036] (2) Prepare MES (2-morpholinoethanesulfonic acid) buffer, EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) solution and NHS (N-hydroxysuccinimide) solution. Add EDC solution and NHS solution to centrifuge tubes, mix well and activate. After activation, wash with MES buffer and remove supernatant.

[0037] (3) Add KL-6 coated antibody to centrifuge tube, couple, magnetically separate and remove supernatant, add magnetic bead blocking solution for blocking, wash after blocking and remove supernatant;

[0038] (4) Add magnetic bead preservation solution to wash, remove supernatant, and then resuspend in magnetic bead preservation solution to obtain magnetic bead coated working solution.

[0039] Preferably, the acrid ester-labeled antibody comprises the following steps:

[0040] (1) Take KL-6 labeled antibody and add acridine ester at a molar ratio of antibody to acridine ester of 1:5-1:15. The final concentration of KL-6 labeled antibody is 1-3 mg / mL. React at 25℃ for 2-3 h.

[0041] (2) Add lysine at a molar ratio of acridine ester to lysine of 1:120-1:160 for blocking, and react for 15-30 min;

[0042] (3) After the blocking is completed, use a 40-60KD dialysis bag to replace the buffer solution. Dialyze 3-5 times in total, each time for 2-3 hours. After dialysis, add glycerol to the final concentration of KL-6 labeled antibody to 0.3-0.8mg / mL and set aside.

[0043] (4) When using, add acridinium ester preservation solution to make the final antibody concentration 6-10 μg / mL to obtain acridinium ester labeled antibody working solution.

[0044] Preferably, the sample pretreatment includes mixing the sample to be tested with the sample processing solution at a volume ratio of 1:15 to 1:25.

[0045] Specifically, the sample pretreatment includes mixing the sample to be tested with the sample processing solution at a volume ratio of 1:19.

[0046] Preferably, the detection of KL-6 content in the sample includes the following steps:

[0047] (1) Take the pretreated sample, add the magnetic bead coating working solution, mix well and incubate at 37°C for 3-10 min, then separate by magnetic separation, wash, remove the supernatant to obtain the magnetic bead-antigen complex.

[0048] (2) Add acridine ester labeled antibody working solution to the reaction cup containing magnetic bead-antigen complex, mix well and incubate at 37°C for 3-10 min, then perform magnetic separation, wash, remove supernatant to obtain magnetic bead-antigen-detection antibody complex;

[0049] (3) Add the pre-excitation solution and the excitation solution to the reaction cup containing the magnetic bead-antigen-detection antibody complex, mix well and then measure the maximum luminescence intensity;

[0050] (4) A standard curve is fitted based on the luminescence intensity detected by the standard and the concentration of the standard, and the KL-6 content in the sample to be tested is calculated through the standard curve.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The kit for detecting the salivary glycan antigen KL-6 provided by this invention comprises magnetic beads, KL-6 antibody, acridine ester, magnetic bead preservation solution, acridine ester preservation solution, sample processing solution, pre-activation solution, and activation solution; the sample processing solution contains, by mass fraction, 0.03%-0.3% polyoxyethylene fatty acid ester and 0.3%-2.0%...

[0053] Bovine serum albumin, 0.3%-2.0% raffinose, 0.3%-2.0% sodium hydroxide, 0.01%-1.5% NaN3 and 0.3%-1.5% sodium chloride, with the balance being purified water at pH 8-10; this kit can be used to determine the salivary glycoprotein KL-6 antigen in serum with high accuracy and strong anti-interference ability. Detailed Implementation

[0054] Unless otherwise specified, all raw materials used in this invention are commercially available products and their sources are not specifically limited.

[0055] Example 1: Preparation and Application of the Reagent Kit

[0056] (1) Preparation of basic solution

[0057] 1.1 Preparation of magnetic bead preservation solution: 79.8% 0.01M phosphate buffer, 10% fetal bovine serum, 10% glycerol, 0.1% Proclin 300 and 0.1% Tween 20 by volume fraction;

[0058] 1.2 Preparation of acridinium ester preservation solution: Prepare 69.9% by volume 0.01M phosphate buffer, 30% glycerol and 0.1% Proclin 300, and then add BSA to make the BSA mass fraction 1%.

[0059] 1.3 Sample processing solution preparation: by mass fraction, it consists of 0.1% polyoxyethylene fatty acid ester, 1% BSA, 1% raffinose, 1% sodium hydroxide, 0.05% NaN3 and 0.9% sodium chloride, with the remainder being purified water at pH 9.5.

[0060] 1.4 Preparation of pre-activation solution: Add 6 g / L hydrogen peroxide and 0.07 M nitric acid to purified water and mix well.

[0061] 1.5. Preparation of activation solution: Add 1.0 mol / L sodium hydroxide and 1.0 g / L Triton X-100 to purified water and mix well.

[0062] 1.6 Preparation of magnetic bead cleaning solution: Prepare 0.2M MES buffer and add 0.02% (v / v) Proclin300.

[0063] 1.7 Preparation of magnetic bead blocking solution: Prepare 0.01M phosphate buffer (99.4% by volume) and Tween 20 (0.6% by volume), then add BSA to make the BSA mass fraction 0.5%.

[0064] In the specific implementation, phosphate buffer refers to PBS buffer.

[0065] (2) Preparation of antibodies coated with magnetic beads

[0066] 2.1 Take 200 μL of magnetic beads (EM1-100 / 40 (high carboxyl) magnetic microspheres, catalog number 23710087) and add them to a 2 mL centrifuge tube. Perform magnetic separation for 3 min and then discard the supernatant.

[0067] 2.2 Add 400 μL of magnetic bead cleaning solution to the centrifuge tube, shake to mix, magnetically separate to remove the supernatant, and wash twice.

[0068] 2.3 Weigh a certain amount of EDC and add a certain volume of 0.02M MES buffer to prepare a 50mg / ml EDC solution; weigh a certain amount of NHS and add a certain volume of 0.02M MES buffer to prepare a 50mg / ml NHS solution.

[0069] 2.4 Add 100 μL of EDC solution and 100 μL of NHS solution to the centrifuge tube, and shake to mix.

[0070] 2.5 Place the two centrifuge tubes on a mixer and activate for 30 minutes. Adjust the speed appropriately to ensure the liquid flows steadily downwards when inverted.

[0071] 2.6 After activation, wash twice with 2 volumes of 0.02M MES buffer, remove the supernatant, and obtain the activated magnetic beads.

[0072] 2.7 Add 120 μg of KL-6 coated antibody (purchased from Nanjing Oukai Biotechnology, catalog number k72c8) to the activated magnetic beads, and adjust the volume to 300 μL with 0.01 M phosphate buffer. Couple for 3 h.

[0073] 2.8 Wash twice with 600 μL of magnetic bead blocking solution and remove the supernatant.

[0074] 2.9 Add 600 μL of magnetic bead blocking solution to the centrifuge tube, shake to mix for 30 min, and discard the supernatant.

[0075] 2.10 Wash twice with 600 μL of magnetic bead preservation solution, remove the supernatant, and then transfer to 30 mL of magnetic bead preservation solution to obtain the working solution of magnetic bead coating.

[0076] (3) Preparation of acridinium ester labeled antibody

[0077] 3.1 Take 200 μg of KL-6 labeled antibody (purchased from Shenzhen Youpin Biotechnology, catalog number YP100267) and dissolve it in 0.01M phosphate buffer. Add acridine ester (9 mg / mL NSP-SA-NHS, dissolved in DMSO), and add KL-6 labeled antibody to make the final concentration 2 mg / mL. The final molar ratio of KL-6 labeled antibody to acridine ester is 1:10.

[0078] 3.2. React at 25°C in the dark on a constant temperature shaker for 3 hours;

[0079] 3.3 Add lysine (acridinium ester: lysine = 1:140, molar concentration ratio), block and react in the dark for 20 min.

[0080] 3.4 After the reaction was completed, the buffer solution was replaced using a 50KD dialysis bag. The dialysis buffer was 0.01M phosphate buffer. Dialysis was performed 4 times in total, with each dialysis lasting 3 hours.

[0081] 3.5 After dialysis, add glycerol to the final antibody concentration of 0.5 mg / mL to obtain acridine ester labeled antibody solution.

[0082] 3.6 The acridine ester-labeled antibody solution was diluted with acridine ester preservation solution to a final antibody concentration of 8 μg / mL to obtain the working solution of acridine ester-labeled antibody.

[0083] (4) Sample preprocessing steps:

[0084] Mix 10 μL of the test sample with 190 μL of the sample processing solution (volume ratio 1:19) and mix well.

[0085] (5) Detection and calculation of KL-6 content in the sample to be tested

[0086] The detection was performed using a fully automated chemiluminescence analyzer, and the specific operation is as follows:

[0087] 5.1 Take 100 μL of the pretreated sample, add 50 μL of the magnetic bead coating working solution, mix well, incubate at 37℃ for 5 min, then perform magnetic separation, wash to remove unbound substances, and remove the supernatant to obtain the magnetic bead-antigen complex.

[0088] 5.2 Add 50 μL of acridine ester labeled antibody working solution to the reaction vessel containing the magnetic bead-antigen complex, mix well, incubate at 37°C for 5 min, then perform magnetic separation, wash to remove unbound substances, and remove the supernatant to obtain the magnetic bead-antigen-detection antibody complex.

[0089] 5.3 Add 100 μL of pre-excitation solution and 100 μL of excitation solution to the reaction vessel containing the magnetic bead-antigen-detection antibody complex, mix thoroughly, and then measure the maximum luminescence intensity.

[0090] 5.4. Based on the luminescence intensity and concentration of the standard sample, a standard curve is fitted, and the KL-6 content in the sample to be tested is calculated through the standard curve.

[0091] Example 2: Preparation and Application of the Reagent Kit

[0092] The procedure was carried out according to Example 1, except that the mass fraction of polyoxyethylene fatty acid ester in the sample processing solution was 0.03% and the mass fraction of raffinose was 2.0%.

[0093] Example 3: Preparation and Application of the Reagent Kit

[0094] The procedure was carried out according to Example 1, except that the mass fraction of polyoxyethylene fatty acid ester and raffinose in the sample processing solution was 0.3%.

[0095] Comparative Example 1: Preparation and Application of the Reagent Kit

[0096] The procedure was carried out according to Example 1, except that polyoxyethylene fatty acid esters were not added to the sample processing solution.

[0097] Comparative Example 2: Preparation and Application of the Reagent Kit

[0098] The procedure was carried out according to Example 1, except that raffinose was not added to the sample processing solution.

[0099] Comparative Example 3: Preparation and Application of the Reagent Kit

[0100] The procedure was carried out according to Example 1, except that polyoxyethylene fatty acid esters and raffinose were not added to the sample processing solution.

[0101] Comparative Example 4: Preparation and Application of the Reagent Kit

[0102] The procedure was carried out according to Example 1, except that the mass fraction of polyoxyethylene fatty acid ester in the sample processing solution was 0.4%.

[0103] Comparative Example 5: Preparation and Application of the Reagent Kit

[0104] The procedure was carried out according to Example 1, except that the mass fraction of raffinose in the sample processing solution was 3.0%.

[0105] Comparative Example 6: Preparation and Application of the Reagent Kit

[0106] The procedure was carried out according to Example 1, except that the polyoxyethylene fatty acid ester in the sample processing solution was replaced with an equal amount of Brij35.

[0107] Comparative Example 7: Preparation and Application of the Reagent Kit

[0108] The procedure was carried out according to Example 1, except that raffinose in the sample processing solution was replaced with an equal amount of sucrose.

[0109] Example 1: Validation of the reagent kit's detection efficacy

[0110] In this invention, KL-6 antigen (purchased from Nanjing Oukai Biotechnology, catalog number C1552) was used as a standard, and phosphate buffer containing 10% fetal bovine serum was used as a matrix solution. KL-6 antigen was diluted at a certain ratio to obtain a series of quality control products of different concentrations. The kits prepared in the examples and comparative examples were tested for the following items.

[0111] The luminescence intensity was measured using different quality control samples (50, 200, 500, 1500, 3000, and 6000 U / mL, respectively). A standard curve was plotted with the quality control sample concentration (U / mL) on the x-axis and the corresponding luminescence intensity (RLU) on the y-axis. The r values ​​of the standard curves obtained from different kits were analyzed. 2 All values ​​are greater than 0.98; the sample measured values ​​are calculated based on the luminescence intensity obtained from the sample measurements and the fitted standard curve.

[0112] Data analysis was performed using SPSS 21.0 software. Statistical analysis was conducted using t-tests, and P < 0.05 was considered statistically significant.

[0113] (1) Accuracy

[0114] Accuracy tests were performed on three quality control samples (500, 1000, and 3000 U / mL). The measurements were repeated three times and the average value was taken. The relative deviation from the theoretical concentration was calculated (relative deviation = [(measured average value - theoretical value) / theoretical value] × 100%). The results and analysis are shown in Table 1. It can be seen that the relative deviation of the measurements in Examples 1-3 of this invention is smaller and the accuracy is higher.

[0115] Table 1. Accuracy determination of different reagent kits

[0116]

[0117]

[0118] (2) Anti-interference capability

[0119] High blood lipids, high hemoglobin, and jaundice samples can affect test results, necessitating analysis of the kit's anti-interference capabilities. To examine the impact of high blood lipids on the kit, this example uses triglyceride samples of varying concentrations to assess their effect. Bilirubin, the substance causing jaundice, is orange-yellow in color. When the bilirubin concentration in the sample exceeds a certain value, it affects the sample's measurement results, thus impacting the test performance. To examine the impact of jaundice on the kit, this example uses bilirubin samples of varying concentrations to assess their effect. High concentrations of hemoglobin can affect antigen-antibody reactions, thus influencing chemiluminescence reactions. To examine the impact of high hemoglobin on the test results, this example uses hemoglobin samples of varying concentrations to assess their effect.

[0120] Experimental Materials and Methods

[0121] Blank clinical serum (with KL-6 removed) was used as the matrix solution. A certain mass of interfering substances (triglycerides, hemoglobin, and bilirubin) was added to the matrix solution to prepare basic samples with different concentrations of interfering substances. A certain amount of KL-6 antigen was dissolved in the matrix solution to prepare a high-concentration antigen solution. An equal volume of basic sample was added to an equal volume of high-concentration antigen solution to prepare interfering sample for detection. The specific interfering substance concentrations and theoretical antigen concentrations are shown in Table 2-4. Each concentration of interfering sample was tested three times consecutively, and the average value was taken (the CV value of the three measurements for different kits was less than 5%). The relative deviation from the theoretical antigen concentration was calculated (relative deviation = [(average measured value - theoretical value) / theoretical value] × 100%).

[0122] The test results are shown in Tables 2-4. It can be seen that the combined use of polyoxyethylene fatty acid ester and raffinose in the pretreatment solution provided by the present invention (Examples 1-3) significantly reduces the relative deviation under high concentration of interfering substances compared with the use of polyoxyethylene fatty acid ester (Comparative Example 1) and raffinose (Comparative Example 2) alone, and has a synergistic effect.

[0123] Table 2. Effects of different concentrations of triglycerides on sample detection.

[0124]

[0125] Table 3. Effects of different hemoglobin concentrations on sample detection

[0126]

[0127]

[0128] Table 4. Effects of different bilirubin concentrations on sample detection

[0129]

[0130] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A kit for detecting sialylated sugar chain antigen KL-6, characterized by, The kit comprises magnetic beads, KL-6 antibody, acridinium ester, magnetic bead storage solution, acridinium ester storage solution, sample processing solution, pre-priming solution and priming solution; the sample processing solution comprises, by mass fraction, 0.03%-0.3% polyoxyethylene fatty acid ester, 1% bovine serum albumin, 0.3%-2.0% raffinose, 1% sodium hydroxide, 0.05% NaN3 and 0.9% sodium chloride, with the balance being purified water and a pH of 9.

5.

2. The kit of claim 1, wherein The magnetic bead storage solution comprises, by volume fraction, 70%-80% phosphate buffer, 5%-15% calf serum, 5%-15% glycerol, 0.05%-0.15% Proclin 300 and 0.05%-0.15% Tween 20.

3. The kit of claim 2, wherein The magnetic bead storage solution comprises, by volume fraction, 79.8% phosphate buffer, 10% calf serum, 10% glycerol, 0.1% Proclin 300 and 0.1% Tween 20.

4. The kit of claim 1, wherein The acridinium ester storage solution comprises, by volume fraction, 60%-80% phosphate buffer, 20%-40% glycerol and 0.05%-0.15% Proclin 300, and further comprises, by mass fraction, 0.5%-1.5% BSA.

5. The kit of claim 4, wherein The acridinium ester storage solution comprises, by volume fraction, 69.9% phosphate buffer, 30% glycerol and 0.1% Proclin 300, and further comprises, by mass fraction, 1.0% BSA.

6. The kit according to any one of claims 2 to 5, characterized in that, The phosphate buffer has a concentration of 0.005-0.1M.

7. The kit of any one of claim 6, characterized in that, The phosphate buffer has a concentration of 0.01M.

8. The kit of claim 1, wherein The pre-priming solution comprises 4-8g / L hydrogen peroxide and 0.05-0.10M nitric acid.

9. The kit of claim 8, wherein The pre-priming solution comprises 6g / L hydrogen peroxide and 0.07M nitric acid.

10. The kit of claim 1, wherein The priming solution comprises 0.5-1.5mol / L sodium hydroxide and 0.3-3.0g / L Triton X-100.

11. The kit of claim 10, wherein The priming solution comprises 1.0mol / L sodium hydroxide and 1.0g / L Triton X-100.

12. Use of the kit according to any one of claims 1 to 11 for the detection of a sample in vitro, characterized in that, The application is a non-disease diagnosis or treatment application.

13. Use according to claim 12, characterized in that, The in-vitro sample comprises a body fluid sample.

14. Use according to claim 13, characterized in that, The body fluid sample comprises serum or plasma.

Images