Kit for detecting leukemia inhibitory factor and detection method

Abstract

The present disclosure relates to a kit and a detection method for detecting leukemia inhibitory factor. The kit includes: a first reagent containing a stationary phase coated with a first anti-human leukemia inhibitory factor (LIF) antibody; a second reagent containing a second anti-human LIF antibody with a label, wherein the second anti-human LIF antibody is different from the first anti-human LIF antibody; a luminescent substrate solution containing a luminescent substrate capable of reacting with the label to generate chemiluminescence; and an anti-interference agent containing mouse IgG and Tween 20. This kit can effectively eliminate interference in samples and obtain accurate results.

Description

Technical Field

[0001] The present disclosure relates to the field of blood analysis, and particularly to a kit for detecting leukemia inhibitory factor and a method for detecting leukemia inhibitory factor. Background Art

[0002] Human leukemia inhibitory factor (hLIF) is an interleukin-6 type cytokine with multiple biological activities and affects different cell types, and is a polypeptide with 202 amino acids. Similar to other members of the interleukin family, leukemia inhibitory factor (LIF) binds to the receptor complex containing GP130 and activates similar signal cascades, such as the phosphorylation pathway of STAT3. Existing studies have shown that high expression levels and high serum levels of LIF have a certain auxiliary role in the poor prognosis judgment of various cancers. In pancreatic ductal adenocarcinoma, LIF is a key paracrine factor that activates pancreatic stellate cells and activates STAT3 phosphorylation in cancer cells. Pharmacological LIF blockade and genetic receptor deletion can significantly slow down tumor progression and enhance the effectiveness of chemotherapy.

[0003] However, there is no commercially available immunological assay kit for detecting LIF.

[0004] Therefore, it is necessary to provide a kit and a method for accurately detecting LIF using immunological methods. Summary of the Invention

[0005] In view of this, the present disclosure aims to provide a kit and a method for detecting leukemia inhibitory factor. The kit can more accurately detect leukemia inhibitory factor using the immunocapture method, and can effectively resist interference, especially in the presence of interference by heterophilic antibodies (HA) and / or autoantibodies, to avoid false results or inaccurate results that are too high or too low.

[0006] The first aspect of the present disclosure provides a kit for detecting human leukemia inhibitory factor, wherein the kit includes:

[0007] A first reagent containing a stationary phase coated with a first anti-human leukemia inhibitory factor LIF antibody;

[0008] A second reagent containing a second anti-human LIF antibody with a label, wherein the second anti-human LIF antibody is different from the first anti-human LIF antibody;

[0009] A luminescent substrate solution containing a luminescent substrate capable of reacting with the label to produce chemiluminescence; and

[0010] An anti-interference agent containing mouse IgG and Tween 20.

[0011] According to one embodiment, the first reagent and the anti-interference agent are provided in the kit in a mixed form as a mixed reagent.

[0012] In the mixed reagent, the concentration of the murine IgG is 100-1000 μg / mL, and the concentration of the Tween 20 is 100-1000 μg / mL. Preferably, in the mixed reagent, the concentration of the murine IgG is 300-700 μg / mL, and the concentration of the Tween 20 is 300-700 μg / mL.

[0013] According to one embodiment, the stationary phase is superparamagnetic microparticles.

[0014] According to one embodiment, the label is selected from biotin, digoxin, alkaline phosphatase, horseradish peroxidase, and fluorescein. Preferably, the label is alkaline phosphatase.

[0015] According to a preferred embodiment, the label is alkaline phosphatase, and the chemiluminescent substrate is AMPPD.

[0016] The second aspect of the present disclosure provides a method for detecting human leukemia inhibitory factor LIF, comprising:

[0017] Mixing a certain amount of the first reagent, the anti-interference agent, the second reagent, and the sample uniformly to obtain a first reaction solution, and incubating for 3-10 minutes;

[0018] Separating the stationary phase;

[0019] Adding a certain amount of chemiluminescent substrate solution to the stationary phase and mixing uniformly to obtain a second reaction solution, and incubating for 10-20 minutes;

[0020] Detecting the number of photons generated in the second reaction solution, wherein the measured number of photons is proportional to the content of LIF in the sample.

[0021] Wherein the first reagent, the second reagent, the anti-interference agent, and the chemiluminescent substrate solution are respectively defined as above.

[0022] According to one embodiment, the method further comprises: comparing the measured number of photons with a standard curve to obtain the content of LIF in the sample.

[0023] The sample is serum or plasma.

[0024] The kit provided by the present disclosure includes two antibodies that can bind to different epitopes of human LIF, and can more accurately detect LIF in a sample through the double antibody sandwich method. In addition, the kit also includes an anti-interference agent, and the combination of mouse IgG and Tween 20 can well eliminate the influence of interfering substances in the sample on the detection, and has excellent effects especially on the interference of heterophilic antibodies and autoantibodies. Therefore, the kit can accurately detect LIF in samples of different subjects. Detailed Embodiments

[0025] The technical solutions of the present disclosure will be clearly and completely described below in conjunction with the embodiments and examples. Obviously, the specific embodiments described are only a part of the embodiments, rather than all of the embodiments. Based on these embodiments, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0026] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention belongs. In case of contradiction, this specification shall prevail.

[0027] In the text, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a method or product comprising a series of elements includes not only those elements expressly recited, but also other elements not expressly listed, or also elements inherent to the implementation of the method or product.

[0028] Unless otherwise specified, the singular forms "a / an" and "the" used herein include the plural of the nouns referred to.

[0029] Unless otherwise specified, the % used herein refers to mass percentage.

[0030] Based on the various physiological activities of leukemia inhibitory factor (LIF), it is expected that inhibiting LIF can produce anti-tumor effects. Therefore, in the research of therapeutic drugs for various diseases, especially for cancer, the research related to LIF is one of the hot research topics. The typical plasma or serum concentration of LIF in normal healthy individuals is between 0 - 10 pg / ml. However, since the clearance of the antibody / ligand complex is slower than that of the free ligand, once the cytokine binds to the antibody, the total LIF level in the plasma may increase. Accurately detecting the LIF present in the blood can assist in the research of related diseases and monitor the therapeutic effect.

[0031] The inventor detected LIF in numerous samples and found that the detection of LIF is susceptible to interference from contaminants in the samples, resulting in false results, or results that are significantly too high or too low, affecting clinical applications. Therefore, the inventor proposed a LIF detection kit capable of anti-interference. This kit uses a double-antibody sandwich immunoassay and utilizes a chemiluminescence system for detection, which can effectively resist interference and obtain accurate results.

[0032] According to the present disclosure, there is provided a kit for detecting human leukemia inhibitory factor. The kit includes: a first reagent containing a stationary phase coated with a first anti-human leukemia inhibitory factor (LIF) antibody; a second reagent containing a second anti-human LIF antibody with an enzyme label, wherein the second anti-human LIF antibody is different from the first anti-human LIF antibody; a luminescent substrate solution containing a luminescent substrate capable of reacting with the label to produce chemiluminescence; and an anti-interference agent containing murine IgG and Tween 20.

[0033] According to a preferred embodiment, the anti-interference agent is provided in the kit in the form of a mixed reagent with the first reagent.

[0034] The kit includes two anti-human LIF antibodies that can bind to different immunogenic sites or epitopes of LIF to form an antibody-LIF-antibody sandwich structure. Specifically, the first anti-human LIF antibody binds to the stationary phase for easy separation in subsequent steps; the second anti-human LIF antibody is linked to a label that can subsequently bind to the luminescent substrate in the luminescent substrate solution to produce chemiluminescence for easy detection. The kit is suitable for immuno-sandwich detection. First, the sample is mixed with the stationary phase coated with the first anti-human LIF antibody and the second anti-human LIF antibody with an enzyme label, and incubated at, for example, 37 °C for a certain period of time. The analyte LIF in the sample binds to the first anti-human LIF antibody on the stationary phase and the second anti-human LIF antibody with an enzyme label, forming an antibody-LIF-antibody sandwich structure. The stationary phase is separated from the reaction solution and the stationary phase is washed, so that only the second anti-human LI antibody bound to LIF is separated with the stationary phase, while the unreacted second anti-human LI antibody remains in the reaction solution. The separated stationary phase is mixed with the luminescent substrate solution for reaction (for example, incubated at 37 °C for a certain period of time), and by detecting the chemiluminescence signal and referring to the standard curve, the content of LIF in the sample can be measured. The principle of this method is well-known to those skilled in the art, however, this detection is susceptible to interference from various factors, such as triglycerides, bilirubin, hemoglobin, total protein, biotin, and antibodies, especially interference from heterophilic antibodies and autoantibodies. The kit effectively solves the above interference problem by further including an anti-interference agent.

[0035] According to the present disclosure, the anti-interference agent contains murine IgG and Tween 20. The inventors have found that adding murine IgG alone to the reaction solution of the sample and the antibody has a certain anti-interference effect, and to a certain extent, corrects the detection results of abnormal samples, but there is still a certain deviation from the accurate results. However, surprisingly, after further adding Tween 20, various common interferences can be almost completely eliminated, and accurate detection results can be obtained for abnormal samples, while Tween 20 itself has no anti-interference effect. In addition, other surfactants, even other series products of Tween, such as Tween 80, have a certain auxiliary anti-interference effect on some interfering substances when added together with murine IgG, but none of them have the comprehensive synergistic anti-interference effect of Tween 20. Thus, the inventors have proposed the above-mentioned kit capable of accurately detecting LIF in various blood samples.

[0036] The murine IgG may be mouse IgG. When the anti-interference agent is provided in a mixed form with the first reagent, the concentration of murine IgG in the mixed reagent is 100 - 1000 μg / mL, preferably 300 - 700 μg / mL, and most preferably 500 μg / mL.

[0037] When the anti-interference agent is provided in a mixed form with the first reagent, the concentration of Tween 20 in the mixed reagent is 100 - 1000 μg / mL, preferably 300 - 700 μg / mL, and most preferably 500 μg / mL.

[0038] When the anti-interference agent is used as a separate reagent, it may further contain necessary additives such as preservatives, buffers, and osmotic pressure regulators. The present disclosure has no particular limitation on each additive, and they can be added as needed. The preservative can be selected from the ProClin series, sodium azide, etc., but is not limited thereto. The buffer can be selected from phosphoric acid and its salts, citric acid and its salts, acetic acid and its salts, 2-(N-morpholino)ethanesulfonic acid, etc., but is not limited thereto. The osmotic pressure regulator can be a salt, such as NaCl, etc. The present disclosure has no particular limitation on the addition amounts of the preservative, buffer, osmotic pressure regulator, and bovine serum albumin in the first reagent. However, since it needs to be mixed and used with the first reagent and the second reagent, the addition amounts of each additive can be the same as those in the first reagent and the second reagent.

[0039] When the anti-interference agent is used as a separate reagent, the concentrations of murine IgG and Tween 20 can be appropriately increased according to different mixing ratios, and the present disclosure has no particular limitation on this.

[0040] The first reagent contains a first anti-human LIF antibody, wherein the first anti-human LIF antibody is coated on a stationary phase. The stationary phase can be magnetic beads, a streptavidin-coated well plate, etc. According to a preferred embodiment, the stationary phase is preferably magnetic beads, and more preferably superparamagnetic microparticles. The surface of the magnetic beads can be modified with functional groups for coupling with the first anti-human LIF antibody. The present disclosure has no particular limitation on the modified functional groups, which can be, for example, carboxyl groups, hydroxyl groups, amino groups, etc., but are not limited thereto, and among them, hydroxyl groups are more preferred. The particle size of the magnetic beads can be in the range of 0.5 - 4 μm, preferably 2 μm.

[0041] The present disclosure has no particular limitation on the first anti-human LIF antibody, and those known anti-human LIF antibodies can all be used in the present invention. Monoclonal antibodies are preferably used. For example, the first anti-human LIF antibody can be selected from Anti-LIF antibody, LIF monoclonal antibody, LIF antibody, etc.

[0042] In the first reagent, the content of magnetic beads coated with the first LIF antibody is 0.05 - 0.20%, preferably 0.10%.

[0043] The first reagent further includes necessary additives such as preservatives, buffers, osmotic pressure regulators, blocking agents (such as bovine serum albumin), etc. The present disclosure has no particular limitation on each additive, and they can be added as needed. The preservative can be selected from the ProClin series, sodium azide, etc., but is not limited thereto. The buffer can be selected from phosphoric acid and its salts, citric acid and its salts, acetic acid and its salts, 2-(N-morpholino)ethanesulfonic acid, etc., but is not limited thereto. The osmotic pressure regulator can be salts such as NaCl, etc. The present disclosure has no particular limitation on the addition amounts of the preservative, buffer, osmotic pressure regulator, and bovine serum albumin in the first reagent, and they can be added as needed. According to a specific embodiment, the first reagent includes the following components:

[0044] Magnetic microparticles coated with anti-human LIF antibody 0.05 - 0.20%;

[0045] Buffer 0.1 - 2%;

[0046] Preservative 0.05 - 0.5%; and

[0047] The balance of water to 100%.

[0048] Preferably, the first reagent includes the following components:

[0049] Superparamagnetic microparticles coated with anti-human LIF antibody 0.10%;

[0050] 2-(N-morpholino)ethanesulfonic acid (MES) 0.58%;

[0051] ProClin300 0.05%;

[0052] Sodium azide 0.09%; and

[0053] Water 99.18%.

[0054] The second reagent contains a second anti-human LIF antibody, wherein the second anti-human LIF antibody is different from the first anti-human LIF antibody. A label is conjugated to the second anti-human LIF antibody. The label can be any substance that can be detected by chemiluminescence. Examples of the label include, but are not limited to, biotin, digoxin, alkaline phosphatase, horseradish peroxidase, fluorescein, etc. Preferably, the label is alkaline phosphatase.

[0055] The present disclosure places no particular limitation on the second anti-human LIF antibody, and those anti-human LIF antibodies known in the art can be used in the present invention. Monoclonal antibodies are preferably used. The specific types of the second anti-human LIF antibody available for selection can be the same as those of the first anti-human LIF antibody, provided that the first and second anti-human LIF antibodies are different, so that they can bind to different antigenic epitopes of LIF respectively.

[0056] In the second reagent, the content of the second anti-human LIF antibody conjugated with a label is 0.005 - 0.02%, preferably 0.01%.

[0057] Similar to the first reagent, the second reagent also includes necessary additives such as preservatives and buffers, which will not be elaborated here.

[0058] According to a specific embodiment, the second reagent includes the following components:

[0059] Anti-human LIF antibody-label 0.005 - 0.02%;

[0060] Buffer 0.1 - 2%;

[0061] Preservative 0.05 - 1%; and

[0062] The balance of water to 100%.

[0063] The second reagent includes:

[0064] Anti-human LIF antibody-alkaline phosphatase label 0.01%

[0065] 2-(N-morpholino)ethanesulfonic acid (MES) 0.98%

[0066] ProClin300 0.05%;

[0067] Sodium azide 0.09%;

[0068] Water 98.87%.

[0069] The second reagent may also include necessary additives such as preservatives, buffers, osmotic pressure regulators, blocking agents (such as bovine serum albumin), etc. The types and amounts of these additives may be the same as those of the first reagent and will not be elaborated here.

[0070] The luminogenic substrate solution contains a luminogenic substrate that can react with the label to produce chemiluminescence. Corresponding to different labels, the kit includes luminogenic substrate solutions containing different chemiluminescent substrates. For example, corresponding to the alkaline phosphatase label, the luminogenic substrate solution may contain disodium p-nitrophenyl phosphate (PNPP), acridinium ester, luminol, isoluminol, AMPPD, tripropylamine, tetramethylbenzidine, and other enzyme-catalyzed chemiluminescent substrate solutions, etc., preferably AMPPD. When AMPPD is mixed with alkaline phosphatase, AMPPD is decomposed by alkaline phosphatase and a phosphate group is removed to generate an unstable intermediate product. This intermediate product generates methyl o-carboxybenzoate anion through intramolecular electron transfer. When the excited methyl o-carboxybenzoate anion returns from the excited state to the ground state, chemiluminescence is generated and used for detection.

[0071] The luminogenic substrate solution contains the luminogenic substrate of the luminescent label in the second reagent. The present disclosure has no particular limitation on the concentration of the luminogenic substrate in the luminogenic substrate solution. The luminogenic substrate can also be dissolved in a suitable solvent as long as the solvent does not interfere with the reaction and is easy to mix with other reagents. The kit can be used to detect human serum or plasma samples.

[0072] The above kit is used to detect LIF in a sample. The detection can be carried out on any suitable chemiluminescence detection device. The present disclosure has no particular limitation on the specific steps of the detection method, and appropriate detection steps can be selected according to specific circumstances. Exemplarily, the method for detecting LIF includes the following steps.

[0073] Mix a certain amount of the first reagent, anti-interference agent, and the second reagent, and add the sample and mix evenly to obtain a first reaction solution, and incubate at about 37 °C for 3 - 10 minutes; separate the stationary phase; add a certain amount of the luminogenic substrate solution to the stationary phase and mix evenly to obtain a second reaction solution, and incubate at about 25 °C for 10 - 20 minutes; and detect the number of photons generated in the second reaction solution, where the measured number of photons is proportional to the content of LIF in the sample.

[0074] Wherein the first reagent, the second reagent, the anti-interference agent, and the luminogenic substrate solution are as described above.

[0075] The method further includes the step of comparing the measured number of photons with a standard curve to obtain the content of LIF in the sample. The standard curve is usually the standard information (main curve information) built into the detection device.

[0076] According to a specific embodiment, the sample is serum or plasma.

[0077] Exemplarily, 50 μL each of a mixed reagent containing a first reagent and an anti-interference agent and a second reagent can be aspirated and added to a reaction cup. 10 μL of a sample to be tested (serum or plasma) and 350 μL of a cleaning solution are aspirated and added to the reaction cup and mixed well with the reagent. The mixture is incubated at about 37 °C for 5 minutes. The stationary phase (such as magnetic bead particles) is separated out, 100 μL of a substrate solution is added, and after mixing, it is incubated at about 25 °C for 15 minutes. The number of photons generated in the reaction is measured by a photomultiplier tube, and the number of photons generated is proportional to the concentration of LIF antigen in the sample. Further, by comparing with a standard curve previously stored in the detection device, the content of LIF in the sample to be tested can be obtained.

[0078] The present invention will be further described below through specific examples. Unless otherwise specified, the reagents are all commercially available conventional reagents.

[0079] Example

[0080] Example 1

[0081] Prepare a human LIF detection kit, which includes the following reagents:

[0082] Prepare a mixed reagent containing a first reagent and an anti-interference agent, and mix the following components to form a mixed reagent, and the concentration is the concentration after mixing:

[0083] Superparamagnetic particles coated with a first anti-human LIF antibody, wherein the first anti-human LIF antibody is a Milipore monoclonal antibody, the superparamagnetic particles are magnetic bead particles (Thermo Fisher), with a particle size of 2 μm, stored in Proclin-300 buffer, and added in appropriate amounts according to the instructions; mouse IgG with a concentration of 500 μg / mL, Tween 20 with a concentration of 500 μg / mL; sodium azide with a concentration of 1 g / L; MES buffer (pH = 6.0); 0.5 M NaCl; 0.5% bovine serum albumin (BSA).

[0084] Prepare a second reagent: mix the following components to prepare a second reagent, and the concentration is the concentration after mixing.

[0085] A second anti-human LIF antibody with an alkaline phosphatase label, wherein the second anti-human LIF antibody is an Invirogen monoclonal antibody, stored in Proclin-300 buffer, and added in appropriate amounts according to the instructions; containing sodium azide with a concentration of 1 g / L; MES buffer (pH = 6.0); 0.5 M NaCl; and 0.5% BSA.

[0086] Preparation of luminescent substrate solution: 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxetane (AMPPD).

[0087] Example 2

[0088] The kit was prepared in the same manner as in Example 1, except that the anti-interference agent contained 100 μg / mL of Tween 20.

[0089] Example 3

[0090] The kit was prepared in the same manner as in Example 1, except that the anti-interference agent contained 300 μg / mL of Tween 20.

[0091] Example 4

[0092] The kit was prepared in the same manner as in Example 1, except that the anti-interference agent contained 700 μg / mL of Tween 20.

[0093] Example 5

[0094] The kit was prepared in the same manner as in Example 1, except that the anti-interference agent contained 1000 μg / mL of Tween 20.

[0095] Comparative Example 1

[0096] The kit was prepared in the same manner as in Example 1, except that the anti-interference agent contained mouse IgG and did not contain Tween 20.

[0097] Comparative Example 2

[0098] The kit was prepared in the same manner as in Example 1, except that the anti-interference agent contained Tween 20 and did not contain mouse IgG.

[0099] Comparative Example 3

[0100] The kit was prepared in the same manner as in Example 1, except that the anti-interference agent replaced Tween 20 with Brij 35 at a concentration of 500 μg / mL.

[0101] Comparative Example 4

[0102] The kit was prepared in the same manner as in Example 1, except that the anti-interference agent replaced Tween 20 with CHEMAL at a concentration of 8 mg / mL.

[0103] Comparative Example 5

[0104] The kit was prepared in the same manner as in Example 1, except that the anti-interference agent replaced Tween 20 with Tween 80 at a concentration of 500 μg / mL.

[0105] Comparative Example 6

[0106] The kit was prepared in the same manner as in Example 1, except that Tween 20 in the anti-interference agent was replaced with Triton X-100 at a concentration of 500 μg / mL.

[0107] Comparative Example 7

[0108] The kit was prepared in the same manner as in Example 1, except that Tween 20 in the anti-interference agent was replaced with CHAPS at a concentration of 10 mg / mL.

[0109] Comparative Example 8

[0110] The kit was prepared in the same manner as in Example 1, except that Tween 20 in the anti-interference agent was replaced with betaine at a concentration of 500 μg / mL.

[0111] Comparative Example 9

[0112] The kit was prepared in the same manner as in Example 1, except that Tween 20 in the anti-interference agent was replaced with ammonium sulfate at a concentration of 0.1 mol / L.

[0113] Comparative Example 10

[0114] The kit was prepared in the same manner as in Example 1, except that Tween 20 in the anti-interference agent was replaced with sodium lauroyl sarcosinate at a concentration of 1 mg / mL.

[0115] Control Example

[0116] The kit was prepared in the same manner as in Example 1, except that the kit did not include an anti-interference agent.

[0117] Preparation of abnormal samples

[0118] Using the kit prepared in the control example, the dilution recovery rate of LIF in multiple human serum and EDTA-anticoagulated plasma samples was detected. Specifically, each serum and plasma sample contained different concentrations of LIF. The blood samples with different concentration ranges were diluted 10-fold with physiological saline and then detected using the above kit. The recovery rate was calculated as the ratio of the LIF concentration in the original sample to the LIF concentration measured after dilution (the ratio of the measured value after dilution × 10 to the measured value before dilution), as shown in Table 1 below. It can be seen that the dilution recovery rate of most samples was within 100% ± 15%, but there were individual samples with relatively high recovery rates, approaching or exceeding 200%.

[0119] Table 1: Dilution recovery rates of samples with different LIF concentrations

[0120]

[0121]

[0122] The serum-2 and plasma-4 samples with abnormal recovery rates were further subjected to PEG precipitation test and HBT incubation test respectively to determine the interfering substances therein. Specifically, for the PEG precipitation test, the sample was mixed with PEG solution (25% polyethylene glycol solution) at a ratio of 1:1, allowed to stand for several minutes, centrifuged at 3500 rpm for 10 min, and the supernatant was taken and retested with the above-mentioned kit. For the parallel control group, pure water was mixed with the sample at a ratio of 1:1 and the same operation was carried out. For the HBT incubation test, the sample (≤500 μL) was added to the HBT tube and incubated at room temperature for 30 - 60 min and then retested with the above-mentioned kit. The content of LIF in the original sample measured by the above-mentioned kit and the content of LIF in the test after PEG precipitation or HBT incubation are shown in Table 2 below, and the deviation between the two tests was calculated.

[0123] Table 2: Results of PEG precipitation and HBT incubation tests

[0124]

[0125] The PEG precipitation method was used to test samples with significantly high or low test results and poor dilution recovery rate. If the test deviation is large, it indicates the interference of autoantibodies and macromolecular proteins in the sample. And HBT is a heterophilic antibody blocker. Therefore, the HBT incubation method was used to test samples with significantly high test results and poor dilution recovery rate. If the test deviation is large, it indicates the interference of heterophilic antibodies in the sample. From the results in Table 2 above, it can be seen that there is obvious interference of autoantibodies in the serum-2 sample, and there is obvious interference of heterophilic antibodies in the plasma-4 sample, and there is also some interference of autoantibodies at the same time.

[0126] Test examples

[0127] Test example 1

[0128] The kits of Example 1, Comparative Example 1, Comparative Example 2 and Control Example were used to detect LIF in four samples of abnormal serum-2, abnormal plasma-4, normal serum-4 and normal plasma-8 respectively. The specific test method is as follows:

[0129] Mix 10 μL of the sample with 50 μL of the mixed reagent and 50 μL of the second reagent in the kit. The mixed reagent contains, in the control example: superparamagnetic microparticles coated with the first anti-human LIF antibody, a preservative, and MES buffer; in Comparative Example 1: superparamagnetic microparticles coated with the first anti-human LIF antibody, mouse IgG at a concentration of 500 μg / mL, a preservative, and MES buffer; in Comparative Example 2: superparamagnetic microparticles coated with the first anti-human LIF antibody, Tween 20 at a concentration of 500 μg / mL, a preservative, and MES buffer; in Example 1: superparamagnetic microparticles coated with the first anti-human LIF antibody, Tween 20 at a concentration of 500 μg / mL, mouse IgG at a concentration of 500 μg / mL, a preservative, and MES buffer. That is, except for mouse IgG and Tween 20, the other components and their contents are the same in the mixed reagents of each kit.

[0130] Add 350 μL of the washing solution and mix well, then incubate at 37 °C for 5 minutes. Separate the superparamagnetic microparticles, add 100 μL of the AMPPD substrate solution, mix well and incubate at 25 °C for 15 minutes; use a CL-2000i chemiluminescence instrument for detection, measure the number of photons generated in the reaction through the photomultiplier tube in the instrument, and calculate the LIF concentration in the sample according to the built-in master curve information (i.e., the standard curve information) in the instrument. The test results of each test and the results of calculating the test deviation based on the test results of the control example are shown in Table 3 below.

[0131] Table 3: Detection results and deviations of abnormal and normal samples using the kits of Example 1, Comparative Examples 1-2, and the control example

[0132]

[0133] As can be seen from the above table, the deviations of the test results of each kit for samples without interference (serum-4 and plasma-8) are very small, while there are significant differences in the deviations of the test results for abnormal samples with interference (serum-2 and plasma-4). Specifically, the kit of Comparative Example 1 containing only murine IgG has a certain anti-interference effect on abnormal samples, resulting in a relatively large deviation from the test results of the control samples. The kit of Comparative Example 2 containing only Tween 20 has a very weak anti-interference effect on abnormal samples, and the deviation from the test results of the control samples is very small (i.e., there is no obvious correction for abnormal results). In contrast, the kit of Example 1 containing an anti-interference agent of murine IgG and Tween 20 significantly eliminates different interferences, resulting in a deviation of more than -80%, even more than -90% from the test results of the control samples. This result is consistent with the dilution recovery rate shown in Table 1 above (about 200% for abnormal samples). Moreover, the test results of the kit of Example 1 for normal samples are basically the same as those of the kit of the control example, indicating that the anti-interference agent has no adverse effect on the test.

[0134] Test Example 2

[0135] To investigate the anti-interference effects of different combinations of surfactants and murine IgG, the kits of Examples 1 to 5 and Comparative Examples 3 to 10 were used to perform LIF detection on serum samples added with different interfering substances (3.5 g / dL triglyceride, 70 mg / dL bilirubin, 3.5 g / dL hemoglobin, 15 g / dL human total protein, 5 μg / mL biotin) according to the same test method as in Test Example 1. The results are shown in Table 4 below, where the sample without any interfering substance was used as the blank control.

[0136] Table 4: Test Results of the Kits of Examples 1 to 5 and Comparative Examples 3 to 10 for Samples with Added Interfering Substances

[0137]

[0138] As can be seen from Table 4 above, different combinations of surfactants and murine IgG all have a certain anti-interference effect on different interfering substances, but the anti-interference abilities for different interfering substances are not consistent. The anti-interference ability for a certain interfering substance is stronger, while that for another interfering substance is weaker. Tween 20 has good anti-interference effects on different interfering substances, so it can be used for samples in various situations to obtain accurate test results. In addition, different concentrations of Tween 20 combined with murine IgG all have a certain anti-interference effect on different interfering substances, and Tween 20 at 500 μg / mL has the best effect.

[0139] The above are only examples of some embodiments of the present invention, and do not limit the patent scope of the present invention. Any equivalent structural transformation made by using the content of the specification of the present invention under the inventive concept of the present invention, or any direct / indirect application in other related technical fields shall be included within the patent protection scope of the present invention.

Claims

1. A kit for detecting human leukemia inhibitory factor, wherein the kit comprises: A first reagent, the first reagent containing a stationary phase coated with a first anti-human leukemia inhibitory factor LIF antibody, the stationary phase being superparamagnetic microparticles; A second reagent, the second reagent containing a second anti-human LIF antibody with a label, wherein the second anti-human LIF antibody is different from the first anti-human LIF antibody; A luminescent substrate solution, wherein the luminescent substrate solution contains a luminescent substrate capable of reacting with the label to produce chemiluminescence; And An anti-interference agent, the anti-interference agent being mouse IgG and Tween 20, for anti-interference of heterophilic antibodies and / or autoantibodies, wherein the first reagent and the anti-interference agent are provided in the kit in a mixed form as a mixed reagent, and in the mixed reagent, the concentration of mouse IgG is 300 - 700 μg / mL, and the concentration of Tween 20 is 300 - 700 μg / mL.

2. The kit according to claim 1, wherein the label is selected from biotin, digoxin, alkaline phosphatase, horseradish peroxidase, and fluorescein.

3. The kit according to claim 1, wherein the label is alkaline phosphatase.

4. The kit according to claim 1, wherein the label is alkaline phosphatase, and the luminescent substrate is AMPPD.

5. A method for detecting human leukemia inhibitory factor LIF, comprising: Mixing a certain amount of the first reagent, the anti-interference agent, the second reagent, and a sample uniformly to obtain a first reaction solution, and incubating for 3 - 10 minutes; Separating the stationary phase; Adding a certain amount of the luminescent substrate solution to the stationary phase and mixing uniformly to obtain a second reaction solution, and incubating for 10 - 20 minutes; Detecting the number of photons generated in the second reaction solution, wherein the measured number of photons is proportional to the content of LIF in the sample, wherein the first reagent contains a stationary phase coated with a first anti-human leukemia inhibitory factor LIF antibody, the stationary phase being superparamagnetic microparticles; The second reagent contains a second anti-human LIF antibody with a label, wherein the second anti-human LIF antibody is different from the first anti-human LIF antibody; The anti-interference agent is mouse IgG and Tween 20, for anti-interference of heterophilic antibodies and / or autoantibodies; and The luminescent substrate solution contains a luminescent substrate capable of reacting with the label to produce chemiluminescence, wherein the first reagent and the anti-interference agent are used as a mixed reagent in a mixed form, and in the mixed reagent, the concentration of mouse IgG is 300 - 700 μg / mL, and the concentration of Tween 20 is 300 - 700 μg / mL.

6. The method according to claim 5, wherein the label is selected from biotin, digoxin, alkaline phosphatase, horseradish peroxidase, and fluorescein.

7. The method according to claim 5, wherein the label is alkaline phosphatase.

8. The method according to claim 5, wherein the label is alkaline phosphatase, and the luminescent substrate is AMPPD.

9. The method according to any one of claims 5-8, wherein the method further comprises: Comparing the measured number of photons with a standard curve to obtain the content of LIF in the sample.

10. The method according to any one of claims 5-8, wherein the sample is serum or plasma.

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