Kit and method for simultaneously detecting CEA, CA125 and CA153 based on flow cytometry

Abstract

The invention provides a kit and a method for simultaneously detecting CEA, CA125 and CA153 based on a flow cytometry, which are characterized in that proper microspheres and antibody markers are specially arranged for CEA, CA125 and CA153 antibodies, a 1.5-step incubation method and a biotin system are combined, and proper blocking agents and confining liquid are added into a reaction buffer solution, so that the CA153 in a sample is better combined with the microspheres and the antibody markers, and the detection accuracy is improved. According to the present invention, the cross influence of the CA153 on the CEA and the CA125 is reduced, such that the detection accuracy can be significantly improved when the three tumor markers such as the CEA, the CA125 and the CA153 are simultaneously detected based on the flow cytometry, the detection sensitivity of the CEA can achieve 0.3 ng / mL, the detection sensitivity of the CA125 can achieve 0.5 U / mL, and the detection sensitivity of the CA153 can achieve 0.5 U / mL so as to meet the clinical requirements.

Description

[0001] This application is a divisional application of parent case 2023110440986, with the application date of August 18, 2023. Technical Field

[0002] The present invention relates to the technical field of flow cytometry, and in particular to a kit and method for simultaneously detecting three tumor markers based on flow cytometry. Background Art

[0003] Tumor marker testing is a screening item that has been added to physical examinations in recent years. It detects tumor diseases by testing the body's response to cancer cells or substances released by cancer cells during tumor formation. The significance of tumor marker testing lies in the early detection of tumors; tumor surveys and screening; diagnosis, differential diagnosis and staging of tumors; monitoring of the efficacy of surgery, chemotherapy and radiotherapy for tumor patients; indicators of tumor recurrence; prognosis of tumors; and finding the primary lesions of metastatic tumors of unknown origin. Tumor markers used for clinical diagnosis include six major categories: carcinoembryonic antigens, enzymes, hormones, glycoproteins, oncogenes and cell surface tumor antigens. Among them, carcinoembryonic antigen (CEA), cancer antigen 125, cancer antigen 153, etc. can all be used for auxiliary diagnosis of tumors, such as for the detection of breast cancer.

[0004] Carcinoembryonic Antigen (CEA) is a complex acidic glycoprotein that mainly exists in adult cancer tissues and fetal gastrointestinal tissues. It is a broad-spectrum tumor marker.

[0005] Carbohydrate Antigen 125 (CA125) is a large molecular glycoprotein discovered by the monoclonal antibody OC125 prepared using the ovarian serous cystadenocarcinoma cell line (OVCA433) as an antigen. It exists in epithelial ovarian cancer tissue and is currently a commonly used tumor marker for detecting ovarian cancer in clinical practice.

[0006] Carbohydrate Antigen 153 (CA153) is a glycoprotein-specific antigen produced by tumor cells. After entering the blood, elevated CA153 levels can be detected in the blood of tumor patients. CA153 is the most important specific marker for breast cancer. 30%-50% of breast cancer patients have significantly elevated CA153 levels. The change in its content is closely related to the therapeutic effect. It is the best indicator for diagnosing and monitoring postoperative recurrence and observing therapeutic effects in breast cancer patients. Dynamic measurement of CA153 is helpful for early detection of recurrence after treatment of stage II and stage III breast cancer patients. When CA153 is greater than 100U / mL, it can be considered that there is metastatic lesion.

[0007] Since the same tumor can contain one or more markers, and different or different tissue types of the same tumor can have both common and different markers. Therefore, selecting some tumor markers with higher specificity to jointly measure a certain tumor is conducive to improving the sensitivity and specificity of tumor detection. A large number of studies have shown that the combined detection of multiple tumor marker proteins can improve the sensitivity and specificity of tumor diagnosis. For example, the combined detection of CEA, CA125, and CA153 can improve the diagnosis of breast cancer.

[0008] Flow cytometry can detect the level of tumor markers at the cellular level. By using flow cytometric microsphere technology and spherical matrix as a carrier and flow cytometry as a detection platform, large-scale detection of biological molecules can be performed in a relatively short period of time. This system is mainly based on flow fluorescence-encoded microspheres, which can achieve simultaneous detection of multiple targets and has a relatively broad application space. Therefore, it is expected that the three-item joint detection of CEA, CA125, and CA153 can be achieved through flow cytometry. However, the current multi-target joint detection based on flow cytometry still has the problem of low sensitivity.

[0009] Therefore, there is an urgent need to find a kit and a detection method based on flow cytometry that can simultaneously detect the three tumor markers CEA, CA125, and CA153 with high sensitivity and high accuracy, so as to avoid the above-mentioned problems existing in the prior art. Summary of the invention

[0010] In view of the problems existing in the prior art, the present invention provides a kit and method for simultaneously detecting three tumor markers based on flow cytometry, by setting appropriate microspheres and antibody markers specifically for CEA, CA125 and CA153 antibodies, combining a 1.5-step incubation method and a biotin system, and adding appropriate blocking agents and blocking liquids to the reaction buffer, so that CA153 in the sample can be better combined with the microspheres and antibody markers, and the cross-influence of CA153 on CEA and CA125 can be reduced, so that when the three tumor markers CEA, CA125 and CA153 are simultaneously detected by flow cytometry, the detection accuracy can be significantly improved, and the detection sensitivity of CEA reaches 0.3 ng / mL, the detection sensitivity of CA125 reaches 0.5 U / mL, and the detection sensitivity of CA153 reaches 0.5 U / mL, which meets clinical needs.

[0011] In one aspect, the present invention provides a kit for simultaneously detecting three tumor markers based on flow cytometry, the kit comprising:

[0012] The first solution: a microsphere solution coupled with an antibody, wherein the microsphere solution coupled with an antibody includes a microsphere solution coupled with a CEA antibody, a microsphere solution coupled with a CA125 antibody, and a microsphere solution coupled with a CA153 antibody; the particle size of the microsphere coupled with the CA153 antibody is larger than the particle size of the microsphere coupled with the CEA antibody and CA125; the microsphere coupled with the CA153 antibody has a carboxyl modified functional group; the microsphere coupled with the CEA antibody and CA125 has an amino modified functional group;

[0013] Second solution: a solution containing labeled CA153 antibody;

[0014] The third solution: a solution labeled with CEA antibody and a solution labeled with CA125 antibody;

[0015] Fourth solution: reaction buffer;

[0016] The three tumor markers include CEA, CA125 and CA153.

[0017] In some embodiments, the first solution, the second solution, the third solution, and the fourth solution are separately packaged and stored.

[0018] The present invention has been found through research that although the conventional flow cytometer technology can also realize the simultaneous detection of CEA, CA125 and CA153 to a certain extent, the detection sensitivity is usually very low, usually only reaching the level of CEA 2ng / mL, CA125 5U / mL, CA153 4U / mL, and there is a very serious cross-influence, which is prone to false positives; when CEA and CA125 are detected simultaneously, the sensitivity is significantly improved, which shows that the cross-influence may be mainly caused by CA153.

[0019] CA153 is a soluble fragment of mucin 1 (MUC-1) lost in the blood. Under normal circumstances, it is only expressed at the top of the plasma membrane, with normal or only a small amount of glycosylation. When cancer occurs, MUC-1 expression increases and polarity disappears, thus extending to the entire plasma membrane surface and even to the cytoplasm. At this time, glycosylation is serious, resulting in a decrease in detection accuracy. Therefore, even when tested alone, the requirements for the kit are relatively high. When CA153 and other tumor markers (such as CEA and CA125) are tested together based on flow cytometry, due to CA153, the higher the concentration of CA153 (the more serious the glycosylation), the greater the accuracy and precision of the test results (although CA125 also has glycosylation, its effect on the test results is not obvious). The performance indicators of the combined detection of the three markers are difficult to reach the performance indicators of the Roche kit for detecting a single tumor marker, thus limiting the preparation of multi-test kits for promotion and application. In breast cancer, the CA153 antigen is often abnormally glycosylated, overexpressed, and released into the blood circulation. The present invention speculates that it may be because CA153 is severely glycosylated, which may lead to shielding of antigen epitopes and difficulty in antibody binding. When CEA, CA125 and CA153 are tested jointly based on flow cytometry, it is difficult to ensure the accuracy of the test results.

[0020] Although CA125 also has glycosylation, a large number of experimental results have shown that its effect on the test results is not obvious and can basically be ignored.

[0021] Therefore, when simultaneously detecting CEA, CA125 and CA153 based on flow cytometry, in order to improve the detection sensitivity, both the detection reagents and the detection process must be specifically treated to eliminate the impact and improve the detection sensitivity.

[0022] Therefore, on the one hand, the present invention specifically selects and designs microspheres coupled to CEA antibodies, CA125 antibodies and CA153 antibodies, respectively, so that the particle size of the microspheres coupled to CA153 antibodies is larger than the particle size of the microspheres coupled to CEA antibodies and CA125, so that when detected by flow cytometry, they can be distinguished by the two aspects of microsphere particle size + different intensities of fluorescence carried by the microspheres, thereby improving the accuracy and sensitivity of detection.

[0023] The microspheres coupled with CEA, CA125 and CA153 antibodies have different fluorescence intensities, and can be distinguished by flow cytometry. However, due to the interference of highly glycosylated CA153 antibodies, the effect of distinguishing by fluorescence intensity is not ideal. Since flow cytometry can also distinguish microspheres of different sizes, the microspheres coupled with CA153 antibodies can be set to a different size from the microspheres coupled with CEA and CA125 antibodies, so that CA153 can be completely distinguished from CEA and CA125, and the accuracy of CA153 detection results can be improved.

[0024] At the same time, diluting the test sample can also eliminate the influence of CA153, but diluting the sample will cause the CEA and CA125 contents to decrease at the same time, thereby reducing the detection sensitivity of CEA and CA125. Therefore, it is necessary to select a suitable sample dilution, which can reduce the influence of CA153 and minimize the influence on the detection sensitivity of CEA and CA125. At the same time, it is also necessary to further optimize the detection reagents and process flow to improve the detection sensitivity. The present invention compares the functional groups coupled on the microspheres, and finds that the microspheres with carboxyl modified functional groups coupled to CA153 antibodies, and the microspheres with amino modified functional groups coupled to CEA antibodies and CA125 antibodies can significantly improve the sensitivity of simultaneous detection of CEA, CA125 and CA153, and reduce cross-effects.

[0025] Furthermore, the reaction buffer in the fourth solution contains a blocking agent, and the blocking agent includes mouse IgG, goat IgG, TRU-Block (purchased from meridian, product number A66800H), TRU-Block3 (purchased from meridian, product number A66803H), and MAK33 (purchased from Roche, model number 11939661103).

[0026] Adding blocking agents to the reaction buffer can effectively prevent the interference of endogenous antibodies in blood samples or other binding proteins in patient samples, polyreactive antibodies, autoantibodies (heterophilic antibodies) and human anti-animal antibodies, thereby reducing the interference of interfering factors on the test results and eliminating false positives.

[0027] However, it is difficult to achieve the ideal detection effect by adding a single blocker. In order to eliminate false positives and improve the accuracy of simultaneous detection of CEA, CA125 and CA153, a more suitable blocker formula must be selected. The present invention selects a combination of multiple blockers and combines them with mouse IgG and goat IgG to ensure more accurate detection of CEA, CA125 and CA153.

[0028] Furthermore, the coupled antibody microsphere solution in the first solution also includes a blocking solution, which includes disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, sodium chloride, ProClin300 buffer, BSA (purchased from Sigma, model A1933) and horse serum (purchased from Sigma, model 12449C).

[0029] Adding blocking solution to the coupled antibody microsphere solution can block some of the antibody binding sites on the microspheres, effectively controlling the antibody coating amount of the microspheres to achieve the optimal antibody amount, thereby improving the detection sensitivity of CEA, CA125 and CA153.

[0030] The invention screens the best blocking solution, controls the antibody coating amount of the microspheres at 85%, and significantly improves the detection sensitivity of CEA, CA125 and CA153.

[0031] In some embodiments, the kit provided by the present invention is not only applicable to blood samples, but also to samples such as urine, saliva, sputum, alveolar lavage fluid, pleural effusion, dialysate, vaginal secretions, and punctured tissue blocks.

[0032] Furthermore, the labeled CA153 antibody in the second solution is labeled with biotin; and the third solution also includes a labeled anti-biotin antibody.

[0033] Since CA153 is severely glycosylated and its content in blood samples is usually high, when CEA, CA125 and CA153 are tested together by flow cytometry, mutual interference is likely to occur, resulting in errors in the test results. The present invention uses biotin to label the labeled CA153 antibody, which can be better distinguished from the labeled CEA antibody and the labeled CA125 antibody, thereby helping to eliminate the interference of the CA153 antibody in the detection process of CEA and CA125, and can help effectively improve the detection accuracy.

[0034] Furthermore, the first solution also contains CA153 antibody.

[0035] In some embodiments, the mass ratio of the microsphere solution coupled to CA153 antibody, the microsphere solution coupled to CA125 antibody, the microsphere solution coupled to CEA antibody, and the CA153 antibody in the first solution is (1-4):1:1:1.

[0036] In a blood sample, once CA153 is contained, the content of CA153 is usually high. When a flow cytometer is used for detection, the false negative or weak positive phenomenon (i.e., HOOK effect) caused by the inappropriate ratio of antigen-antibody involved in the reaction causes the detection result to deviate from the detection result of Roche's single CA153 detection or there is a large deviation from the real CA153 content in the actual sample. In addition, the dilution sample needs to take into account the detection sensitivity of the two indicators of CEA and CA125. The present invention has been proved by research that by adding CA153 antibody to the first solution to neutralize part of CA153, while taking into account the performance of other indicators, it also plays the effect of diluting CA153 separately, so that the CA153 detection result in the blood sample can be closer to the true value (because the concentration of CA153 in the sample is high, the detection value is usually low, and adding a certain amount of CA153 antibody can make the detection result more accurate), which is closer to the detection result of Roche's single CA153 detection.

[0037] Furthermore, the labeled CEA antibody and the labeled CA125 antibody in the third solution are labeled with phycoerythrin; and the anti-biotin antibody is labeled with phycoerythrin.

[0038] In some embodiments, the mass ratio of the labeled CEA antibody solution, the labeled CA125 antibody solution and the labeled anti-biotin antibody solution is (1-10):(1-10):(2-10).

[0039] On the other hand, the present invention provides a pretreatment method for simultaneously detecting three tumor markers in a sample based on flow cytometry, wherein the method uses the above-mentioned kit for pretreatment, and comprises the following steps:

[0040] Step (1): taking a sample to be tested, adding the first solution, the second solution and the fourth solution respectively, and incubating;

[0041] Step (2): adding the third solution and incubating;

[0042] Step (3): Add cleaning solution for cleaning, centrifuge, and wait for detection.

[0043] Furthermore, the sample to be tested in step (1) needs to be diluted 10 to 30 times with a diluent, and the diluent includes tris(hydroxymethyl)aminomethane, sodium chloride, ProClin300 buffer and BSA; and the cleaning solution in step (3) includes BSA and Tween-20.

[0044] In another aspect, the present invention provides a method for simultaneously detecting three tumor markers based on flow cytometry, using the above-mentioned kit for detection, comprising the following steps:

[0045] (1) taking the diluted sample to be tested, adding the first solution, the second solution and the fourth solution respectively, and incubating;

[0046] (2) adding the third solution and incubating;

[0047] (3) Add cleaning solution for washing, centrifuge, and detect by flow cytometry.

[0048] The method for simultaneously detecting three tumor markers based on flow cytometry provided by the present invention requires a 1.5-step incubation method before the sample is detected by flow cytometry, so as to ensure the accuracy of the detection result.

[0049] The present invention has tried various incubation methods during sample pretreatment, including:

[0050] One-step incubation method: the first solution, the second solution, the third solution and the sample are all incubated together;

[0051] Two-step incubation method: the first solution, the second solution and the sample are first incubated, washed, and then the third solution is added, incubated, and washed;

[0052] 1.5-step incubation method: incubate the first solution, the second solution and the sample first, then add the third solution, incubate and wash.

[0053] The present invention has been proved through a large number of experimental studies that the 1.5-step incubation method can obtain better sample pretreatment effect and more accurate detection results. The reason may be that the 1.5-step incubation method can complete the incubation of CA153 in advance, reducing the cross-influence on CEA and CA125.

[0054] Furthermore, the sample to be tested in step (1) needs to be diluted 10 to 30 times with a diluent, and the diluent includes tris(hydroxymethyl)aminomethane, sodium chloride, ProClin300 buffer and BSA; and the cleaning solution in step (3) includes BSA and Tween-20.

[0055] By selecting the appropriate sample dilution multiple, we can reduce the impact of CA153 and minimize the impact on the detection sensitivity of CEA and CA125.

[0056] Furthermore, the incubation time is 30 minutes.

[0057] The present invention compares different cleaning solutions and screens out the most suitable cleaning solution formula, which is helpful to improve the detection effect.

[0058] Since CA153 needs to be diluted with sample diluent before testing, selecting an appropriate dilution factor can obtain more accurate test results while also ensuring the detection sensitivity of CEA and CA125.

[0059] On the other hand, the present invention provides a system for simultaneously detecting three tumor markers based on flow cytometry, the system comprising a marker detection module, a data input interface, a data output interface and a data analysis module; the marker detection module uses the above-mentioned kit, or adopts the above-mentioned method for detection to obtain the detection value of the marker, the data input interface is used to input the detection value of the three markers, and the data analysis module is used to analyze the detection value of the marker, the markers are CEA, CA125, and CA153; after analysis by the data analysis module, the data output interface is used to output the concentration of CEA, CA125, and CA153 in the sample to be tested.

[0060] On the other hand, the present invention provides a use of a blocking solution in preparing a preparation for improving the detection sensitivity of CEA, CA125 and CA153 detected simultaneously by flow cytometry, wherein the blocking solution comprises disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, sodium chloride, ProClin300 buffer, BSA and horse serum.

[0061] In another aspect, the present invention provides a use of a blocker for preparing a preparation for simultaneously detecting three tumor markers in a blood sample by flow cytometry and eliminating false positives, wherein the blocker comprises mouse IgG, goat IgG, TRU-Block, TRU-Block3 and MAK33, wherein the three tumor markers are CEA, CA125 and CA153.

[0062] In another aspect, the present invention provides a use of a blocking agent for preparing a preparation for simultaneously detecting three tumor markers by flow cytometry and eliminating false positives caused by interference of rheumatoid factor 1500U / mL and HAMA 2000IU / mL, wherein the blocking agent comprises mouse IgG, goat IgG, TRU-Block, TRU-Block3 and MAK33, wherein the three tumor markers are CEA, CA125 and CA153.

[0063] In yet another aspect, the present invention provides a use of biotin in preparing a preparation for improving the detection sensitivity of simultaneously detecting CEA, CA125 and CA153 based on flow cytometry.

[0064] In another aspect, the present invention provides a use of biotin in preparing a method for improving the accuracy and precision of CA153 detection.

[0065] The kit and detection method for simultaneously detecting three tumor markers, CEA, CA125 and CA153, based on flow cytometry constructed by the present invention have the following beneficial effects:

[0066] 1. Realized high-sensitivity detection of CEA, CA125 and CA153 based on flow cytometry, increasing the detection sensitivity of CEA from 2ng / mL to 0.3ng / mL, the detection sensitivity of CA125 from 5U / mL to 0.5U / mL, and the detection sensitivity of CA153 from 4U / mL to 0.5U / mL;

[0067] 2. Make the size of the microspheres coupled to CA153 antibodies different from that of the microspheres coupled to CEA and CA125 antibodies, so that the flow cytometer can completely distinguish CA153 from CEA and CA125 by the size of the microspheres, eliminate the interference of CA153 on CEA and CA125, and improve the accuracy of CA153 detection results;

[0068] 3. Select microspheres with different group modifications for CEA, CA125 and CA153 antibodies respectively. The microspheres coupled with CA153 antibodies have carboxyl modified functional groups, and the microspheres coupled with CEA antibodies and CA125 have amino modified functional groups, so as to improve the sensitivity of simultaneous detection of CEA, CA125 and CA153 based on flow cytometry;

[0069] 4. By adding blocking agents to the reaction buffer, the interference of interfering factors on the test results can be reduced, and the detection sensitivity and accuracy can be improved;

[0070] 5. Adding CA153 antibody to the first solution helps improve the accuracy of the test results of high-concentration samples, which is closer to the test results of Roche's single CA153 test;

[0071] 6. Add blocking solution to the antibody-coupled microsphere solution to effectively control the antibody coating amount of the microspheres and improve the detection sensitivity of CEA, CA125 and CA153;

[0072] 7. Use biotin to label CA153 antibody and improve the detection sensitivity and accuracy of CA153;

[0073] 8. Dilute the sample with sample diluent before testing and select the appropriate dilution multiple to obtain more accurate test results while ensuring the detection sensitivity of CA153, CEA and CA125;

[0074] 9. Use the 1.5-step incubation method for sample pretreatment to ensure the accuracy of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 Schematic diagram of the principle of the CEA protein detection process in Example 1, wherein 11 is a first fluorescent microsphere, 12 is a CEA antibody coupled to the microsphere, 13 is a sample to be tested, 14 is a CEA antibody, and 15 is phycoerythrin;

[0076] Figure 2 Schematic diagram of the principle of the CA153 protein detection process in Example 1, wherein 21 is a third fluorescent microsphere, 22 is a CA153 antibody coupled to the microsphere, 13 is a sample to be tested, 24 is a CA153 antibody, 25 is biotin, 15 is phycoerythrin, and 27 is an anti-biotin antibody;

[0077] Figure 3 is the linear regression graph of CEA in Example 1;

[0078] Figure 4 It is the linear regression graph of CA125 in Example 1;

[0079] Figure 5 It is the linear regression graph of CA153 in Example 1;

[0080] Figure 6 is a calibration curve diagram of CEA in Example 1;

[0081] Figure 7 It is the calibration curve diagram of CA125 in Example 1;

[0082] Figure 8 It is the calibration curve diagram of CA153 in Example 1;

[0083] Fig. 9 This is the correlation curve between the values ​​measured by this kit and the values ​​measured by Roche for CEA in Example 1;

[0084] Fig.10 The correlation curve between the CA125 measured by this kit and the Roche measured value in Example 1;

[0085] Fig.11 This is the correlation curve between the CA153 values ​​measured by this kit and the values ​​measured by Roche in Example 1. DETAILED DESCRIPTION

[0086] In order to describe the present invention more specifically, the technical solution of the present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. These descriptions are only to show how the present invention is implemented and cannot limit the specific scope of the present invention. The scope of the present invention is defined in the claims.

[0087] The materials and manufacturers used in the following examples are shown in Table 1.

[0088] Table 1. Materials and manufacturers

[0089]

[0090] Example 1: Kit and detection method provided by the present invention

[0091] 1. Preparation of solutions and calibrants

[0092] The kit provided in this embodiment for simultaneously detecting three tumor markers, CEA, CA125 and CA153, based on flow cytometry comprises a first solution, a second solution, a third solution, a fourth solution, and may further comprise a calibrator.

[0093] 1. Preparation of the first solution

[0094] Take 0.5mL of amino-modified polystyrene microsphere solution with a particle size of 4μm labeled with allophycocyanin (referred to as Y1 horizontal microspheres, purchased from Spherotech, model APAK-3567, fluorescence intensity of 580000), add 0.1mL of CEA antibody solution (CEA antibody coupled to microspheres, containing 100μg of CEA antibody, solvent is PBS), then add 100ugEDC, add 2mL of blocking solution, incubate for 2.5h, and prepare 1mL of microsphere solution coupled with CEA antibody, and control each mL of CEA antibody microsphere solution to contain 1×10 7 Fluorescent microspheres conjugated with CEA antibody.

[0095] Take 0.5mL of amino-modified polystyrene microsphere solution with a particle size of 4μm labeled with allophycocyanin (referred to as Y2 horizontal microspheres, purchased from Spherotech, model APAK-3567, fluorescence intensity of 61000), add 0.1mL of CA125 antibody solution (CA125 antibody coupled to microspheres, containing 100μg of CA125 antibody, solvent is PBS), then add 100ugEDC, add 2mL of blocking solution, incubate for 2.5h, and prepare 1mL of microsphere solution coupled with CA125 antibody, and control each mL of CA125 antibody microsphere solution to contain 1×10 7 Fluorescent microspheres conjugated with CA125 antibody.

[0096] Take 0.5mL of carboxyl-modified polystyrene microsphere solution with a particle size of 5μm labeled with allophycocyanin (referred to as L3 horizontal microspheres, purchased from Spherotech, model CPAK-5067, fluorescence intensity of 190000), add 0.1mL of CA153 antibody (CA153 antibody coupled to microspheres, containing 100μg of CA153 antibody, solvent is PBS) and then add 100ugEDC, add 3mL of blocking solution, incubate for 2.5h, and prepare 1mL of microsphere solution coupled with CA153 antibody, and control each mL of CA153 antibody microsphere solution to contain 1×10 7 Fluorescent microspheres conjugated with CA153 antibody.

[0097] 1 mL of a microsphere solution coupled to a CEA antibody, 1 mL of a microsphere solution coupled to a CA125 antibody, and 1 mL of a microsphere solution coupled to a CA153 antibody were respectively mixed, 197 mL of a reaction buffer and 5-15 μg of a CA153 antibody were added, and 200 mL of a first solution was prepared.

[0098] The preparation method of the blocking solution is as follows: take 36.32 g of disodium hydrogen phosphate dodecahydrate, 2.4 g of sodium dihydrogen phosphate, and 80 g of sodium chloride, add 10 mL of ProClin300 buffer, 25 g of BSA (purchased from Sigma, model A1933) and 3 mg of horse serum (purchased from Sigma, model 12449C), add distilled water to make up to 1000 mL, pH 6.9±0.1.

[0099] 2. Preparation of the second solution

[0100] Take 0.1 mL of CA153 antibody solution (containing 500 μg of CA153 antibody, solvent is PBS), add 5.5659 μL of biotin solution (containing 5.5659 μg of biotin, solvent is DMSO), mix well and incubate with shaking to obtain 0.1055 mL of biotin-labeled CA153 antibody solution, wherein the molar ratio of CA153 antibody to biotin is 3:1; dilute 200 times with diluent to obtain 21.11 mL of the second solution, which contains 8 μg / mL of CA153 antibody.

[0101] 3. Preparation of the third solution

[0102] Take 1 mL of CA125 antibody solution (containing 200 μg of CA125 antibody, solvent is PBS), add 0.5 mL of phycoerythrin solution (containing 600 μg of phycoerythrin, solvent is PBS), mix well and incubate with shaking to obtain 1.5 mL of labeled CA125 antibody solution.

[0103] Take 1 mL of CEA antibody solution (containing 200 μg of CEA antibody, solvent is PBS), add 0.5 mL of phycoerythrin solution (containing 600 μg of phycoerythrin, solvent is PBS), mix well and incubate with shaking to obtain 1.5 mL of labeled CEA antibody solution.

[0104] Take 1 mL of anti-biotin antibody solution (containing 200 μg of anti-biotin antibody, solvent is PBS), add 0.5 mL of phycoerythrin solution (containing 600 μg of phycoerythrin, solvent is PBS), mix well and incubate with shaking to obtain 1.5 mL of labeled anti-biotin antibody solution.

[0105] 1 mL of the labeled CA125 antibody solution, 1 mL of the labeled CEA antibody solution, and 1 mL of the labeled anti-biotin antibody solution were respectively mixed, and 197 mL of the reaction buffer was added to prepare 200 mL of the third solution.

[0106] 4. Preparation of the fourth solution

[0107] Reaction buffer: Take 6 g of tris(hydroxymethyl)aminomethane (Tris for short, purchased from Sigma), 1 mL of ProClin300 reagent (purchased from Sigma), 1 mL of Tween-20 reagent (purchased from Sigma), add 30 ug / mL mouse IgG (purchased from Aegim, model A10004) and 30 ug / mL goat IgG (purchased from LAMPIRE, model 7402505), add 40 ug / mL TRU-Block (purchased from meridian, model number A66800H), add 40 ug / mL TRU-Block3 (purchased from meridian, model number A66803H) and 40 ug / mL MAK33 blocker (purchased from Roche, model number 11939661103), and adjust the volume to 1000 ml after complete dissolution, adjust the pH to 8.0, and obtain reaction buffer.

[0108] 5. Dilution

[0109] Take 3.0275g of tris(hydroxymethyl)aminomethane (Tris) and 9.0g of sodium chloride, dissolve them in 800ml of pure water, add 1ml of ProClin300 buffer (sigma), adjust the pH value to 7.4, add 20g of bovine serum albumin (BSA), and adjust the volume to 1000ml after complete dissolution, adjust the pH value to 7.4, and obtain a diluted solution.

[0110] 6. Cleaning fluid

[0111] Take 0.24g of potassium dihydrogen phosphate, 3.632g of disodium hydrogen phosphate dodecahydrate, 8g of sodium chloride and 0.2g of potassium chloride, dissolve in 800ml of pure water, adjust the pH value to 7.4, add 2.5g of bovine serum albumin (BSA for short), 1ml of ProClin300 buffer and 0.5ml of Tween 20 solution, and after complete dissolution, make the volume to 1000ml, adjust the pH value to 7.4, and obtain a cleaning solution.

[0112] 7. Preparation of Calibrators

[0113] The CEA protein, CA125 protein, and CA153 protein calibrants were prepared into a mixed solution as a stock solution of the calibration working solution, and the calibrants were prepared with pure water to prepare the calibrants.

[0114] CEA protein, CA125 protein, and CA153 protein have 8 series of concentrations (S1 to S8) in the calibrators, as shown in Table 2:

[0115] Table 2. Eight concentration series of CEA, CA125, and CA153 proteins in calibrators

[0116] series CEA (ng / mL) CA125 (U / mL) CA153 (U / mL) S1 0.3 0.5 0.5 S2 1 10 5 S3 10 50 10 S4 20 100 20 S5 100 300 40 S6 280 1100 90 S7 550 2400 210 S8 1000 5000 300

[0117] Preparation of the kit: 2.0 ml of the first solution is loaded into the first packaging bottle, 2.0 ml of the second solution is loaded into the second packaging bottle, 2.0 ml of the third solution is loaded into the third packaging bottle, 50 ml of the fourth solution is loaded into the fourth packaging bottle, 100 ml of the cleaning solution is loaded into the fifth packaging bottle, and the calibrator is loaded into the sixth packaging bottle. The first packaging bottle, the second packaging bottle, the third packaging bottle, the fourth packaging bottle, the fifth packaging bottle, and the sixth packaging bottle are loaded into a packaging box to obtain a kit. The kit is 100 portions / box. The calibrator may or may not be loaded into the kit.

[0118] 2. Detection Method

[0119] The detection method of the kit for simultaneously detecting three tumor markers, CEA, CA125 and CA153, based on flow cytometry provided in this embodiment is as follows:

[0120] (1) Sample dilution: Take 5 μL of the blood sample (serum) to be tested, add 0.1 mL of diluent, mix well, and prepare a diluted sample;

[0121] (2) Take 25 μL of the diluted sample into a sample tube, add 25 μL of the first solution, 25 μL of the second solution, and 25 μL of the reaction buffer, place the sample tube in an oscillator and shake for more than 30 seconds, and incubate at room temperature in the dark for 30 minutes;

[0122] (2) Add 25 μL of the third solution, place the sample tube in an oscillator and shake for more than 30 seconds, and incubate at room temperature in the dark for 30 minutes;

[0123] (3) Add 1000 μL of cleaning solution, wash once, and resuspend the fluorescent microspheres by vortexing (must be fully mixed and shaken for more than 30 seconds); centrifuge at a centrifugal speed of 400 g for 5 minutes, and discard the supernatant; add 150 μL-300 μL of cleaning solution to the sample tube, resuspend the fluorescent microspheres by vortexing (must be fully mixed and shaken for more than 30 seconds), and finally place the sample tube into a flow cytometer (purchased from Beckman Coulter Biotech (Suzhou) Co., Ltd., model DxFLEX) for detection.

[0124] In the detection method provided in this embodiment, the principle of the detection process of CEA protein in the sample is as follows: Figure 1As shown, the microsphere-coupled CEA antibody 12 is loaded on the first fluorescent microsphere 11 , and the CEA antibody 14 is labeled with phycoerythrin 15 . The microsphere-coupled CEA antibody 12 first specifically binds to the sample to be tested 13 , and then specifically binds to the CEA antibody 14 labeled with phycoerythrin 15 .

[0125] The principle of the CA125 protein detection process in the sample is similar to that of the CEA protein detection process, wherein the fluorescent microspheres loaded with CA125 protein are the second fluorescent microspheres.

[0126] The principle of the CA153 protein detection process in the sample is as follows Figure 2 As shown, the CA153 antibody 22 coupled to the microspheres is loaded on the third fluorescent microspheres 21, and the CA153 antibody 24 is labeled with biotin 25. First, the CA153 antibody 22 coupled to the microspheres specifically binds to the sample 13 to be tested, and the CA153 antibody 24 labeled with biotin 25 binds to the anti-biotin antibody 27 labeled with phycoerythrin 15, and then the CA153 antibody 24 labeled with biotin 25 specifically binds to the sample 13 to be tested, and the cascade amplification of the CA153 signal is achieved through biotin 25.

[0127] 3. Analysis of test results

[0128] 1. Linear verification

[0129] The CEA sample containing 1000ng / mL, the CA125 sample containing 5000U / mL, and the CA153 sample containing 300U / mL were diluted into 8 samples of different concentrations according to Table 2. Each concentration was tested 3 times, and the mean of the test results was calculated. The linear regression equation was calculated with the dilution concentration as the independent variable and the mean of the test result as the dependent variable. The correlation coefficient of the linear regression was calculated. The linear regression equation is shown in Figure 3-Figure 5 ,in Figure 3 is the linear regression graph of CEA, Figure 4 is the linear regression graph of CA125, Figure 5 This is the linear regression graph of CA153.

[0130] Among them, the linear regression equation of CEA is y=0.9813x-0.128, R 2 =0.9989; the linear regression equation of CA125 is y=1.0118x-20.027, R 2 =0.998; the linear regression equation of CA153 is y=1.0374x-1.3699, R 2 =0.9988.

[0131] 2. Batch Difference Test

[0132] Three batches of test kits were taken, namely I, II and III, with 10 copies in each batch. According to the steps in the instructions, the same reference product was tested repeatedly, and the mean value M and standard deviation SD of the 30 measurement results were calculated, and the coefficient of variation CV was calculated. The test results of inter-batch differences are detailed in Table 3.

[0133] Table 3. Results of batch difference test

[0134] CEA (ng / mL) CA125 (U / mL) CA153 (U / mL) I-1 298.5 1098.5 197.5 I-2 304.58 989.5 196.5 I-3 300.46 996.5 193.6 I-4 32.56 997.65 197.56 I-5 305.45 998.56 199.6 I-6 306.54 1016.1 195.6 I-7 308.54 1018.2 198.6 I-8 302.41 1002.2 200.6 I-9 305.1 1012.45 204.6 I-10 301.2 996.5 199.3 II-1 287.5 989.2 205.6 II-2 297.36 1063.2 207.5 II-3 325.4 1034.1 206.5 II-4 316.45 1006.5 185.6 II-5 308.6 968.5 198.5 II-6 315.6 1063.2 195.4 II-7 316.9 1085.6 193.2 II-8 305.62 992.3 206.5 II-9 312.4 1032.1 204.6 II-10 319.6 1047.6 203.2 III-1 309.2 1039.4 206.5 III-2 289.3 1046.5 189.5 III-3 305.1 1046 199.5 III-4 332.5 1041.2 199.5 III-5 306.4 1037.2 196.5 III-6 311.6 1056.5 189.4 III-7 316.2 1036.2 207.5 III-8 308.5 997.5 205.6 III-9 306.7 997.2 202.1 III-10 307 995.6 198.6 M 304.109 1029.06 197.83 SD 20.52 37.17 9.1 CV 6％ 4％ 5％

[0135] As shown in Table 3, the coefficient of variation (CV) of the inter-batch differences of CEA, CA125 and CA153 of the three batches of kits were all within 10%.

[0136] 3. Preparation of calibration curve

[0137] Prepare the calibration curve samples according to Table 2, test each concentration 3 times, and calculate the mean of the test results. Use the calibration curve sample concentration as the independent variable and the fluorescence intensity as the dependent variable to prepare the calibration curve by logarithmic fitting. Calculate the correlation coefficient of the calibration curve. Figure 6-Figure 8 ,in Figure 6 is the calibration curve of CEA, Figure 7 is the calibration curve of CA125, Figure 8 The calibration curve of CA153 is shown in FIG.

[0138] Reference Figure 6-Figure 8 , Linear correlation R of CEA calibration curve 2 The linear correlation R of CA125 calibration curve was 0.9989. 2 The linear correlation R of CA153 calibration curve was 0.9993. 2 is 0.9996.

[0139] 4. Repeatability verification

[0140] The kit and the detection method provided in this embodiment were used to detect 2 reference samples at different concentration levels. Ten parallel tests were performed according to the reference sample test steps, and the coefficient of variation was calculated. The test results of repeatability are shown in Table 4.

[0141] Table 4. Repeatability verification

[0142]

[0143] It can be seen from Table 4 that the coefficients of variation of the reference substances at high and low concentration levels are all within 4%.

[0144] 5. Comparison with Roche test

[0145] The Roche test kit is a high-quality test kit for testing tumor markers in this field. However, the Roche test kit cannot detect the three tumor markers CEA, CA125 and CA153 simultaneously through a chemiluminescence analyzer, because the simultaneous detection of CEA, CA125 and CA153 is prone to cross-influence, and there are very few combined detection test kits on the market. The Roche test kit can only set up a test kit for CEA, CA125 and CA153 to detect the tumor marker CEA alone (model: Elecsys CEA; REF: 11731629322; LOT: (10) 62773003), a test kit for CA125 alone (model: Elecsys CA125Ⅱ; REF: 11776223190; LOT: (10) 61059703) and a test kit for CA153 alone (model: Elecsys CA153Ⅱ; REF:03045838122; LOT: (10)67129401).

[0146] For the same sample, the kit provided in this example and the Roche kit were used for detection, and the detection results are shown in Table 5.

[0147] Table 5. Comparison of test results between the kit provided in this example and the Roche kit

[0148]

[0149]

[0150]

[0151] The correlation curve between the values ​​measured by this kit and the values ​​measured by Roche is drawn using the values ​​in Table 5. Figures 9 to 11 ,in, Fig. 9 This is the correlation curve between the CEA values ​​measured by this kit and those measured by Roche; Fig.10 This is the correlation curve between the values ​​measured by this kit and the values ​​measured by Roche; Fig.11 This is the correlation curve between the values ​​measured by this kit and the values ​​measured by Roche.

[0152] according to Fig. 9 For the tumor marker CEA, the correlation between the values ​​measured by this kit and those measured by Roche is R 2 is 0.9961; according to Fig.10 For tumor marker CA125, the correlation between the values ​​measured by this kit and those measured by Roche is R 2 is 0.9946; according to Fig.11 For tumor marker CA153, the correlation between the values ​​measured by this kit and those measured by Roche is R 2 It is 0.9752.

[0153] It can be seen that the correlation between the measured values ​​of tumor markers CEA, CA125 and CA153 by the kit provided in this embodiment and the measured values ​​of tumor markers CEA, CA125 and CA153 by different kits of Roche are all above 0.9, indicating that the performance indicators of the kit provided by the present invention are very similar to those of the Roche kit, and the three tumor markers can be detected simultaneously with high sensitivity, thereby improving the detection efficiency.

[0154] 6. Comparison with mass spectrometry results

[0155] For the same sample, the kit and mass spectrometer provided in this embodiment were used for detection, the mass spectrometer (purchased from Shimadzu, model LC-MS / 8040), using 0.1% formic acid water and 0.1% formic acid acetonitrile as the mobile phase, using ESI ion source, nebulizing gas pressure (GS1): 45Psi, auxiliary gas pressure: 45Psi, air curtain pressure: 35Psi, temperature: 650°C, spray voltage: 5000V (positive ion mode), gradient elution, the detection results are shown in Table 6.

[0156] Table 6. Comparison of the kit and mass spectrometry results provided in this example

[0157]

[0158]

[0159] Example 2: Effect of Blockers on Improving Accuracy

[0160] When no blocker is added, there will be a more serious non-specific binding problem. In order to reduce the false positive or false negative test results caused by non-specific binding in the sample and improve the accuracy and specificity of the test. It is necessary to add a blocker to the reaction buffer. In this embodiment, the kit provided in Example 1 is used to add interfering substances (rheumatoid factor 1500IU / mL, HAMA 1000ng / mL) to samples of CEA, CA125 and CA153 close to the blank limit (PBS buffer without the three tumor markers), and the three tumor markers of CEA, CA125 and CA153 are detected at the same time. In the reaction buffer of the fourth solution, different blockers as shown below are used respectively: the effect of different blockers on the blank limit detection results is investigated, and each group is repeated three times. The test results are shown in Tables 7-1 and 7-2.

[0161] Table 7-1. Blocker interference detection results (blocker concentration according to Example 1)

[0162]

[0163] Note: or / means sometimes it cannot be detected.

[0164] Conclusion: As can be seen from Table 7-1, the test is inaccurate when the blocker is not added. After adding the blocker, the blank matrix sample value decreases, close to the Roche value, and consistent with the mass spectrometry value. After adding mouse IgG, goat IgG, TRU-Block, TRU-Block3 and MAK33, the effect is the best and the test result is the most accurate.

[0165] Compared with the blank control without adding blockers, adding blockers to the reaction buffer can effectively reduce the measured value of blank matrix samples and improve the detection accuracy of the three tumor markers CEA, CA125 and CA153; at the same time, the selection of different blockers will also directly affect the test results. The reason may be that different blockers have different blocking effects on different impurities. More importantly, different combinations of blockers can help eliminate false positives and eliminate the cross-influence caused by CA153 glycosylation in high-concentration blood samples. In order to minimize the impact of interfering factors in blood samples on CEA, CA125 and CA153, and minimize the cross-influence caused by CA153 glycosylation, and prevent false detection and missed detection, it is necessary to select appropriate blockers to improve the accuracy of the test results.

[0166] Compared with a single blocker, using different combinations of blockers can also improve the effect of eliminating false positives and improving accuracy, but not any combination of blockers can achieve the ideal effect of eliminating interference and false positives. This embodiment verifies the detection results of blockers in different combinations. The results show that for blank limit samples, the combination of mouse IgG+goat IgG+TRU-Block+TRU-Block3+MAK33 is used. When jointly detecting the blank limit of three tumor markers CEA, CA125 and CA153, it has the effect of significantly improving the accuracy of the detection results and eliminating false positives.

[0167] It should also be noted that for blank samples, it is sometimes difficult to detect when close to the minimum detection limit, and the result is " / ", and sometimes there will be a detection value.

[0168] This example also uses the kit provided in Example 1 to simultaneously detect three tumor markers, CEA, CA125 and CA153, in non-specific samples (blood samples), wherein different blocking agents as shown below were used in the reaction buffer of the fourth solution: The effects of different blocking agents on the test results were investigated, and each group was repeated three times. The test results are shown in Table 7-2.

[0169] Table 7-2. Effects of blocking agents on non-specific sample detection results

[0170]

[0171] Conclusion: As can be seen from Table 7-2, when the sample is tested directly without adding a blocker, the measured value is abnormally high. After adding the blocker, the sample value is reduced, close to the Roche value, and consistent with the mass spectrometry value. After adding mouse IgG, goat IgG, TRU-Block, TRU-Block3 and MAK33, the effect is best and the test result is most accurate. Compared with the samples in Table 7-1, blood has more interfering factors due to its complex composition. In addition to the above-mentioned rheumatoid factor 1500U / mL and HAMA 2000IU / mL, it may also contain other interfering factors.

[0172] Example 3: Effect of microspheres on detection sensitivity

[0173] 1. Influence of microsphere particle size

[0174] Since there is a significant cross-influence in the joint detection of CEA, CA125 and CA153, the reason may be caused by CA153. Therefore, this embodiment considers using different microspheres coupled with CA153 antibodies, and using flow cytometry to preferentially distinguish microspheres of different sizes, thereby increasing the discrimination of CEA, CA125 and CA153 and reducing cross-influence. This embodiment uses the following 4 methods to select microspheres:

[0175] 1) CEA antibody, CA125 antibody and CA153 antibody were all coupled with the same microspheres (particle size 4 μm, Y microspheres, coupled with carboxyl modified functional groups, purchased from Thermo, model FM4CR01B), and distinguished by the different intensities of fluorescence carried by the microspheres;

[0176] 2) CA153 antibody-coupled microspheres (particle size 5 μm, L microspheres, coupled with carboxyl-modified functional groups, purchased from Spherotech, model CPAK-5067), CA125 and CEA antibody-coupled microspheres (particle size 4 μm, Y microspheres, coupled with carboxyl-modified functional groups, purchased from Thermo, model FM4CR01B), which are distinguished by the particle size of the microspheres and the different intensities of fluorescence carried by the microspheres;

[0177] 3) CA125 antibody-coupled microspheres (particle size 5 μm, L microspheres, coupled with carboxyl-modified functional groups, purchased from Spherotech, model CPAK-5067), CA153 antibody and CEA antibody-coupled microspheres (particle size 4 μm, Y microspheres, coupled with carboxyl-modified functional groups, purchased from Thermo, model FM4CR01B), which are distinguished by the particle size of the microspheres and the different intensities of fluorescence carried by the microspheres;

[0178] 4) CEA antibody-coupled microspheres (particle size 5 μm, L microspheres, coupled with carboxyl-modified functional groups, purchased from Spherotech, model CPAK-5067), CA153 antibody and CA125 antibody-coupled microspheres (particle size 4 μm, Y microspheres, coupled with carboxyl-modified functional groups, purchased from Thermo, model FM4CR01B), which are distinguished by the particle size of the microspheres and the different intensities of fluorescence carried by the microspheres;

[0179] The low concentration calibrator (S3) was used for testing, and the test results are shown in Tables 8-1 and 8-2.

[0180] Table 8-1. Effect of microspheres

[0181]

[0182] Table 8-2, Effect of microspheres

[0183]

[0184] Note: The blank value (MFI) is the detection value of the blank matrix, and the signal-to-noise ratio is the ratio of the test value (MFI) to the blank value (MFI).

[0185] It can be seen from Tables 8-1 and 8-2 that, compared with the first method, when the second method is used, the use of microspheres with different particle sizes coupled to CA153 antibodies can significantly improve the signal-to-noise ratio of CA153 (the signal-to-noise ratio difference is large and easy to distinguish), significantly improve the detection sensitivity or specificity, and better distinguish CA153 from CEA and CA125, reduce cross-influence, and better ensure the accuracy of the test results.

[0186] When the third method is used to better distinguish CA125 from CEA and CA153, it cannot improve the signal-to-noise ratio and cannot reduce the cross-influence; when the fourth method is used to better distinguish CEA from CA125 and CA153, it cannot improve the signal-to-noise ratio and cannot reduce the cross-influence. It can be seen that when the three items are tested simultaneously, CA153 has obvious interference, which may be caused by the high glycosylation of CA153.

[0187] 2. Influence of microsphere coupling functional groups

[0188] Since the CA153 test sample needs to be diluted, the detection sensitivity of CEA and CA125 is reduced. It is necessary to optimize the detection sensitivity of CEA and CA125. According to the second method mentioned above, microspheres with corresponding particle sizes are selected, and then microspheres coupled with different functional groups are further selected to optimize the detection sensitivity of CEA and CA125. The coupling groups of CEA microspheres and CA125 microspheres are adjusted from carboxyl modification to amino modification (particle size 4 μm, purchased from Spherotech, model APAK-3567), and the coupling groups of CA153 microspheres are adjusted from carboxyl modification to hydroxyl modification functional group (particle size 5 μm, purchased from Sigma, model 15647). The calibration substance (S3) is used for detection, and the fluorescence values ​​of the detection results are shown in Table 9.

[0189] Table 9. Detection sensitivity of different microspheres

[0190]

[0191] From the fluorescence values ​​of CEA, CA125, and CA153 when different microspheres are used to detect the same sample, it can be seen that the functional groups modified by different microspheres will directly affect the signal-to-noise ratio; when CEA uses amino-modified microspheres, the signal-to-noise ratio is higher and the sensitivity is higher; when CA125 uses amino-modified microspheres, the signal-to-noise ratio is higher and the sensitivity is higher; when CA153 uses carboxyl-modified microspheres, the signal-to-noise ratio is higher and the sensitivity is higher. Therefore, CEA uses amino-modified microspheres, CA125 uses amino-modified microspheres, and CA153 uses carboxyl-modified microspheres.

[0192] Example 4: Biotin labeling improves the detection sensitivity of CA153

[0193] At the beginning of the present invention, CA153 antibody, CEA antibody and CA125 antibody were all labeled with phycoerythrin. It was found that there would be serious interference when CEA, CA125 and CA153 were detected simultaneously, especially in high-concentration blood samples, resulting in significantly low detection results of CA153. Therefore, biotin was tried to cascade amplify the CA153 antibody signal, thereby significantly improving the detection sensitivity of CA153. This embodiment adopts the above two different labeling methods, and detects high-concentration samples and low-concentration samples respectively according to the method provided in Example 1. Each sample is tested 5 times repeatedly, and the average value is taken. The test results are shown in Table 10.

[0194] Table 10. Effect of biotin labeling on test results

[0195]

[0196] As can be seen from Table 10, when testing low-concentration blood samples, the test results are not much different whether phycoerythrin is directly used for labeling or biotin is used for labeling; when testing high-concentration blood samples, the CA153 test result is significantly lower when phycoerythrin is used for labeling, which is less than half of the test result of the Roche kit. The reason may be that CA153 is severely glycosylated, which leads to serious cross-influence when three tests are performed, causing the test result to be seriously low; when biotin is used for cascade signal amplification, the accuracy of CA153 test results can be significantly improved. Therefore, it is preferred to use biotin to label CA153, so that the signal can be cascaded amplified, ensuring the accuracy of the test results and eliminating the influence of CA153 glycosylation.

[0197] Example 5: Effect of adding antibodies to microsphere solution

[0198] This example is tested according to the method provided in Example 1, wherein CA153 antibody is added to the first solution, and CA153 antibody is not added, and samples are used for testing. Each sample is tested 3 times, and the average value is taken to investigate the effect of adding CA153 antibody on the CA153 detection effect. Table 11-1 shows the concentration values ​​of the calibration curve and the corresponding fluorescence values, and Table 11-2 shows the effect of adding antibodies on the CA153 detection results.

[0199] Table 11-1. Calibration curve concentrations and fluorescence values

[0200]

[0201] Table 11-2. Effect of adding antibodies on CA153 test results

[0202]

[0203]

[0204] According to Table 11-2, adding antibodies can help improve the accuracy of high-concentration sample test results. The reason is that when the high-concentration glycosylated CA153 is wrapped, it is easy to be difficult to effectively detect. When a small amount of CA153 antibodies is added, part of the CA153 can be neutralized, which improves the problem of high-concentration glycosylated CA153 wrapping, and can increase the CA153 fluorescence signal value in the blood sample. Through the calibration curve equation, the test results are closer to the true value. It can be seen from Tables 11-1 and 11-2 that adding CA153 antibodies has no effect on the accuracy of the test results of low-concentration samples. The reason is that although the fluorescence signal value of the low concentration is reduced, the test results are still close to the accurate value through the standard curve equation.

[0205] Example 6: Effects of different blocking solutions on test results

[0206] In the process of coupling microspheres with antibodies, since there are remaining unoccupied binding sites on the surface of the microspheres, in order to prevent the binding of non-specific substances, it is necessary to add a blocking solution to the coupled antibody microsphere solution to block the remaining antibody binding sites on the microspheres, effectively control the binding of non-specific substances, and thus improve the detection sensitivity and specificity of CEA, CA125 and CA153. In this example, the same sample was tested for three tumor markers according to the method provided in Example 1, and 5 blocking solutions as shown in Table 12 were used respectively, and no blocking solution was added as a negative control. Each sample was tested 5 times, and the average value was taken to investigate the effect of different blocking solutions on the test results.

[0207] There are three methods for preparing homemade blocking solution:

[0208] 1) Containing horse serum: Take 36.32g of disodium hydrogen phosphate dodecahydrate, 2.4g of sodium dihydrogen phosphate and 80g of sodium chloride, dissolve in 800ml of pure water, add 10ml of ProClin300 buffer (sigma), adjust the pH value to 6.9, add 25g of bovine serum albumin (BSA for short), 2ml of horse serum, and make up to 1000ml after complete dissolution, adjust the pH value to 6.9, and obtain blocking solution.

[0209] 2) Without horse serum: Take 36.32g of disodium hydrogen phosphate dodecahydrate, 2.4g of sodium dihydrogen phosphate and 80g of sodium chloride, dissolve in 800ml of pure water, add 10ml of ProClin300 buffer (sigma), adjust the pH to 6.9, add 25g of bovine serum albumin (BSA), and adjust the volume to 1000ml after complete dissolution, adjust the pH to 6.9, and obtain the blocking solution.

[0210] 3) Containing goat serum (Biosharp, BL210A) but without horse serum: 36.32 g disodium hydrogen phosphate dodecahydrate, 2.4 g sodium dihydrogen phosphate and 80 g sodium chloride were dissolved in 800 ml pure water, and 10 ml ProClin300 buffer (Sigma) was added, and the pH value was adjusted to 6.9. 25 g bovine serum albumin (BSA) and 2 ml goat serum were added, and the volume was adjusted to 1000 ml after complete dissolution, and the pH value was adjusted to 6.9 to obtain a blocking solution.

[0211] CE210 is a blocking solution purchased from Beijing Bolma Biotechnology Co., Ltd., model CE210, and CE510 is a blocking solution purchased from Beijing Bolma Biotechnology Co., Ltd., model CE510.

[0212] Table 12. Effects of different blocking solutions on CA153 detection results

[0213]

[0214]

[0215] MFI: fluorescence value

[0216] As can be seen from Table 12, adding a blocking solution to the microsphere solution helps to improve the detection sensitivity and accuracy, because adding a blocking solution to the coupled antibody microsphere solution can block the remaining antibody binding sites on the microsphere, effectively control the binding of non-specific substances, and improve the detection sensitivity and accuracy. However, the effects of different blocking solutions are different, and whether the blocking solution contains horse serum also has a certain impact on the sensitivity of the test results. After calculation, when the homemade 1) blocking solution is used, the CA153 content in the blood sample is 36.2U / mL, the CEA content is 6.5ng / mL, and the CA125 content is 40.7U / mL, which is almost consistent with the Roche kit test results (the specific content is confirmed by mass spectrometry). When the homemade 1) blocking solution is not added, the test results are as follows: CA153 content is 10.2U / mL, CEA content is 1.5ng / mL, and CA125 content is 12.7U / mL (the specific experimental process is omitted). Compared with other blocking solutions, it can significantly improve the detection sensitivity and accuracy. Therefore, the most preferred blocking solution is homemade 1) blocking solution, which contains horse serum, which can make the test results more accurate and significantly improve the detection sensitivity of CA153, CEA, and CA125.

[0217] Example 7: 1.5-step detection process selection

[0218] In this example, according to the method provided in Example 1, in the process of simultaneously detecting three tumor markers based on flow cytometry, for S8 in the calibration series concentration, three treatment methods, namely, one-step incubation method, two-step incubation method and 1.5-step incubation method, were used for sample treatment.

[0219] The one-step incubation method refers to incubating the first solution, the second solution, the third solution and the sample together;

[0220] Two-step incubation method: refers to incubating the first solution, the second solution and the sample first, washing, and then adding the third solution, incubating, and washing, requiring two washes in total;

[0221] 1.5-step incubation method: incubate the first solution, the second solution and the sample first, then add the third solution, incubate and wash.

[0222] Then flow cytometry was performed to investigate the effects of different pretreatment methods on the detection results of CEA, CA125 and CA153.

[0223] The test results of the three treatment methods are shown in Table 13.

[0224] Table 13. Effects of different treatment methods on test results

[0225] Treatment CEA (ng / mL) CA125 (U / mL) CA153 (U / mL) One-step incubation 932.58 3155.68 209.18 Two-step incubation method 983.22 4965.57 253.36 1.5-step incubation method 998.65 4998.32 299.35

[0226] According to Table 13, when the one-step incubation method is used for detection, the test results are obviously biased low, there is obvious interference, and it is difficult to detect CEA, CA125 and CA153 at the same time; when the two-step incubation method and the 1.5-step incubation method are used, since the CA153 antibody is incubated first and incubated separately from the CEA antibody and CA125 antibody, the binding process of the CA153 antibody and CA153 is completed first, which has less impact on the binding process of CEA and CA125 with the antibody, thereby reducing cross-influence and interference, and the test results are more accurate.

[0227] Compared with the two-step incubation method and the 1.5-step incubation method, the two-step incubation method performs two washes, and the steps are more complicated, but the detection results are reduced. Therefore, the 1.5-step incubation method is most preferably used for processing.

[0228] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A kit for simultaneously detecting CEA, CA125, and CA153 based on flow cytometry, characterized in that: include: The first solution: an antibody-coupled microsphere solution, wherein the antibody-coupled microsphere solution includes a CEA antibody-coupled microsphere solution, a CA125 antibody-coupled microsphere solution, and a CA153 antibody-coupled microsphere solution; The microspheres coupled to the CA153 antibody have a carboxyl modified functional group; The microspheres coupled to CEA antibody and CA125 antibody have amino-modified functional groups; Second solution: a solution containing labeled CA153 antibody; The third solution: a solution labeled with CEA antibody and a solution labeled with CA125 antibody; The fourth solution includes a reaction buffer; The three tumor markers include CEA, CA125 and CA153.

2. The kit according to claim 1, characterized in that The particle size of the microspheres coupled to the CA153 antibody in the first solution is larger than the particle size of the microspheres coupled to the CEA antibody and the CA125 antibody.

3. The kit according to claim 1, characterized in that The first solution also includes a blocking solution, which includes disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, sodium chloride, ProClin 300 buffer, BSA and horse serum.

4. The kit according to claim 1, characterized in that The reaction buffer in the fourth solution contains a blocking agent, and the blocking agent includes mouse IgG, goat IgG, TRU-Block, TRU-Block3 and MAK33.

5. A method for preparing a kit for simultaneously detecting CEA, CA125, and CA153 based on flow cytometry, characterized in that: The following steps are involved: (1) Preparation of the first solution: preparing a microsphere solution coupled to CEA antibody, adding a blocking solution, and incubating; Prepare a microsphere solution coupled with CA125 antibody, add blocking solution, and incubate; Prepare a microsphere solution coupled with CA153 antibody, add blocking solution, and incubate; Mixing a microsphere solution coupled to CEA antibody, a microsphere solution coupled to CA125 antibody, and a microsphere solution coupled to CA153 antibody; (2) Preparation of the second solution: preparing a solution of labeled CA153 antibody; (3) preparing a third solution: preparing a solution labeled with CEA antibody and a solution labeled with CA125 antibody; (4) Preparation of the fourth solution: prepare a reaction buffer containing a blocking agent, wherein the blocking agent includes mouse IgG, goat IgG, TRU-Block, TRU-Block3 and MAK33.

6. The preparation method according to claim 5, characterized in that: The blocking solution in step (1) includes disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate, sodium chloride, ProClin 300 buffer, BSA and horse serum.

7. The preparation method according to claim 6, characterized in that: The microspheres coupled to the CA153 antibody in step (1) have carboxyl modified functional groups; the microspheres coupled to the CEA antibody and the CA125 antibody have amino modified functional groups.

8. The preparation method according to claim 7, characterized in that: The particle size of the microspheres coupled to the CA153 antibody in step (1) is larger than the particle size of the microspheres coupled to the CEA antibody and the CA125 antibody.

9. Use of a blocking agent in the preparation of a preparation for improving the detection sensitivity of a flow cytometer for simultaneously detecting three tumor markers in a blood sample, characterized in that: The blockers include mouse IgG, goat IgG, TRU-Block, TRU-Block3 and MAK33, wherein the three tumor markers are CEA, CA125 and CA153.

10. Use of a blocking agent in preparing a preparation for simultaneously detecting three tumor markers by flow cytometry and eliminating false positives caused by interference of rheumatoid factor 1500U / mL and HAMA 2000IU / mL, characterized in that: The blockers include mouse IgG, goat IgG, TRU-Block, TRU-Block3 and MAK33, wherein the three tumor markers are CEA, CA125 and CA153.

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