Dried blood spot tumor marker detection system, kit and use thereof

Abstract

Disclosed in the present invention are a dried blood spot tumor marker detection system, a kit and the use thereof. The system comprises the following modules: S1, a sample collection module, wherein the module is used for collecting a blood sample using a blood sample collection card to obtain a dried blood spot sample; S2, a tumor marker extraction module, wherein the module is used for extracting a tumor marker using an extraction buffer; specifically, the dried blood spot sample is placed into the extraction buffer and shaken, and the supernatant is collected to obtain a test solution; S3, a tumor marker detection module, wherein the module is used for detecting the tumor marker using a detection reagent; specifically, the test solution is brought into contact with the detection reagent, and detection is performed using a flow fluorescence analyzer to obtain a detected concentration; and S4, a data processing module, wherein the module is used for performing data processing on the detected concentration; specifically, the detected concentration is multiplied by a serum-to-dried blood spot correlation coefficient to obtain a final concentration. The system is particularly suitable for the quantitative analysis of multiple tumor markers in dried blood spots, with a high accuracy and a good repeatability of detection results.

Description

Dried blood spot tumor marker detection system, kit and application thereof

[0001] This application claims priority to Chinese Patent Application No. 2024108572939, filed on June 28, 2024. This application incorporates the entirety of the aforementioned Chinese Patent Application. TECHNICAL FIELD

[0002] The present application relates to a dried blood spot tumor marker detection system, kit and application thereof. BACKGROUND

[0003] Tumor markers refer to specific molecules or cells that can be detected in individuals with tumors, such as carcinoembryonic antigen (CEA), carcinoembryonic antigen (CA125), etc. They are mainly divided into serum markers, tissue markers, urine markers, etc. The presence or level of tumor markers can be used for early diagnosis of tumors, prognosis evaluation, disease monitoring, and treatment response monitoring, etc. With the development of liquid biopsy technology, it is possible to use markers in blood and other body fluids for tumor screening and monitoring.

[0004] The existing detection methods for tumor markers include serum marker detection, tissue marker detection, urine marker detection, saliva marker detection, and liquid biopsy. Among them, serum marker detection mainly collects blood samples from patients and uses biochemical analysis methods to detect the level of specific markers in serum to achieve the purpose of detection. The commonly used serum marker detection methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), etc. Serum marker detection has high accuracy and stability, but it needs to be based on traditional venous blood collection. Traditional blood collection has the characteristics of high invasiveness, large wound, high pollution risk, etc. Moreover, serum samples are not easy to store for a long time and need special cold chain for transportation, which is costly.

[0005] Dried blood spot technology (DBS) refers to dropping blood from the fingertips or heel of the subject on the relevant sample collection card, and naturally drying at room temperature to form a dried blood spot. Compared with traditional venous blood collection, dried blood spot technology has natural technical advantages, such as small invasiveness, minimal invasion, low pollution risk, etc. Dried blood spot samples have good stability, are easy to store for a long time, and do not need special cold chain for transportation. Dried blood spot preparation is relatively simple and does not require special operating skills, making it suitable for self-sampling.

[0006] However, there is no report on the application of the dried blood spot detection technology combined with the flow fluorescence immunoassay technology to detect tumor markers. This is mainly because in the prior art, when the dried blood spot detection technology is applied to detect tumor markers, especially multiple tumor markers, the accuracy, detection limit and repeatability of the detection results are difficult to achieve the effect of the detection technology based on serology due to the various components in the blood sample and the structural characteristics of different tumor markers, which limits the application of the dried blood spot technology in the field of tumor detection. SUMMARY

[0007] To overcome the above-mentioned defects existing in the prior art, the present application provides a dried blood spot tumor marker detection system, a kit and an application thereof. The system can realize quantitative analysis of tumor markers in dried blood spots (peripheral blood samples), especially simultaneous detection of multiple tumor markers, and the results obtained are equivalent to direct serum detection.

[0008] To achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0009] One of the technical solutions of the present application is: a dried blood spot tumor marker detection system, which comprises the following modules:

[0010] S1, a sample collection module: this module is used for collecting blood samples using a blood sample collection card to obtain dried blood spot samples;

[0011] S2, a tumor marker extraction module: this module is used for extracting tumor markers using an extraction buffer, placing the dried blood spot sample in the extraction buffer, shaking, and taking the supernatant to obtain a test solution; wherein the shaking time is 1-1.5h; the shaking rate is 300-1000rpm;

[0012] S3, a tumor marker detection module: this module is used for detecting tumor markers using a detection reagent, contacting the test solution with the detection reagent, and detecting using a flow fluorescence instrument to obtain a detected concentration;

[0013] S4, a data processing module: this module is used for data processing of the detected concentration, multiplying the detected concentration by a serum and dried blood spot correlation coefficient to obtain a final concentration; wherein the serum and dried blood spot correlation coefficient is obtained by a linear model algorithm and / or an average coefficient algorithm.

[0014] The application combines the dry blood spot technology and the flow fluorescence technology in tumor marker detection. The dry blood spot technology provides the convenience of collection, preservation and transportation which cannot be provided by the traditional sampling method. The flow fluorescence technology makes up for the defects of the dry blood spot technology, such as small sample requirement and only suitable for detection methods with high sensitivity. The flow fluorescence technology (also known as Lumine x xMAP technology and liquid suspension chip technology) is praised as "the real clinical application type biochip". It was certified by the FDA of the United States in 2001 and is the first and only multi-index detection technology platform approved by the FDA of the United States for clinical diagnosis. The technology can simultaneously detect multiple physiological and pathological indexes in the same reaction system, improving the detection efficiency. The ability of multi-index joint detection can detect dozens to hundreds of different analytes in a single experiment, significantly reducing the experimental time and cost. The flow fluorescence technology has small sample requirement and high sensitivity. The detection lower limit is extremely low, which can detect extremely low concentration of analytes, so it has significant advantages in early disease diagnosis and micro-substance analysis. The flow fluorescence technology has a wide linear range, which can cover from extremely low to extremely high concentration range, providing great flexibility for quantitative analysis. Meanwhile, it also has good accuracy and repeatability. This multi-marker detection method can effectively solve the problems of low specificity and low sensitivity in tumor marker detection, which is suitable for tumor marker detection, making the early screening and monitoring of tumors more accurate.

[0015] However, there are still many technical difficulties in combining the above three technologies from theory to practice. The inventors of the application have broken through the technical difficulties existing in the combination of the three technologies through continuous research. In the sample collection, the blood sample collection card is used, and the structure of the blood sample collection card is further optimized. In the extraction of tumor markers, the extraction conditions are optimized, and the above purposes of the application are finally achieved.

[0016] The sample collection module (module S1)

[0017] In some embodiments, in module S1, the blood sample collection card comprises a first board paper, an adsorption filter paper and a second board paper which are sequentially stacked from top to bottom, the upper surface of the second board paper is provided with an information collection area and a sample collection area, and the adsorption filter paper is adhered to the sample collection area.

[0018] The adsorption filter paper is provided with a collection hole, and the collection hole is provided with a collection paper piece, and the outer circumferential side of the collection paper piece is connected with the hole wall of the collection hole through a weak part which is easy to break.

[0019] The first board paper is provided with a first through hole corresponding to the collection hole, and the hole wall of the first through hole is located on the outer circumferential side of the hole wall of the collection hole.

[0020] In the present application, the blood sample collection card realizes quantitative and accurate collection of blood samples, especially peripheral blood samples, through the preferred structure form, and maximally avoids the problems of large deviation of tumor marker detection results and poor stability caused by operation differences of different collection methods and diffusion of effective components, and has excellent feasibility for single tumor marker detection or simultaneous detection of multiple tumor markers.

[0021] The above structure form is adopted, the collection hole is prefabricated on the adsorption filter paper, and the collection paper sheet is connected through the weak part which is easy to break. On the one hand, sampling is convenient, and the collection paper sheet can be taken off to realize sampling by using tweezers, without the need of a punch or scissors, thereby reducing sample loss caused in sample acquisition. On the other hand, the prefabricated collection paper sheet can effectively reduce the interference of the hematocrit problem. The prefabricated collection paper sheet can effectively block the diffusion in the serum component diffusion process in the blood, and limit the effective solute in the collection paper sheet. In addition, a layer of first board paper is stacked on the upper surface of the adsorption filter paper to prevent the user from directly touching the adsorption filter paper and causing pollution to the adsorption filter paper when using the blood sample collection card. The first board paper is provided with a first through hole corresponding to the position of the collection paper sheet, which can also remind the user of the position of the collection paper sheet, thereby improving the collection efficiency.

[0022] In some embodiments, in module S1, the number of collection holes is multiple, and the multiple collection holes are arranged at intervals along the length and / or width direction of the adsorption filter paper. By arranging multiple collection holes and corresponding collection paper sheets, the number of samples is increased.

[0023] In some embodiments, in module S1, the diameter of the collection hole is 0.4cm-1.6cm, and the diameter of the first through hole is 0.9cm-2.1cm.

[0024] In some embodiments, in module S1, the shape of the collection paper sheet and the collection hole is circular. The circular collection paper sheet and collection hole are convenient to manufacture.

[0025] In some embodiments, in module S1, the number of weak parts is multiple, and the multiple weak parts are uniformly arranged at intervals along the circumferential direction of the collection paper sheet. The connection points between the collection paper sheet and the weak part are uniformly located on the circumferential side of the collection paper sheet, which is convenient for tweezers to clamp the collection paper sheet.

[0026] In some embodiments, in module S1, the second sheet paper is provided with a second through hole corresponding to the collection hole, and the hole wall of the second through hole is located at the outer circumferential side of the hole wall of the collection hole. With the above structure, after blood is added on the collection paper sheet from the front surface of the second sheet paper, the penetration of the blood on the collection paper sheet can be observed through the second through hole to determine whether the blood collection amount on the collection paper sheet is appropriate, so that a suitable collection paper sheet can be selected as a sample for the next detection according to the penetration.

[0027] In some embodiments, in module S1, the blood sample collection card further comprises a cover, and the second sheet paper is provided with a lock structure configured to fix the cover to cover the sample collection area. With the above structure, after blood collection is completed, the cover is limited by cooperation between the cover and the lock structure, so that the cover can cover the collection paper sheet to prevent the collection paper sheet from being contaminated.

[0028] In some embodiments, in module S1, the end of the cover is connected to the end of the second sheet paper close to the sample collection area, and a folding line is provided between the cover and the second sheet paper.

[0029] The cover has a first position and a second position, and the cover is configured to support the second sheet paper when the cover is located at the first position, and the lock structure fixes the cover when the cover is flipped upward relative to the second sheet paper to the second position.

[0030] With the above structure, the cover is folded along the folding line to the front surface or the back surface of the second sheet paper. When the cover is in the first position, the cover is at an angle relative to the second sheet paper to support the second sheet paper and prevent the blood sample from overflowing and contacting the table top during the sample adding process. By flipping the cover upward to cover the collection paper sheet, the collection paper sheet can be prevented from being contaminated, which is convenient for operation.

[0031] In some embodiments, in module S1, the lock structure is located between the information collection area and the sample collection area, and the second sheet paper is cut to form a trapezoidal cutout as the lock structure, and the trapezoidal cutout is used for inserting or pulling out the cover. With the above structure, the trapezoidal cutout is directly provided on the second sheet paper, and the cover is inserted into the trapezoidal cutout to limit the cover, which is convenient for operation.

[0032] In some embodiments, in module S1, the number of lock structures is multiple, and the multiple lock structures are arranged at intervals along the length direction of the adsorption filter paper. The cover is limited by the multiple lock structures, and the limiting effect is good.

[0033] In some embodiments, in module S1, the blood loading amount of the dried blood spot sample is 10-40 μL per dried blood spot sample, for example, 20 μL per dried blood spot sample.

[0034] In some embodiments, in module S2, before placing the dried blood spot sample in the extraction buffer, the step further comprises a step of room temperature standing of the dried blood spot sample obtained in step S1; preferably, the time of the room temperature standing is 20 min.

[0035] Extraction module of tumor markers (module S2)

[0036] In the present application, in module S2, the extraction buffer can be a conventional extraction buffer for extracting tumor markers in the art.

[0037] In some embodiments, in module S2, the extraction buffer comprises the following components: 0.4%-0.6% (w / v) BSA, 1.5%-2.5% (v / v) donkey serum, 8%-12% (v / v) lowcross buffer, 0.08%-0.12% (v / v) Proclin 300 and PBS, pH 7.0-7.6.

[0038] In a specific embodiment, the extraction buffer comprises the following components: 0.4% (w / v) BSA, 1.5% (v / v) donkey serum, 8% (v / v) lowcross buffer, 0.08% (v / v) Proclin 300 and PBS, pH 7.4.

[0039] In a specific embodiment, the extraction buffer comprises the following components: 0.6% (w / v) BSA, 2.5% (v / v) donkey serum, 12% (v / v) lowcross buffer, 0.12% (v / v) Proclin 300 and PBS, pH 7.4.

[0040] In a specific embodiment, the extraction buffer comprises the following components: 0.5% (w / v) BSA, 2% (v / v) donkey serum, 10% (v / v) lowcross buffer, 0.1% (v / v) Proclin 300 and PBS, pH 7.4.

[0041] In some embodiments, in module S2, the amount of the extraction buffer used is 100 μL-1 mL per dried blood spot sample, preferably 200-500 μL per dried blood spot sample, for example, 225 μL per dried blood spot sample.

[0042] In some embodiments, in module S2, the time of shaking is 65 min, 70 min, 75 min, 80 min or 85 min.

[0043] In some embodiments, in module S2, the rate of shaking is 300-600 rpm, for example 500 rpm or 550 rpm.

[0044] In some embodiments, in module S2, the shaking is performed, for example, in a shaker or a mixer.

[0045] Module of detecting tumor markers (module S3)

[0046] In the present application, the tumor marker can be a single tumor marker or multiple tumor markers.

[0047] In some embodiments, the tumor marker is selected from one or more of the common lung cancer related tumor markers, for example, is selected from one or more of P53, PGP9.5, TM4SF1, SOX2, MAGEA4, ESO-1, FXR1, EFHD2, BRAF, GBU4-5, ZNF573, BMI1 and CAGE antigens or autoantibodies binding thereto.

[0048] In the present application, in module S3, the detection reagent can be a reagent conventionally used in the art for detecting the tumor marker as described above.

[0049] In some embodiments, the detection reagent comprises an autoantibody for antigen detection, or an antigen for autoantibody detection. When the tumor marker is an autoantibody, the detection reagent comprises an antigen for autoantibody detection; when the tumor marker is an antigen, the detection reagent comprises an autoantibody for antigen detection.

[0050] When the tumor marker is selected from one or more of P53, PGP9.5, TM4SF1, SOX2, MAGEA4, ESO-1, FXR1, EFHD2, BRAF, GBU4-5, ZNF573, BMI1 and CAGE antigens or autoantibodies binding thereto, the detection reagent is selected from any one of the following groups:

[0051] 1) ESO-1 antigen, TM4SF1 antigen, p53 antigen, BMI1 antigen, FXR1 antigen and EFHD2 antigen;

[0052] 2) ESO-1 antigen, TM4SF1 antigen, p53 antigen, BMI1 antigen, FXR1 antigen, EFHD2 antigen and BRAF antigen;

[0053] 3) ESO-1 antigen, TM4SF1 antigen, p53 antigen, BMI1 antigen, FXR1 antigen, EFHD2 antigen, BRAF antigen, and CAGE antigen;

[0054] 4) BRAF antigen, ESO-1 antigen, EFHD2 antigen, TM4SF1 antigen, ZNF573 antigen, BMI1 antigen, CAGE antigen, FXR1 antigen, PGP9.5 antigen, and P53 antigen;

[0055] 5) BRAF antigen, ESO-1 antigen, EFHD2 antigen, TM4SF1 antigen, ZNF573 antigen, BMI1 antigen, CAGE antigen, FXR1 antigen, PGP9.5 antigen, P53 antigen, and MAGEA4 antigen;

[0056] 6) BRAF antigen, ESO-1 antigen, EFHD2 antigen, TM4SF1 antigen, ZNF573 antigen, BMI1 antigen, CAGE antigen, FXR1 antigen, PGP9.5 antigen, P53 antigen, GBU4-5 antigen, MAGEA4 antigen, and SOX2 antigen.

[0057] When the tumor markers are selected from one or more of P53, PGP9.5, TM4SF1, SOX2, MAGEA4, ESO-1, FXR1, EFHD2, BRAF, GBU4-5, ZNF573, BMI1, and CAGE antigens, the detection reagents are selected from any one of the following groups:

[0058] 1) ESO-1 autoantibody, TM4SF1 autoantibody, p53 autoantibody, BMI1 autoantibody, FXR1 autoantibody, and EFHD2 autoantibody;

[0059] 2) ESO-1 autoantibody, TM4SF1 autoantibody, p53 autoantibody, BMI1 autoantibody, FXR1 autoantibody, EFHD2 autoantibody, and BRAF autoantibody;

[0060] 3) ESO-1 autoantibody, TM4SF1 autoantibody, p53 autoantibody, BMI1 autoantibody, FXR1 autoantibody, EFHD2 autoantibody, BRAF autoantibody, and CAGE autoantibody;

[0061] 4) BRAF autoantibody, ESO-1 autoantibody, EFHD2 autoantibody, TM4SF1 autoantibody, ZNF 573 autoantibody, BMI1 autoantibody, CAGE autoantibody, FXR1 autoantibody, PGP9.5 autoantibody, and P53 autoantibody;

[0062] 5) BRAF autoantibody, ESO-1 autoantibody, EFHD2 autoantibody, TM4SF1 autoantibody, ZNF 573 autoantibody, BMI1 autoantibody, CAGE autoantibody, FXR1 autoantibody, PGP9.5 autoantibody, P53 autoantibody, and MAGEA4 autoantibody;

[0063] 6) BRAF autoantibody, ESO-1 autoantibody, EFHD2 autoantibody, TM4SF1 autoantibody, ZNF 573 autoantibody, BMI1 autoantibody, CAGE autoantibody, FXR1 autoantibody, PGP9.5 autoantibody, P53 autoantibody, GBU4-5 autoantibody, MAGEA4 autoantibody, and SOX2 autoantibody.

[0064] In some embodiments, the antigens or autoantibodies are coupled to magnetic beads, forming coupled magnetic beads.

[0065] In some embodiments, the coupled magnetic beads are stored in the form of lyophilized powder, and are prepared into a lyophilized powder reconstitution solution as a detection reagent when used.

[0066] In some specific embodiments, 13 antigens are indirectly coupled to 13 different coded magnetic beads, respectively. When detection is performed on a Luminex multi-functional flow dot array instrument, the fluorescent coded magnetic beads are sequentially passed through two laser beams in a single column by a sheath flow system, and the fluorescent signals are received and analyzed by the instrument. One laser beam is used to identify the fluorescent code of the magnetic beads, so as to distinguish the categories of the detected autoantibodies, and the other laser beam is used to measure the fluorescent intensity on the magnetic beads, which is positively correlated with the concentration of the autoantibodies in the blood sample. Different concentrations of the prepared calibrators are respectively fitted into a dose-response standard curve according to the corresponding fluorescent signal intensity of the magnetic beads, so as to calculate the concentrations of various autoantibodies in the blood sample by the standard curve equation.

[0067] In some embodiments, in module S3, the reagents used in the detection process further include one or more of a calibrator diluent, a washing solution, an analysis buffer, an anti-human IgG secondary antibody, a calibrator, and a quality control product.

[0068] In some embodiments, the calibrator diluent is a phosphate buffer containing 1% (w / v) BSA, and preferably further contains a preservative.

[0069] In some embodiments, the washing solution is a PBST washing solution containing Tween20, and the pH is 7.4±0.05.

[0070] In some embodiments, the analysis buffer is a phosphate buffer containing 0.2% (w / v) BSA, and preferably further contains a preservative.

[0071] In some embodiments, the anti-human IgG secondary antibody is a RPE-labeled donkey anti-human IgG fluorescent antibody.

[0072] In some embodiments, the calibrator is a recombinant humanized Anti-Myc Tag immunoglobulin containing 1% (w / v) BSA and phosphate buffer; wherein the concentration of the Anti-Myc is preferably 3000 U / mL; wherein the calibrator preferably further contains a protective agent and / or a preservative; wherein the calibrator is preferably prepared as a freeze-dried product.

[0073] In some embodiments, the quality control includes a quality control I and a quality control II, the quality control I has a concentration of 200 U / mL Anti-Myc, 1% (w / v) BSA in phosphate buffer, and the quality control II has a concentration of 7.41 U / mL Anti-Myc, 1% (w / v) BSA in phosphate buffer; wherein the quality control I and the quality control II preferably further contain a protective agent and / or a preservative; wherein the quality control I and the quality control II are preferably prepared as freeze-dried products.

[0074] In some embodiments, the protective agent can be conventional in the art, such as trehalose and / or mannitol.

[0075] In some embodiments, the preservative can be conventional in the art, such as Proclin 300.

[0076] wherein the v / v is volume percentage, and the w / v is mass volume ratio.

[0077] In some embodiments, in module S3, the detection of the tumor marker includes the following steps:

[0078] (1) adding the detection reagent and the sample to be tested in the well plate, mixing, incubating, and washing;

[0079] (2) adding the anti-human IgG secondary antibody to the well plate obtained in step (1), mixing, incubating, and washing;

[0080] (3) adding the analysis buffer to the well plate obtained in step (2), mixing.

[0081] In some embodiments, in step (1), the volume ratio of the detection reagent to the sample to be tested is 1:1.

[0082] In some embodiments, in step (1) and in step (2), the mixing step is independently carried out by shaking, for example at a rate of 1000 rpm, for example for 3 min.

[0083] In some embodiments, in step (1) and in step (2), the incubation step is each independently a static incubation, for example at a temperature of 37°C, for a time of 90 min.

[0084] In some embodiments, in step (1) and in step (2), the well plate is for example a 96-well plate.

[0085] In some embodiments, in step (1) and in step (2), the washing step is each independently comprising the following steps:

[0086] 1) Place the well plate on a magnetic plate, let it stand, spin off the liquid in the well until no liquid drops from the well plate;

[0087] 2) Take the well plate off the magnetic plate, add the washing liquid in the well, mix;

[0088] 3) Repeat steps 1) and 2) for 2 times, spin off the liquid in the well until no liquid drops from the well plate.

[0089] In some embodiments, in step (3), the mixing step is performed by shaking, for example at a rate of 1000 rpm, for a time of 3 min.

[0090] Data processing module (module S4)

[0091] In some embodiments, in module S4, the detected concentration is multiplied by the dilution factor, and then multiplied by the serum-to-dry blood spot correlation coefficient of the tumor marker to obtain the final concentration.

[0092] In some embodiments, in modules S2 and S3, when the tumor marker is selected from P53, PGP9.5, TM4SF1, SOX2, MAGEA4, ESO-1, FXR1, EFHD2, BRAF, GBU4-5, ZNF573, BMI1 and CAGE antigen or autoantibodies binding thereto, in module S4, the serum-to-dry blood spot correlation coefficient of the P53, PGP9.5, TM4SF1, SOX2, MAGEA4, ESO-1, FXR1, EFHD2, BRAF, GBU4-5, ZNF573, BMI1 and CAGE antigen or autoantibodies binding thereto is obtained by a linear model algorithm.

[0093] In some embodiments, in the modules S2 and S3, when the tumor markers are selected from P53, PGP9.5, TM4SF1, SOX2, MAGEA4, ESO-1, FXR1, EFHD2, BRAF, GBU4-5, ZNF573, BMI1 and CAGE antigen or autoantibodies binding thereto, in the module S4, the serum-dry blood spot correlation coefficients of the P53, PGP9.5, TM4SF1, SOX2, MAGEA4, ESO-1, EFHD2, BRAF, GBU4-5 and CAGE antigen or autoantibodies binding thereto are obtained by a linear model algorithm; the serum-dry blood spot correlation coefficients of the BMI1, FXR1 and ZNF573 antigen or autoantibodies binding thereto are obtained by an average coefficient algorithm.

[0094] Diagnostic value calculation module (module S5)

[0095] In some embodiments, the dry blood spot tumor marker detection system further comprises the following modules:

[0096] S5, diagnostic value calculation module: this module is used to calculate the final concentration obtained by the module S4 by a 5p-Logistics regression model to obtain a diagnostic value.

[0097] The second technical solution of the present application is a kit, which comprises a blood sample collection card, an extraction buffer of tumor markers and a detection reagent of tumor markers.

[0098] In some embodiments, the blood sample collection card in the kit according to the second technical solution of the present application is as defined above.

[0099] In some embodiments, the tumor markers in the kit according to the second technical solution of the present application are as defined above.

[0100] In some embodiments, the extraction buffer in the kit according to the second technical solution of the present application is as defined above.

[0101] In some embodiments, the detection reagent in the kit according to the second technical solution of the present application is as defined above.

[0102] In some embodiments, the kit according to the second technical solution of the present application further comprises one or more of a calibrator diluent, a washing solution, an analysis buffer, an anti-human IgG secondary antibody, a calibrator and a quality control product. The calibrator diluent, the washing solution, the analysis buffer, the anti-human IgG secondary antibody, the calibrator and the quality control product are as defined above.

[0103] In some embodiments, the kit according to the second technical solution of the present application further comprises a 96-well plate.

[0104] The third technical solution of the present application is the use of the kit of the second technical solution of the present application in the preparation of a reagent for detecting a tumor marker.

[0105] On the basis of common knowledge in the art, the above-mentioned preferred conditions can be combined in any manner to obtain various preferred embodiments of the present application.

[0106] The reagents and raw materials used in the present application are commercially available.

[0107] The positive progress effect of the present application is that:

[0108] The detection system of the present application combines dry blood spot detection technology and flow cytometry, and is applied to tumor marker detection, especially suitable for simultaneous quantitative analysis of multiple tumor markers in dry blood spots. The results obtained are comparable to direct serum detection, and quantitative analysis of tumor markers can be achieved with only a small amount of sample, with low detection limit, high accuracy, good repeatability, and simpler operation.

[0109] More specifically, compared with other immunological detection techniques, the detection system of the present application based on dry blood spots combined with flow cytometry also has the following advantages:

[0110] 1. Dry blood spot sample collection is simple and safer, and only a few drops of blood are needed to prepare dry blood spots, avoiding the pain and inconvenience of venipuncture. It is more friendly to the elderly or people who are afraid of venipuncture, reduces the psychological stress and discomfort of patients, and can improve the participation and cooperation of patients.

[0111] 2. Dry blood spot samples have strong stability and can be stored at room temperature for several weeks to several months, which is convenient for long-distance transportation and long-term storage.

[0112] 3. The content of biomarkers in dry blood spot samples is relatively stable and less affected by external factors, making the sample analysis more accurate.

[0113] 4. When the detection system of the present application is used for dry blood spot detection, the detection limit is lower, and the minimum limit for protein detection can reach 0.1 pg / mL. The analysis result is the median value of 100 repeated units for each detection index, which is more accurate. It uses a combination of multiple tumor markers (such as 13 kinds), and combines AI algorithm analysis, which has higher sensitivity and specificity. Moreover, 13 markers in one reaction well only need one sample to be tested, which can realize the joint detection of multiple markers, greatly saving the sample amount. Moreover, the system has high-throughput detection advantage when used for dry blood spot detection, multiple indexes can be parallelly detected, and is suitable for joint high-throughput detection of multiple people and multiple autoantibodies, such as one 96-well plate can detect 80 people samples for 13 indexes.

[0114] 5、The filter paper and other materials used in the blood spot sample collection card are easy to handle and recycle, reducing environmental pollution;

[0115] 6、The detection system is used for detecting the dry blood spot, and has faster detection speed, higher detection sensitivity and better repeatability. BRIEF DESCRIPTION OF DRAWINGS

[0116] Fig. 1 is a structural schematic diagram of a blood sample collection card according to an embodiment of the present application.

[0117] Fig. 2 is a structural schematic diagram of another form of a blood sample collection card according to an embodiment of the present application.

[0118] Fig. 3 is a structural schematic diagram of a blood sample collection card according to an embodiment of the present application, in which a cover is in a second position.

[0119] Fig. 4 is a top view of a blood sample collection card according to an embodiment of the present application, in which a cover is in a first position.

[0120] Fig. 5 is a bottom view of a blood sample collection card according to an embodiment of the present application, in which a cover is in a first position.

[0121] Fig. 6 is a structural schematic diagram of a second board paper of a blood sample collection card according to an embodiment of the present application.

[0122] Fig. 7 is a structural schematic diagram of an adsorbing filter paper of a blood sample collection card according to an embodiment of the present application.

[0123] Fig. 8 is a structural schematic diagram of a first board paper of a blood sample collection card according to an embodiment of the present application.

[0124] Fig. 9 is a schematic diagram of a dry blood spot tumor marker detection system according to an embodiment of the present application.

[0125] Fig. 10 is a result of analysis of consistency limit of determination values of serum samples and dry blood spot samples in Example 2 of the present application.

[0126] Fig. 11 is a linear relationship of diagnostic results of dry blood spot samples and serum samples in Example 2 of the present application.

[0127] Fig. 12 is a measured result of MFI values of sample 1 in Verification Example 1 of the present application.

[0128] Fig. 13 is a measured result of MFI values of sample 2 in Verification Example 1 of the present application.

[0129] Fig. 14 is a measured result of MFI values of sample 3 in Verification Example 1 of the present application.

[0130] Fig. 15 is a measured result of MFI values of sample 4 in Verification Example 1 of the present application.

[0131] Fig. 16 is a measured result of MFI values of sample 1 in Verification Example 2 of the present application.

[0132] Figure 17 is a MFI value measurement result of sample 2 in the verification example 2 of the present application.

[0133] Figure 18 is a MFI value measurement result of sample 3 in the verification example 2 of the present application.

[0134] Figure 19 is a MFI value measurement result of sample 4 in the verification example 2 of the present application.

[0135] Specific reference signs are as follows: 1 - first board paper; 11 - first through hole; 2 - adsorption filter paper; 21 - collection hole; 22 - collection paper piece; 23 - weak part; 3 - second board paper; 31 - information collection area; 32 - sample collection area; 33 - second through hole; 34 - lock structure; 341 - trapezoidal cutout; 35 - folding line; 4 - cover; 5 - dried blood spot tumor marker detection system; 51 - sample collection module; 52 - tumor marker extraction module; 53 - tumor marker detection module; 54 - data processing module. DETAILED DESCRIPTION

[0136] The present application is further illustrated by the following examples, but the present application is not limited to the examples. The experimental methods in the following examples, if not otherwise specified, are selected according to the conventional methods and conditions, or according to the commercial instructions.

[0137] In the following examples and comparative examples, the structure of the blood sample collection card used is shown in Figures 1-8, unless otherwise specified.

[0138] The blood sample collection card comprises a first board paper 1, an adsorption filter paper 2 and a second board paper 3 stacked in sequence from top to bottom, the upper surface of the second board paper 3 is provided with an information collection area 31 and a sample collection area 32, and the adsorption filter paper 2 is adhered to the sample collection area 32; the adsorption filter paper 2 is provided with a collection hole 21, and the collection hole 21 is provided with a collection paper piece 22, the outer circumferential side of the collection paper piece 22 is connected to the hole wall of the collection hole 21 through a weak part 23 which is easy to break; the first board paper 1 is provided with a first through hole 11 corresponding to the collection hole 21, and the hole wall of the first through hole 11 is located on the outer circumferential side of the hole wall of the collection hole 21, in other words, the first through hole 11 is larger than the collection hole 21.

[0139] By preforming the collection hole 21 on the adsorption filter paper 2 and connecting the collection paper sheet 22 through the frangible weak part 23, on the one hand, it is convenient to take sample, only need to take down the collection paper sheet 22 to realize sampling with tweezers, without the need of puncher or scissors and other instruments, reducing the sample loss caused by sample acquisition; on the other hand, through the preformed collection paper sheet 22, it can effectively reduce the interference of hematocrit problem, the preformed collection paper sheet 22 can effectively block the diffusion in the serum component diffusion process in the blood, and limit the effective solute in the collection paper sheet 22. In addition, a layer of first board paper 1 is stacked on the upper surface of the adsorption filter paper 2 to prevent the user from directly touching the adsorption filter paper 2 when using the blood sample collection card and causing pollution to the adsorption filter paper 2, the first board paper 1 is provided with a first through hole 11 corresponding to the position of the collection paper sheet 22, which can also remind the user of the position of the collection paper sheet 22, thereby improving the collection efficiency.

[0140] The first board paper 1 and the second board paper 3 adopt ordinary hard board paper, the adsorption filter paper 2 and the collection paper sheet 22 adopt Whatman 903 filter paper with a thickness of 0.5mm, which uses high-purity cotton fiber as raw material, has good filtering performance and uniformity, can effectively remove impurities and particulate matters in blood, and improve the accuracy and reliability of blood detection; at the same time, its material and process make it have high durability and stability, which can ensure the safety and reliability of the blood sample in the process of collection, transportation and detection. The information collection area 31 can record the basic information of the testee, such as name, gender, age, detection item, blood collection date, etc. The side of the second board paper 3 away from the adsorption filter paper 2 is provided with an information instruction area, and the information instruction area can be filled with the instruction manual of the blood sample collection card, so as to facilitate the user to use.

[0141] The adsorption filter paper 2 and the collection paper sheet 22 are integrally formed, and the tooth mark circle is preformed on the adsorption filter paper 2 to form the collection hole 21 and the weak part 23, and the collection paper sheet 22 located inside the collection hole 21.

[0142] As shown in FIG. 1, along the length direction of the adsorption filter paper 2, four collection holes 21 are arranged. The diameter of the collection hole 21 is 0.8cm to meet the 20μL amount of peripheral blood. The shape of the collection paper sheet 22 and the collection hole 21 is circular. The circular collection paper sheet 22 and the collection hole 21 are convenient to manufacture.

[0143] As shown in FIG. 2 and FIG. 3, the lock structure 34 is located between the information collection area 31 and the sample collection area 32, and a trapezoidal cutout 341 is cut on the second board paper 3 as the lock structure 34, which is used for the insertion or extraction of the cover 4. By directly setting the trapezoidal cutout 341 on the second board paper 3, the cover 4 is inserted into the trapezoidal cutout 341 to limit the cover 4, which is convenient to operate. The number of the lock structure 34 is 2, and the 2 lock structures 34 are symmetrically arranged along the length direction of the adsorption filter paper 2. The cover 4 is limited by the plurality of lock structures 34, and the limiting effect is good.

[0144] As shown in FIG. 1 to FIG. 5, the blood sample collection card further comprises a cover 4, and the second board paper 3 is provided with a lock structure 34 configured to fix the cover 4 to cover the sample collection area 32. After the blood collection is completed, the cover 4 is limited by the cooperation between the cover 4 and the lock structure 34, so that the cover 4 can cover the collection paper 22 to prevent the collection paper 22 from being contaminated.

[0145] The end of the cover 4 is connected to one end of the second board paper 3 close to the sample collection area 32, and a fold line 35 is arranged between the cover 4 and the second board paper 3; the cover 4 has a first position and a second position, and the cover 4 is configured to support the second board paper 3 when located at the first position, and the lock structure 34 fixes the cover 4 when the cover 4 is flipped upward to the second position relative to the second board paper 3. The cover 4 is folded along the fold line 35 to the front or back of the second board paper 3, and when the cover 4 is in the first position, as shown in FIG. 1, the cover 4 is at an angle relative to the second board paper 3 to support the second board paper 3 and prevent the blood sample from overflowing and contacting the table top during the dripping process; the cover 4 is flipped upward to cover the collection paper 22, so as to avoid the collection paper 22 from being contaminated, which is convenient to operate.

[0146] As shown in FIG. 5 and FIG. 6, the second board paper 3 is provided with a second through hole 33 corresponding to the collection hole 21, and the hole wall of the second through hole 33 is located on the outer circumferential side of the hole wall of the collection hole 21. After the blood is dripped on the collection paper 22 from the front of the second board paper 3, the penetration of the blood on the collection paper 22 can be observed through the second through hole 33 to determine whether the blood collection amount on the collection paper 22 is appropriate, so as to select appropriate collection paper 22 as a sample for the next detection according to the penetration.

[0147] As shown in FIG. 7, the number of the weak part 23 is 6, and the 6 weak parts 23 are uniformly and interval arranged along the circumferential direction of the collection paper 22. The connecting points between the collection paper 22 and the weak part 23 are uniformly located on the circumferential side of the collection paper 22, which is convenient for the tweezers to pick up the collection paper 22. The weak part 23 is a connecting column, and the two sides of the connecting column are connected with the collection paper 22 and the collection hole 21 respectively.

[0148] The preparation of reagents or solutions used in the examples and comparative examples of the present application is as follows:

[0149] (1) 10x PBS concentrate solution: PBS buffer containing 0.2% (w / v) potassium chloride, 0.2% (w / v) potassium dihydrogen phosphate, 8% (w / v) sodium chloride, and 2.916% (w / v) disodium hydrogen phosphate dodecahydrate. For example, to prepare 1000 mL: weigh 2 g of potassium chloride, 2 g of potassium dihydrogen phosphate, 80 g of sodium chloride, and 29.16 g of disodium hydrogen phosphate dodecahydrate, add purified water and stir to dissolve, make up to 1000 mL, and filter through a 0.45 μm membrane into a clean container. Label the material and store at room temperature.

[0150] (2) Activation buffer: 0.1 M sodium dihydrogen phosphate solution (pH 6.2). For example, to prepare 200 mL: weigh 2.40 g of sodium dihydrogen phosphate, dissolve in 150 mL of purified water, adjust the pH to 6.20 ± 0.05 with 3 mol / L sodium hydroxide, make up to 200 mL, filter through a 0.45 μm membrane into a clean container, fill in and label the material, and store at 2-8°C until use.

[0151] (3) Coupling buffer: 50 mM MES (2-[N-morpholino]ethanesulfonic acid) solution (pH 6.0). For example, to prepare 200 mL: weigh 1.96 g of MES, dissolve in 150 mL of purified water, adjust the pH to 6.00 ± 0.05 with 3 mol / L sodium hydroxide, make up to 200 mL with purified water, filter through a 0.45 μm membrane into a clean container. Fill in and label the material, and store at 2-8°C until use.

[0152] (4) Blocking buffer: 1x PBS buffer (pH 7.4) containing 1% (w / v) BSA and 0.03% (v / v) Proclin 300. For example, to prepare 200 mL: weigh 2 g of BSA, add 150 mL of purified water and stir to dissolve, add 20 mL of 10x PBS solution, and add 60 μL of Proclin 300, stirring until uniform. Adjust the pH to 7.40 ± 0.05 with 3 mol / L sodium hydroxide or 3 mol / L hydrochloric acid, make up to 200 mL with purified water, filter through a 0.45 μm membrane into a clean container. Fill in and label the material, and store at 2-8°C until use.

[0153] (5) Wash buffer: 1 x PBS buffer (pH 7.4) containing 0.05% (v / v) Tween-20, 0.03% (v / v) Proclin 300. For example, for 200 mL: take 20 mL of 10 x PBS solution, add 150 mL of purified water, add 100 μL of Tween-20, 60 μL of Proclin 300, and stir until uniform. Adjust the pH to 7.40 ± 0.05 with 3 mol / L sodium hydroxide or 3 mol / L hydrochloric acid, and dilute to 200 mL with purified water. Filter through a 0.45 μm filter into a clean container. Fill out and affix a material label, and store at 2-8 °C until use.

[0154] (6) Magnetic bead storage buffer / magnetic bead lyophilization buffer: 1 x PBS buffer (pH 7.4) containing 0.5% (w / v) BSA, 3% (w / v) trehalose, 5% (w / v) mannitol, 0.03% (w / v) Proclin 300. For example, for 200 mL: weigh 1 g of BSA, add 150 mL of purified water, stir to dissolve, add 20 mL of 10 x PBS solution, and stir until uniform. Add 6 g of trehalose and 10 g of mannitol, and stir to dissolve. Add 60 μL of Proclin 300, and stir until uniform. Dilute to 200 mL with purified water, and filter through a 0.45 μm filter into a clean container. Fill out and affix a material label, and store at 2-8 °C until use.

[0155] (7) Coating buffer: 1 x PBS solution (pH 7.4) containing 0.02% (w / v) potassium chloride, 0.02% (w / v) potassium dihydrogen phosphate, 0.8% (w / v) sodium chloride, and 0.292% (w / v) disodium hydrogen phosphate dodecahydrate. For example, for 200 mL: take 20 mL of 10 x PBS solution, add 150 mL of purified water, and stir until uniform. Adjust the pH to 7.40 ± 0.05 with 3 mol / L sodium hydroxide or 3 mol / L hydrochloric acid, and dilute to 200 mL with purified water. Filter through a 0.45 μm filter into a clean container. Fill out and affix a material label, and store at 2-8 °C until use.

[0156] (8) 50 mg / mL NHS solution: activation buffer containing 5% (w / v) NHS (N-hydroxysuccinimide). For example, for 5 mL: weigh 0.25 g of NHS, dissolve in 5 mL of activation buffer, aliquot into 500 μL per aliquot, fill out and affix a material label, and store at 2-8 °C until use.

[0157] (9) 50 mg / mL EDC solution: activation buffer containing 5% (w / v) EDC (1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride). For example, for the preparation of 5 mL, 0.25 g of EDC is weighed and dissolved in 5 mL of activation buffer. The reagent is for single use and is prepared fresh.

[0158] (10) Preparation and dispensing of extraction buffer (sample diluent), calibrator diluent, washing solution, analysis buffer, and RPE-donkey anti-human IgG secondary antibody concentrate:

[0159] 1) Extraction buffer (sample diluent): 1x PBS buffer containing 0.5% (w / v) BSA, 2% (v / v) donkey serum, 10% (v / v) Low Cross-Buffer, 0.1% (v / v) Proclin 300, pH = 7.4.

[0160] 2) Calibrator diluent: 1x PBS buffer containing 1% (w / v) BSA, 0.03% (v / v) Proclin 300, pH = 7.4.

[0161] 3) Washing solution: PBST washing solution containing Tween 20, pH = 7.4 ± 0.05.

[0162] 4) Analysis buffer: phosphate buffer containing 0.2% (w / v) BSA, 0.03% (v / v) Proclin 300, pH = 7.4.

[0163] 5) RPE-donkey anti-human IgG secondary antibody concentrate: RPE-labeled donkey anti-human IgG fluorescent antibody concentrate.

[0164] (11) Preparation and dispensing of calibrators and quality controls:

[0165] 1) Preparation of calibrators: recombinant humanized anti-Myc immunoglobulin containing 1% (w / v) BSA, 3% (w / v) trehalose, 5% (w / v) mannitol, 0.03% (v / v) Proclin 300, and phosphate buffer is prepared, and the concentration is prepared into 3000 U / mL calibrator, which is dispensed at 0.5 mL per bottle.

[0166] 2) Preparation and lyophilization of quality control I: high-concentration Anti-Myc; phosphate buffer containing 1% (w / v) BSA, 3% (w / v) trehalose, 5% (w / v) mannitol, 0.03% (v / v) Proclin 300. Anti-Myc is configured into 200 U / mL, which is dispensed at 0.5 mL per bottle and used after quality detection.

[0167] 3) Preparation of quality control product II: Prepare a low concentration Anti-Myc solution containing 1% (w / v) BSA, 3% (w / v) trehalose, 5% (w / v) mannitol, 0.03% (v / v) Proclin 300 in phosphate buffer. Dilute the Anti-Myc raw material labeled with a commercial calibration product to 7.41 U / mL using the calibration product diluent, 0.5 mL per bottle, and store in a small bottle for use after passing the quality test.

[0168] The above-mentioned reagents or solutions are prepared according to the production batch during production, and are prepared separately according to the production instruction formula. The production instruction and formula are strictly audited during the preparation process of each solution, and the production date and expiration date are noted after preparation for future use.

[0169] The product and detection method developed by the present application are suitable for the following instrument: Luminex multifunctional flow dot array instrument.

[0170] The preparation of the coupling magnetic bead mixture used in the examples and comparative examples of the present application is as follows:

[0171] 1. Activation of magnetic beads: The magnetic beads to be coupled are vortexed and ultrasonically mixed. The corresponding magnetic bead suspension is aspirated into a centrifuge tube, and the centrifuge tube is placed on a magnetic separator to magnetically adsorb the magnetic beads, and then the supernatant is carefully removed with a pipette. Remove the magnetic separator, and for example, add 500 μL of activation buffer to the tube for every 0.5 mL of magnetic beads (about 12.5 x 10 6 magnetic beads. The centrifuge tube is placed on a magnetic separator to magnetically adsorb the magnetic beads, and then the supernatant is carefully removed with a pipette. This step is repeated, and the magnetic beads are washed with activation buffer a total of two times. Remove the magnetic separator, and add 400 μL of activation buffer to the tube, vortex and ultrasonically mix the magnetic beads. Add 50 μL of 50 mg / mL NHS solution to the tube, vortex and mix the magnetic beads. Add 50 μL of 50 mg / mL EDC solution to the tube, immediately vortex and mix the magnetic beads; place the centrifuge tube in a rotary mixer, rotate at room temperature, avoid light, 20 rpm for 20 minutes.

[0172] 2. Coupling of Biotin-BSA:

[0173] (1) The centrifuge tube containing the activated magnetic beads from the previous step is placed on a magnetic separator to adsorb the magnetic beads, and then the supernatant is carefully removed with a pipette. Add 0.5 mL of coupling buffer to the tube, vortex and ultrasonically mix the magnetic beads. Repeat this step twice. Wash the magnetic beads with coupling buffer a total of three times. Place the magnetic beads containing the coupling buffer on a magnetic separator to adsorb the magnetic beads, and then carefully remove the supernatant with a pipette.

[0174] (2) Add 970 μL of conjugation buffer to the tube, vortex and mix the beads by sonication. Add 30 μL of BSA-biotin (2 mg / mL) to the tube, vortex and mix the beads. Place the centrifuge tube in a rotator mixer, incubate at room temperature for 2 hours in the dark at 20 rpm.

[0175] (3) Place the centrifuge tube with the incubated beads on a magnetic separator to attract the beads, then carefully remove the supernatant with a pipette. Remove the magnetic separator, add 1 mL of washing solution, vortex and mix the beads by sonication. Place the centrifuge tube on the magnetic separator to attract the beads, then carefully remove the supernatant with a pipette. Remove the magnetic separator and add 1 mL of blocking buffer, vortex and mix the beads by sonication. Place the centrifuge tube in a rotator mixer, incubate at room temperature for 1 hour in the dark at 20 rpm.

[0176] 3. Coating of antigen

[0177] (1) Place the centrifuge tube with the beads from the previous step on a magnetic separator to attract the beads, then carefully remove the supernatant with a pipette. Remove the magnetic separator, add 1 mL of washing solution to the tube, vortex and mix the beads by sonication. Centrifuge the tube briefly to remove the residual liquid on the tube wall, then place the centrifuge tube on a magnetic separator to attract the beads, then carefully remove the supernatant with a pipette.

[0178] (2) Remove the tube from the magnetic separator, add the corresponding antigen (prepared in coating buffer) to the tube. Vortex and mix the beads by sonication. Place the centrifuge tube in a rotator mixer, incubate at room temperature for 1 hour in the dark at 20 rpm.

[0179] (3) Place the centrifuge tube on a magnetic separator to attract the beads, then carefully remove the supernatant with a pipette. Remove the magnetic separator, add 2 mL of washing solution to the centrifuge tube, vortex and mix the beads by sonication. Place the centrifuge tube on a magnetic separator to attract the beads, then carefully remove the supernatant with a pipette. Repeat this step to wash the beads a total of three times.

[0180] (4) Remove the magnetic separator, add 2 mL of bead storage solution to the centrifuge tube, vortex and mix the beads by sonication, and store the centrifuge tube in the dark at 2-8°C. After counting a small amount of beads, adjust the concentration of the beads to 2.0 x 10 6 per mL using the bead storage solution. Store in a -20°C freezer.

[0181] 4. Freeze-drying of the conjugated bead mixture

[0182] (1) Magnetic bead mixture preparation: Take out 13 kinds of magnetic beads intermediates coupled with coated antigens and pass the inspection. Mix according to the appropriate proportion of the volume of each magnetic bead concentration, add magnetic bead freeze-drying buffer, so that the concentration of each detection magnetic bead in the magnetic bead mixture is not less than 3 x 10 4 After shaking and ultrasonic mixing of the magnetic beads, prepare for dispensing.

[0183] (2) Magnetic bead mixture dispensing and freeze-drying: After mixing the magnetic bead mixture, dispense it into freeze-drying bottles according to the indicated amount. Transfer the freeze-drying bottles to the freeze-drying machine box for freeze-drying. After freeze-drying is completed, perform plug pressing, cap screwing, and label pasting.

[0184] Among the 13 kinds of magnetic beads, the coating concentrations of the tumor markers are respectively: ESO-1 (100 μg / mL), FXR1 (200 μg / mL), EFHD2 (200 μg / mL), BRAF (400 μg / mL), TM4SF1 (300 μg / mL), ZNF573 (150 μg / mL), BMI1 (200 μg / mL), CAGE (200 μg / mL), PGP9.5 (300 μg / mL), P53 (200 μg / mL), GBU4-5 (300 μg / mL), MAGE A4 (300 μg / mL), and SOX2 (300 μg / mL).

[0185] Example 1

[0186] This embodiment uses a detection system 5 as shown in FIG. 9 to detect tumor markers in a dried blood spot, which includes the following modules:

[0187] S1, sample collection module 51: This module is used to collect blood samples using a blood sample collection card to obtain a dried blood spot sample.

[0188] S2, tumor marker extraction module 52: This module is used to extract tumor markers using an extraction buffer. Place the dried blood spot sample in the extraction buffer, shake, and take the supernatant to obtain the test solution.

[0189] S3, tumor marker detection module 53: This module is used to detect tumor markers using detection reagents. Contact the test solution with the detection reagents and use a flow cytometer for detection to obtain the detected concentration.

[0190] S4, data processing module 54: This module is used to process the detected concentration by multiplying it by the serum-dried blood spot correlation coefficient to obtain the final concentration.

[0191] Specifically, the steps corresponding to this system are as follows:

[0192] 1. Collection of peripheral dried blood spot samples:

[0193] The dry blood spot sample collection is performed in the following steps:

[0194] 1.1, Information filling: Take out the blood sample collection card and fill in the relevant information of the person to be tested in the information collection area 31 of the second board paper 3 to ensure the accuracy of the information.

[0195] 1.2, Disinfection: Before performing puncture or blood collection operations, use alcohol cotton to wipe and disinfect the puncture point or blood collection site. Reduce the risk of infection and ensure that the sample is not contaminated.

[0196] 1.3, Puncture: Before puncture, push the ring finger 3-5 times from the root, then pinch the fingertip, do not block the blood collection site. This operation can greatly reduce the pain and increase the amount of bleeding. Remove the blood collection needle protective cap, aim the blood collection needle at the blood collection site and press it down. Pop out the blood collection needle and pierce the blood collection site.

[0197] 1.4, Blood collection: Gently push and squeeze the puncture site to make the blood flow out, do not touch the blood collection site to cause contamination; after wiping off the first drop of blood with a cotton swab, use a 20μL quantitative capillary to automatically siphon until it stops, which can obtain a fixed amount of fresh blood. Do not squeeze and block the capillary air bag vent during the blood collection operation.

[0198] 1.5, Spotting: Turn the cover 4 to the first position so that the cover 4 supports the second board paper 3, and the finger blocks the capillary air bag vent. Gently squeeze and drop the collected fresh blood to the center of the collection paper 22 of the blood sample collection card, allowing the blood to spread around. Do not drop outside the circle during the operation.

[0199] 1.6, Drying: Place the blood sample collection card on a clean table and dry naturally for more than 30 minutes before packaging; do not touch the sample collection window to prevent contamination.

[0200] 1.7, Preservation: Turn the cover 4 to the first position and insert it into the trapezoidal cutout 341. Place the dried blood sample collection card into a sealed bag containing desiccant tablets, seal and store or mail to the designated detection point for detection.

[0201] 2, Extraction of tumor markers from dry blood spots

[0202] 2.1, Place the blood sample collection card loaded with dry blood spot samples in step one at room temperature for 20 minutes.

[0203] 2.2, Disinfect the tweezers with 75% alcohol.

[0204] 2.3, Using tweezers after natural drying, take out two pieces of collection paper loaded with dried blood spot samples, and place them in a 2.0 mL EP tube containing 450 μL of extraction buffer; do not touch the sample collection window during operation to prevent interference with the sample.

[0205] 2.4, Fix the EP tube on the test tube rack and place it on the shaker for 1 h of shaking at a speed of 500 rpm.

[0206] 2.5, Take out the supernatant, which is the analysis sample containing tumor markers in dried blood spots required for early tumor screening diagnosis.

[0207] 3, Early tumor screening diagnosis—detection of tumor markers in dried blood spots:

[0208] 3.1, Take out the 96-well plate, vortex 30-60 s to mix the reconstituted solution of tumor marker antigen protein-coupled magnetic bead mixture (specifically, the above-mentioned mixture of 13 tumor marker antigen protein-coupled magnetic beads: P53, PGP9.5, TM4SF1, SOX2, MAGE A4, ESO1, FXR1, EFHD2, BRAF, GBU4-5, ZNF573, BMI1, CAGE) freeze-dried powder reconstituted in 1x PBS buffer containing 0.5% (w / v) BSA, 3% (w / v) trehalose, 5% (w / v) mannitol, and 0.03% (v / v) Proclin 300), and add 50 μL / well to the 96-well plate.

[0209] 3.2, Add sample:

[0210] Add 50 μL of calibrators, quality control I, quality control II, and the analysis sample obtained in step two to the corresponding microwells, respectively, shake the 96-well plate at 1000 rpm for 3 min, apply the sealing film, and incubate at 37°C for 90 min.

[0211] 3.3, Wash the plate:

[0212] 1) Place the incubated 96-well plate on the magnetic plate and secure it, stand for 1 min, keep the 96-well plate fixed on the magnetic plate upward, quickly and forcefully turn the magnetic plate upside down, shake off the liquid in the wells, keep the magnetic plate vertically downward, and shake quickly for 3-4 times until no liquid drops from the 96-well plate;

[0213] 2) Take the 96-well plate off the magnetic plate, add 120 μL of washing solution to each well of the 96-well plate, and place the 96-well plate in the vortex mixer, shake at 1000 rpm for 1 min at room temperature.

[0214] 3.4, Repeat the above 3.3 plate washing operation for a total of 2 times.

[0215] 3.5, Put the 96-well plate on the magnetic plate and buckle it, stand for 1 min, keep the 96-well plate fixed on the magnetic plate upward, quickly and powerfully overturn the magnetic plate downward, shake off the liquid in the hole, keep the magnetic plate vertically downward, shake quickly for 3-4 times until no liquid drops from the 96-well plate.

[0216] 3.6, Add 100 μL RPE-donkey anti-human IgG secondary antibody working solution to each hole, mix evenly at 1000 rpm for 3 min, paste the plate film, and incubate at 37°C for 60 min.

[0217] 3.7, Repeat the above 3.3 plate washing operation for a total of 2 times.

[0218] 3.8, Put the 96-well plate on the magnetic plate and buckle it, stand for 1 min, keep the 96-well plate fixed on the magnetic plate upward, quickly and powerfully overturn the magnetic plate downward, shake off the liquid in the hole, keep the magnetic plate vertically downward, shake quickly for 3-4 times until no liquid drops from the 96-well plate.

[0219] 3.9, Add 100 μL analysis buffer to each hole, shake at 1000 rpm for 3 min, and read on the Luminex 200 multifunctional flow dot array instrument.

[0220] 3.10, Multiply the obtained reading (i.e. detection output concentration) by the correlation coefficient of serum and dry blood spot to obtain the true sample concentration.

[0221] Example 2

[0222] Take dry blood spot samples from 7 volunteers respectively, detect the concentration of tumor markers in the dry blood spot by the flow cytometer under the conditions of Example 1, and obtain the detection output concentration of 13 kinds of tumor markers. Multiply the obtained detection output concentration by the correlation coefficient of serum and dry blood spot to obtain the true sample concentration.

[0223] Further, the true sample concentration of 13 kinds of tumor markers obtained by converting the dry blood spot sample is calculated by a big data binary Logistics regression model to obtain a regression value p (i.e. diagnosis value), and is compared and judged by the optimal Cutoff value (0.70727) of the ROC curve obtained by the big data model (serum sample): the diagnosis value less than or equal to the optimal Cutoff value is negative, and the diagnosis value greater than the optimal Cutoff value is positive.

[0224] Among them, the correlation coefficient (including linear coefficient and average coefficient) of serum and dry blood spot of 13 kinds of tumor markers respectively is shown in Table 1.

[0225] Table 1

[0226] Note: "*" indicates the average fold coefficient of serum / dried blood spot samples.

[0227] As can be seen from Table 1, the fold linear relationship between the dried blood spot samples and the serum samples of each marker is poor (all linear R 2 <0.6), while in the direct linear relationship between the dried blood spot samples and the serum samples, except for the BMI1, FXR1 and ZNF573 markers, the remaining markers all have good linear relationships, and most of the R 2 reach 0.900 or more, which shows that the direct linear model calculation is more accurate than the average coefficient.

[0228] Further, the present application uses the serum sample diagnostic value and the retest value to verify the diagnostic values obtained by different algorithms, and the results are shown in Table 2.

[0229] Table 2

[0230] Note: A: dried blood spot sample linear model algorithm diagnostic value; B: serum sample diagnostic value; C: serum sample retest diagnostic value; D: dried blood spot average coefficient algorithm diagnostic value; "*" positive and negative determination is divided by the best Cutoff value (0.70727) obtained by the ROC curve for comparison.

[0231] As can be seen from Table 2, the dried blood spot sample adopts the linear model algorithm to output the diagnostic value, and the serum sample diagnostic value has certain difference, but the determination results are consistent; the diagnostic value and the blood sample retest diagnostic value have high consistency.

[0232] As can also be seen from Figure 10, the diagnostic values of the linear model algorithm and the average coefficient algorithm both have differences with the serum sample, but these differences are within the 95% consistency limit, which is acceptable in clinical practice, meaning that the two methods have good consistency and can be used interchangeably. However, the diagnostic value obtained by the average coefficient algorithm has a significant gap with the serum sample diagnostic value, and the final determination result is consistent with the serum sample retest determination result, but the critical value sample diagnostic value is lower (0.68558), and the accuracy is relatively lower than the linear model algorithm.

[0233] Further, in combination with the linear relationship of the diagnostic value in Figure 11, the k -1 of the linear model of the dried blood spot linear model algorithm diagnostic value VS the serum sample diagnostic value (B-A) is basically the same as the k -1 of the serum sample retest diagnostic value VS the serum sample diagnostic value (B-C), which are 1.048 and 1.046, respectively, while the k -1The average coefficient algorithm is used to verify the three markers (BMI1, FXR1 and ZNF573) with a diagnostic value of 1.130, which is obviously different from the linear model algorithm, further indicating that the accuracy of the average coefficient algorithm is lower than that of the linear model algorithm.

[0234] Considering that the linear fitting results of the BMI1, FXR1 and ZNF573 markers are poor, R 2 less than 0.700, the three markers are verified by using the average coefficient algorithm, and the results are shown in Table 3. As can be seen from the results in Table 3, the results obtained by using the average coefficient algorithm for the BMI1, FXR1 and ZNF573 markers and the linear model algorithm for the remaining markers are basically consistent with the determination results of using the linear model algorithm for all markers, and the diagnostic values are slightly lower than those of using the linear model algorithm for all markers. The reason for such a result may be that the proportion of the three markers in the Logistics regression model is relatively low, and the numerical change range is relatively high, so that the final diagnostic value does not change significantly.

[0235] Table 3

[0236] Note: A: linear model diagnostic value of dry blood spot sample; E: except that the BMI1, FXR1 and ZNF573 markers are calculated by using the average coefficient algorithm, the remaining markers are calculated by using the linear model algorithm; "*" is the determination of yin and yang divided by the best Cutoff value (0.70727) of the ROC curve.

[0237] In summary, the linear model algorithm is used to output the diagnostic values of all markers of the dry blood spot sample, or except that the BMI1, FXR1 and ZNF573 markers are calculated by using the average coefficient algorithm, the remaining markers are calculated by using the linear model algorithm, which can achieve detection results comparable to serum samples. However, overall, the linear model algorithm is used to output the diagnostic values of all markers of the dry blood spot sample.

[0238] Example 3

[0239] On the basis of Example 1, other conditions remain unchanged, only the shaking time in step two S4 is changed to 90 min.

[0240] Example 4

[0241] On the basis of Example 1, other conditions remain unchanged, only the shaking speed in step two S4 is changed to 550 rpm.

[0242] Comparative Example 1

[0243] On the basis of Example 1, other conditions remain unchanged, only the shaking time in step two S4 is changed to 30 min.

[0244] Comparative Example 2

[0245] On the basis of Example 1, other conditions remain unchanged, only change S4 in step two: fix the EP tube on the test tube rack, placed in the cell disruptor for ultrasonic, conventional low frequency (20-100 kHz) ultrasound 1 min.

[0246] Comparative Example 3

[0247] On the basis of Example 1, other conditions remain unchanged, only change the shaking speed in S4 of step two to 160 rpm.

[0248] Example 5

[0249] Kit production process

[0250] The kit is composed of sample diluent, calibrator diluent, washing solution, analysis buffer, RPE-donkey anti-human IgG secondary antibody concentrate, calibrator, quality control, coupled magnetic beads, 96-well plate and other components. Each component is prepared separately, independently packaged, and then assembled into a box. The preparation process is summarized as follows:

[0251] (1) Preparation and packaging of sample diluent, calibrator diluent, washing solution, analysis buffer, and RPE-donkey anti-human IgG secondary antibody concentrate:

[0252] 1) Sample diluent: 1x PBS buffer containing 0.5% (w / v) BSA, 2% (v / v) donkey serum, 10% (v / v) LowCross-Bu ffer, 0.1% (v / v) Proclin300, pH = 7.4.

[0253] 2) Calibrator diluent: phosphate buffer containing 1% (w / v) BSA, 0.03% (v / v) Proclin300, pH = 7.4.

[0254] 3) Washing solution: PBST washing solution containing Tween20, pH = 7.4 ± 0.05.

[0255] 4) Analysis buffer: phosphate buffer containing 0.2% (w / v) BSA, 0.03% (v / v) Proclin300, pH = 7.4.

[0256] 5) RPE-donkey anti-human IgG secondary antibody concentrate: RPE-labeled donkey anti-human IgG fluorescent antibody concentrate.

[0257] (2) Preparation, packaging and freeze-drying of calibrator and quality control:

[0258] 1) Preparation and freeze-drying of the calibrator: the recombinant humanized anti-Myc immunoglobulin containing 1% (w / v) BSA, 3% (w / v) trehalose, 5% (w / v) mannitol, 0.03% (v / v) Proclin 300 and phosphate buffer was prepared into a calibrator with a concentration of 3000 U / mL, and was divided into 0.5 mL per bottle and placed in a freeze dryer for freeze-drying.

[0259] 2) Preparation and freeze-drying of the quality control I: high-concentration Anti-Myc; freeze-dried product containing 1% (w / v) BSA, 3% (w / v) trehalose, 5% (w / v) mannitol, 0.03% (v / v) Proclin 300 and phosphate buffer. Anti-Myc was prepared into 200 U / mL, and was divided into 0.5 mL per bottle and placed in a freeze dryer for freeze-drying. The freeze-dried quality control I was used after passing the quality test.

[0260] 3) Preparation and freeze-drying of the quality control II: low-concentration Anti-Myc; freeze-dried product containing 1% (w / v) BSA, 3% (w / v) trehalose, 5% (w / v) mannitol, 0.03% (v / v) Proclin 300 and phosphate buffer. The Anti-Myc raw material calibrated by the enterprise-level calibrator was diluted to 7.41 U / mL using the calibrator diluent, 0.5 mL per bottle, and was divided into small bottles and placed in a freeze dryer for freeze-drying. The freeze-dried quality control I was used after passing the quality test.

[0261] (3) Magnetic bead coupling, dispensing and freeze-drying of 13 kinds of related antigen proteins:

[0262] Freeze-dried product of 13 kinds of magnetic bead mixtures coupled with lung cancer related antigens, containing 0.5% (w / v) BSA, 3% (w / v) trehalose, 5% (w / v) mannitol, 0.03% (v / v) Proclin 300 and phosphate buffer solution freeze-dried powder.

[0263] (4) 96-well plate (empty plate) bagging and labeling.

[0264] (5) Kit assembly.

[0265] Verification Example 1

[0266] Dry blood spot samples from 4 volunteers were taken, and the MFI (mean fluorescence intensity) values were detected by flow cytometry under the conditions of Example 1, Example 3 and Comparative Example 1. The results are shown in Figures 12-15.

[0267] As can be seen from the figure, the MFI values of the extraction time 30 min (comparative example 1) are lower than those of 60 min (example 1) and 90 min (example 3) as a whole, and the heat map presents a clear dividing line between the extraction time 30 min and 60 min, indicating that there is a clear difference between the two.

[0268] Specifically, as can be seen from the scatter plot and the heat map, when the extraction time is 30 min, the MFI values of the TM4SF1, p53, pGP9.5, SOX2, BMI1, FXR1, MAGE A4, ESO-1, ZNF573, BRAF, GUB4-5, EFHD2 and CAG E markers are significantly lower than those of 60 min and 90 min, while the MFI values of the thirteen markers between 60 min and 90 min have a small difference, with a skewness degree of 2.60% to 6.35%, which is within the deviation degree of 1.42% to 8.47% of the retest samples. Therefore, it can be considered that there is no significant difference between the extraction time 60 min and 90 min.

[0269] Verification example 2

[0270] The dried blood spot samples from four volunteers were taken to detect the MFI (mean fluorescence intensity) values by flow cytometry under the conditions of example 4, comparative example 2 and comparative example 3. The results are shown in Figures 16-19.

[0271] As can be seen from the figure, the extraction effect of shaking 1 h (550 rpm) (example 4, corresponding to processing method A in Figures 16-19) is slightly better than that of ultrasonic 1 min (comparative example 2, corresponding to processing method B in Figures 16-19), and significantly better than that of shaking 1 h (160 rpm) (comparative example 3, corresponding to processing method C in Figures 16-19).

[0272] Specifically, the deviation amplitude between ultrasonic 1 min and shaking 1 h (550 rpm) is 10.34% to 12.94%, the deviation amplitude between shaking 1 h (160 rpm) and shaking 1 h (550 rpm) is 13.01% to 22.18%, and the average deviation amplitude of the retest samples is 1.42% to 8.47%.

Claims

1. A dried blood spot tumor marker detection system, comprising: The dry blood spot tumor marker detection system comprises the following modules: S1, a sample collection module: the module is used for collecting blood samples using a blood sample collection card to obtain a dry blood spot sample; S2, a tumor marker extraction module: the module is used for extracting a tumor marker using an extraction buffer, placing the dry blood spot sample in the extraction buffer, shaking, and taking the supernatant to obtain a test solution; wherein the shaking time is 1-1.5 h; the shaking rate is 300-1000 rpm; S3, a tumor marker detection module: the module is used for detecting a tumor marker using a detection reagent, contacting the test solution with the detection reagent, and detecting using a flow cytometer to obtain a detected concentration; S4, a data processing module: the module is used for data processing of the detected concentration, multiplying the detected concentration by a serum-dry blood spot correlation coefficient to obtain a final concentration; wherein the serum-dry blood spot correlation coefficient is obtained by a linear model algorithm and / or an average coefficient algorithm.

2. The dried blood spot tumor marker detection system of claim 1, wherein, In module S1, the blood sample collection card comprises a first board paper, an adsorption filter paper and a second board paper stacked in sequence from top to bottom, the upper surface of the second board paper is provided with an information collection area and a sample collection area, and the adsorption filter paper is attached to the sample collection area; a collection hole is formed in the adsorption filter paper, a collection paper sheet is arranged in the collection hole, and the outer circumferential side of the collection paper sheet is connected to the hole wall of the collection hole through a weak part that is easy to break; a first through hole corresponding to the collection hole is formed in the first board paper, and the hole wall of the first through hole is located on the outer circumferential side of the hole wall of the collection hole; Preferably, the number of collection holes is multiple, and the multiple collection holes are arranged at intervals along the length and / or width direction of the adsorption filter paper; and / or, the diameter of the collection hole is 0.4-1.6 cm, and the diameter of the first through hole is 0.9-2.1 cm.

3. The dried blood spot tumor marker detection system of claim 1, wherein, In module S1, the blood loading amount corresponding to the dry blood spot sample is 10-40 μL per dry blood spot sample, for example, 20 μL per dry blood spot sample.

4. The dried blood spot tumor marker detection system of claim 1, wherein, In module S2, the extraction buffer comprises the following components: 0.4-0.6% (w / v) BSA, 1.5-2.5% (v / v) horse serum, 8-12% (v / v) lowcross buffer, 0.08-0.12% (v / v) Proclin 300 and PBS, and the pH is 7.0-7.6; and / or, in module S2, the amount of the extraction buffer is 100 μL-1 mL per dry blood spot sample, preferably 200-500 μL per dry blood spot sample, for example, 225 μL per dry blood spot sample; and / or, in module S2, the shaking time is 65 min, 70 min, 75 min, 80 min or 85 min; and / or, in module S2, the shaking rate is 300-600 rpm, for example, 500 rpm or 550 rpm; and / or, in module S2, the shaking is performed in a shaking bed or a homogenizer, for example.

5. The dried blood spot tumor marker detection system of claim 1, wherein, the tumor markers are selected from one or more of lung cancer related tumor markers, for example one or more of P53, PGP9.5, TM4SF1, SOX2, MAGEA4, ESO-1, FXR1, EFHD2, BRAF, GBU4-5, ZNF573, BMI1 and CAGE antigens or autoantibodies binding thereto; and / or the detection reagents comprise autoantibodies for antigen detection or antigens for autoantibody detection; Preferably, the detection reagents are selected from any of the following groups: 1) ESO-1 antigen, TM4SF1 antigen, p53 antigen, BMI1 antigen, FXR1 antigen and EFHD2 antigen; 2) ESO-1 antigen, TM4SF1 antigen, p53 antigen, BMI1 antigen, FXR1 antigen, EFHD2 antigen and BRAF antigen; 3) ESO-1 antigen, TM4SF1 antigen, p53 antigen, BMI1 antigen, FXR1 antigen, EFHD2 antigen, BRAF antigen and CAGE antigen; 4) BRAF antigen, ESO-1 antigen, EFHD2 antigen, TM4SF1 antigen, ZNF573 antigen, BMI1 antigen, CAGE antigen, FXR1 antigen, PGP9.5 antigen and P53 antigen; 5) BRAF antigen, ESO-1 antigen, EFHD2 antigen, TM4SF1 antigen, ZNF573 antigen, BMI1 antigen, CAGE antigen, FXR1 antigen, PGP9.5 antigen, P53 antigen and MAGEA4 antigen; 6) BRAF antigen, ESO-1 antigen, EFHD2 antigen, TM4SF1 antigen, ZNF573 antigen, BMI1 antigen, CAGE antigen, FXR1 antigen, PGP9.5 antigen, P53 antigen, GBU4-5 antigen, MAGEA4 antigen and SOX2 antigen; or the detection reagents are selected from any of the following groups: 1) ESO-1 autoantibody, TM4SF1 autoantibody, p53 autoantibody, BMI1 autoantibody, FXR1 autoantibody and EFHD2 autoantibody; 2) ESO-1 autoantibody, TM4SF1 autoantibody, p53 autoantibody, BMI1 autoantibody, FXR1 autoantibody, EFHD2 autoantibody and BRAF autoantibody; 3) ESO-1 autoantibody, TM4SF1 autoantibody, p53 autoantibody, BMI1 autoantibody, FXR1 autoantibody, EFHD2 autoantibody, BRAF autoantibody and CAGE autoantibody; 4) BRAF autoantibody, ESO-1 autoantibody, EFHD2 autoantibody, TM4SF1 autoantibody, ZNF573 autoantibody, BMI1 autoantibody, CAGE autoantibody, FXR1 autoantibody, PGP9.5 autoantibody and P53 autoantibody; 5) BRAF autoantibody, ESO-1 autoantibody, EFHD2 autoantibody, TM4SF1 autoantibody, ZNF573 autoantibody, BMI1 autoantibody, CAGE autoantibody, FXR1 autoantibody, PGP9.5 autoantibody, P53 autoantibody and MAGEA4 autoantibody; 6) BRAF autoantibody, ESO-1 autoantibody, EFHD2 autoantibody, TM4SF1 autoantibody, ZNF573 autoantibody, BMI1 autoantibody, CAGE autoantibody, FXR1 autoantibody, PGP9.5 autoantibody, P53 autoantibody, GBU4-5 autoantibody, MAGEA4 autoantibody and SOX2 autoantibody. 5) BRAF autoantibody, ESO-1 autoantibody, EFHD2 autoantibody, TM4SF1 autoantibody, ZNF573 autoantibody, BMI1 autoantibody, CAGE autoantibody, FXR1 autoantibody, PGP9.5 autoantibody, P53 autoantibody, and MAGEA4 autoantibody; 6) BRAF autoantibody, ESO-1 autoantibody, EFHD2 autoantibody, TM4SF1 autoantibody, ZNF573 autoantibody, BMI1 autoantibody, CAGE autoantibody, FXR1 autoantibody, PGP9.5 autoantibody, P53 autoantibody, GBU4-5 autoantibody, MAGEA4 autoantibody, and SOX2 autoantibody.

6. The dried blood spot tumor marker detection system of claim 1, wherein, In module S3, the reagents used in the detection process further include one or more of calibrator diluent, washing solution, analysis buffer, anti-human IgG secondary antibody, calibrator, and quality control; Preferably, in module S3, the detection of the tumor markers comprises the following steps: (1) adding the detection reagent and the sample to be tested in a well plate, mixing, incubating, and washing; (2) adding the anti-human IgG secondary antibody to the well plate obtained in step (1), mixing, incubating, and washing; (3) adding the analysis buffer to the well plate obtained in step (2), mixing; In step (1), the volume ratio of the detection reagent to the sample to be tested is preferably 1:1; In step (1) and step (2), the mixing step is preferably independently performed by oscillation, for example, at a rate of 1000 rpm for a time period of, for example, 3 min; In step (1) and step (2), the incubation step is preferably independently static incubation, for example, at a temperature of 37°C for a time period of, for example, 90 min; In step (1) and step (2), the well plate is, for example, a 96-well plate.

7. The dried blood spot tumor marker detection system of claim 1, wherein, In modules S2 and S3, when the tumor markers are selected from P53, PGP9.5, TM4SF1, SOX2, MAGEA4, ESO-1, FXR1, EFHD2, BRAF, GBU4-5, ZNF573, BMI1, and CAGE antigen or autoantibody binding thereto, in module S4, the correlation coefficients of the serum of P53, PGP9.5, TM4SF1, SOX2, MAGEA4, ESO-1, FXR1, EFHD2, BRAF, GBU4-5, ZNF573, BMI1, and CAGE antigen or autoantibody binding thereto with the dried blood spot are obtained by a linear model algorithm; Or, in the modules S2 and S3, when the tumor markers are selected from P53, PGP9.5, TM4SF1, SOX2, MAGEA4, ESO-1, FXR1, EFHD2, BRAF, GBU4-5, ZNF573, BMI1 and CAGE antigen or self-antibodies binding thereto, in the module S4, the serum and dried blood spot correlation coefficients of the P53, PGP9.5, TM4SF1, SOX2, MAGEA4, ESO-1, EFHD2, BRAF, GBU4-5 and CAGE antigen or self-antibodies binding thereto are obtained by linear model algorithm; the serum and dried blood spot correlation coefficients of the BMI1, FXR1 and ZNF573 antigen or self-antibodies binding thereto are obtained by average coefficient algorithm; And / or, the dried blood spot tumor marker detection system further comprises the following modules: S5, a diagnostic value calculation module: this module is used for calculating the final concentration obtained by the module S4 by using 5p-Logistics regression model to obtain a diagnostic value.

8. A kit characterized in that, The kit comprises a blood sample collection card, an extraction buffer of tumor markers and a detection reagent of tumor markers.

9. The kit of claim 8, wherein The blood sample collection card is as defined in claim 2, the extraction buffer is as defined in claim 4, the detection reagent is as defined in claim 5, and the tumor markers are as defined in claim 5. Preferably, the kit further comprises one or more of a calibrator diluent, a washing solution, an analysis buffer, an anti-human IgG secondary antibody, a calibrator and a quality control; and / or, the kit further comprises a 96-well plate.

10. Use of the kit of claim 8 or 9 in the preparation of a reagent for detecting tumor markers.

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