An enzyme-linked immunofluorescence detection kit and its application in detecting protein content

Abstract

The present invention discloses an enzyme-linked immunofluorescence detection kit and its application in detecting protein content. The present invention adopts the double antibody sandwich method principle design, the capture antibody is coated on a solid phase substrate or medium, and after adding a sample containing an object to be detected and a detection antibody labeled with alkaline phosphatase, a double antibody sandwich structure complex is formed, and after washing to remove free components, an aggregation-induced luminescence type fluorescent substrate is added, and the fluorescent signal generated by the enzymatic reaction is proportional to the concentration of the object to be detected in the sample. The detection kit developed by the present invention includes a microplate, a magnetic bead solution (or a pre-coated microplate), a calibrator, a quality control product and an alkaline phosphatase-labeled antibody, an aggregation-induced luminescence type fluorescent substrate, a detection buffer, and a washing solution. The detection kit of the present invention adopts a double antibody sandwich enzyme-linked immunofluorescence method, and the detection kit prepared by the preparation method of the present invention has good sensitivity and precision when used.

Description

Technical Field

[0001] The invention belongs to the technical field of medical testing, and in particular relates to an enzyme-linked immunofluorescence detection kit and an application thereof in detecting protein content. Background Art

[0002] The application market of traditional colorimetric enzyme-linked immunosorbent assay is shrinking due to time-consuming operation, insufficient sensitivity or narrow detection range, and is gradually being replaced by chemiluminescence.

[0003] Ferritin (Fer) is a type of iron storage protein widely found in animals and plants. It is most abundant in the liver and spleen of mammals. Its outer diameter is about 12-14nm, and the diameter of the cyst cavity is about 6nm. The outer shell (i.e., apoferritin) is composed of 24 subunits, each of which contains about 163 amino acid residues. Each molecule can bind up to 4500 iron atoms. The molecular weight is about 450KDa. Iron-bound Fer is "soluble" in water, and the concentration of plasma Fer is proportional to the iron stored in the body. Normal human serum contains a small amount of Fer, and the normal average is about 15-200μg / L for men and about 12-150μg / L for women. Fer content determination is currently the earliest and most accurate indicator for diagnosing latent anemia, with a diagnostic compliance rate of more than 95%.

[0004] In clinical practice, Fer is not only an important indicator for diagnosing iron deficiency anemia, but also one of the markers of malignant tumors. Fer determination is suitable for understanding the situation of iron metabolism in the body. Testing ferritin at the beginning of treatment can reflect the iron reserves in the body at that time, and can detect the deficiency of iron storage at an early stage. Under normal circumstances, stored iron can be used for the synthesis of hemoglobin. If it is lower than 12ng / mL, it is judged as latent iron deficiency. If the Fer level is high and the possibility of abnormal iron supply is excluded, it reflects the situation of excessive iron in the body. In addition, Fer is also a broad-spectrum tumor marker and can be used for auxiliary diagnosis of tumors such as liver cancer and lung cancer. The upper limit of Fer determination is 400ng / mL. Some tumors often rise and exceed this value, which is common in acute leukemia, Hodgkin's disease, lung cancer, colon cancer, liver cancer and prostate cancer. Fer detection has diagnostic value for liver metastatic tumors. 76% of patients with liver metastasis have Fer levels higher than 400ng / mL. Combined detection with AFP, especially for liver cancer patients with normal AFP, can improve the accuracy of diagnosis.

[0005] Long Qi et al. studied the relationship between brain tissue T2* and iron overload in children with severe β-thalassemia and divided the subjects into a mild group (serum ferritin level between 1000 and 2500 μg / mL) and a severe group (serum ferritin level>2500 μg / mL) according to the serum ferritin level (Long Qi, Wang Wuni, Chen Guangfu. T2* in brain tissue of children with severe β-thalassemia *Research on the relationship between ferritin and iron overload and its pathogenesis [J]. Modern Hospital, 2019, 19(01): 70-73.). The latest version of the industry standard for ferritin quantitative detection kit requires the linear range of detection to be no narrower than [10,500] ng / mL (YYT 1456-2016 Ferritin quantitative detection reagent (kit)). This indicates that a wider detection linear range is required in practical applications.

[0006] CA15-3 is a breast tissue antigen protein that is "Y" shaped and secreted by normal or cancerous breast cells. Its molecular weight is about 400KDa (300-450KDa). CA15-3 is a glycoprotein of mucin 1 (MUC-1). Under normal circumstances, MUC-1 is only expressed at the top of the plasma membrane and has normal glycosylation. When cancer occurs, MUC-1 expression increases and its polarity disappears, thus extending to the entire plasma membrane surface, being recognized by the human immune system and forming a tumor antigen. In addition, the extracellular region of MUC-1 also loses its adhesion power under the action of proteolysis and merges into the blood, or MUC-1 undergoes alternative splicing during cancer and loses its transmembrane region and dissolves into the blood, which is the CA15-3 detected clinically.

[0007] CA15-3 exists in many adenocarcinoma cells and is the most important specific marker for breast cancer. The change in its content is closely related to the treatment effect. It is the best indicator for diagnosing and monitoring postoperative recurrence and observing the efficacy of breast cancer patients. Generally, the reference value of serum CA15-3 is <30U / mL.

[0008] Breast cancer often has elevated CA15-3. The sensitivity is low in the early stage of breast cancer, about 60%, while the positive rate of metastatic breast cancer can reach 80%. In Europe, CA15-3 is often used as an auxiliary diagnostic indicator for breast cancer, and is also used for postoperative follow-up, monitoring tumor recurrence and metastasis. However, other tumors such as lung cancer, kidney cancer, colon cancer, pancreatic cancer, ovarian cancer, liver cancer, etc. may also have different degrees of elevation. The rise and fall of the patient's serum CA15-3 level is parallel to the changes in breast cancer. It is an important signal of recurrence and metastasis, and this signal is issued earlier than the appearance of clinical symptoms and the time of detection of recurrence and metastasis by ultrasound, X-ray or CT. According to analysis and research, there is a consistency between the changes in serum CA15-3 levels of breast cancer patients and their local lymph nodes and distant metastasis. In particular, those with distant metastases have significantly increased CA15-3 expression levels and positive rates. Therefore, CA15-3 has the function of monitoring breast cancer metastasis. If its serum level continues to rise, chemotherapy, radiotherapy or endocrine therapy should be started or strengthened, or switched to endocrine therapy.

[0009] Therefore, achieving high-sensitivity and precision protein content detection is the focus of current research and has important application prospects. Summary of the invention

[0010] In view of the shortcomings of traditional (colorimetric) enzyme-linked immunosorbent assay technology, the purpose of the present invention is to provide a (fluorescence) enzyme-linked immunosorbent assay kit and its application in detecting protein content; the present invention is based on a new type of fluorescent molecule of "aggregation-induced emission", which has the characteristics of strong resistance to photobleaching and high luminescence efficiency, so the present invention adopts the principle of enzyme-linked immunosorbent assay and uses the "aggregation-induced emission" type fluorescent molecule as a substrate to construct an enzyme-linked immunosorbent assay method.

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention discloses a (fluorescent) enzyme-linked immunofluorescence detection kit and its application in detecting the content of one or more proteins in blood or other tissue fluids. The method is designed based on the principle of double antibody sandwich method. The capture antibody is coated on a solid phase substrate or medium. After adding a sample containing an object to be detected and a detection antibody labeled with alkaline phosphatase, a complex of a double antibody sandwich structure is formed. After washing to remove free components, an aggregation-induced luminescent fluorescent substrate is added. The fluorescent signal generated by the enzymatic reaction is proportional to the concentration of the object to be detected in the sample. The substrate used to coat the capture antibody includes a polystyrene microplate and magnetic beads. The detection kit developed using this method presents two forms due to the different selection of the capture antibody solid phase medium.

[0013] An enzyme-linked immunofluorescence assay kit includes two forms:

[0014] (1) comprising a pre-coated microplate, a calibrator, a quality control product, an alkaline phosphatase labeled antibody, an aggregation-induced luminescence fluorescent substrate solution, a detection buffer, and a washing solution; the microplate is pre-coated with a capture antibody for capturing the object to be detected in the sample; the calibrator and the quality control product are prepared from genetically engineered recombinant proteins of the object to be detected or extracted human natural proteins of different concentrations, the calibrator includes at least 5 concentrations, and the quality control product includes at least 1 concentration; or

[0015] (2) It includes a pre-treated microplate / reaction tube / reaction cup, a magnetic bead solution, a calibrator, a quality control product, an alkaline phosphatase-labeled antibody, an aggregation-induced luminescence fluorescent substrate solution, a detection buffer, and a washing solution; the magnetic beads are pre-coated with a capture antibody for capturing the object to be detected in the sample; the calibrator and the quality control product are prepared from genetically engineered recombinant proteins of the object to be detected or extracted human natural proteins of different concentrations, the calibrator includes at least 5 concentrations, and the quality control product includes at least 1 concentration.

[0016] Preferably, the titer range of the alkaline phosphatase labeled antibody is 1:(400-1000); further preferably, the titer range of the alkaline phosphatase labeled antibody is 1:400.

[0017] Preferably, the pre-coated capture antibody and the alkaline phosphatase-labeled detection antibody should have different binding sites for the object to be detected.

[0018] Preferably, the aggregation-induced emission type fluorescent substrate is TPE-phos or AE-phos; the aggregation-induced emission type fluorescent substrate is catalyzed by alkaline phosphatase to generate new product molecules, which then aggregate to form micro-aggregates, emitting strong fluorescence under excitation light. Further preferably, the working concentration range of the substrate solution TPE-phos is 80 μmol / L to 100 μmol / L, and the working concentration range of the substrate solution AE-phos is 400 μmol / L to 500 μmol / L.

[0019] Preferably, the substrate solution is ready-to-use, or is prepared from a substrate stock solution via a substrate diluent.

[0020] Preferably, the capture antibody is a mouse monoclonal antibody to the protein.

[0021] Preferably, the concentration of the calibrator is 0-1000 ng / mL; the concentration of the quality control product is 50-500 ng / mL.

[0022] Preferably, the preparation of the pre-coated microplate comprises the following steps:

[0023] 1) Take the capture antibody and dilute it with CBS buffer;

[0024] 2) Add the diluted antibody solution to each well of the microplate, let it stand in a refrigerator at 2-8°C or at room temperature at 18-25°C for 8-16 hours, then discard the solution in the wells, add 200 μL of 1% BSA solution to each well, let it stand at room temperature for 1-2 hours, then discard it, dry it, and seal it in an aluminum foil bag.

[0025] More preferably, the concentration of the antibody solution obtained after dilution is in the range of 5.0 to 10.0 μg / mL; more preferably 5.0 μg / mL.

[0026] Preferably, the preparation of the magnetic beads pre-coated with capture antibodies comprises the following steps:

[0027] 1) Take the carboxyl magnetic bead solution, retain the beads by magnetic force and discard the supernatant; vortex and wash with pre-cooled MES buffer at least 3 times;

[0028] 2) Discard the supernatant of the magnetic beads, add NHS solution, and oscillate to disperse evenly; then add EDC solution and oscillate to mix; continue mixing on a four-dimensional mixer at room temperature for 20 to 40 minutes, oscillating and mixing once every 2 to 5 minutes during this period;

[0029] 3) Magnetic retention of magnetic beads, discard the supernatant, add pre-cooled MES buffer and shake to wash at least 2 times, and finally resuspend in pre-cooled MES buffer;

[0030] 4) Transfer the magnetic bead suspension obtained in 3) to the pre-cooled capture antibody solution dissolved in MES buffer at least 3 times. After each addition of the magnetic bead suspension, oscillate and mix immediately. After completion, let it stand for 10 to 15 minutes, and mix it again every 2 to 4 minutes. Finally, continue to gently oscillate and mix the suspension at room temperature for 90 to 120 minutes.

[0031] 5) Magnetic retention of magnetic beads and discarding of supernatant;

[0032] 6) Add blocking solution, gently shake and mix, and react at room temperature for 20 to 40 minutes;

[0033] 7) Discard the supernatant after magnetic separation, add washing solution, and wash at least 3 times with a pipette;

[0034] 8) Discard the supernatant after magnetic separation, add preservation solution to adjust the concentration of antibody-coupled magnetic beads to 2-3 mg / mL, gently shake and mix, and store at 2-8°C for later use.

[0035] The application of the above-mentioned enzyme-linked immunofluorescence detection kit in detecting protein content.

[0036] Preferably, the protein is one or more proteins in blood or other tissue fluids.

[0037] Preferably, the detection method includes a one-step method and a two-step method.

[0038] Preferably, the protein is ferritin, glycoprotein CA15-3, etc.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The invention relates to a protein detection kit, which adopts a double antibody sandwich enzyme-linked immunofluorescence method. The protein detection kit prepared by the preparation method of the invention has good sensitivity and precision when used. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a 3D diagram of the inner and outer packaging of the product of the present invention.

[0042] Figure 2 It is a detection standard curve diagram of the kit in Example 1 of the present invention.

[0043] Figure 3 It is a detection standard curve diagram of the kit in Example 4 of the present invention.

[0044] Figure 4This is a detection standard curve diagram of the kit in Example 7 of the present invention.

[0045] Figure 5 is the structural formula of the aggregation-induced emission fluorescent substrate TPE-phos used in the examples of the present invention. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0047] The preparation method of the enzyme-linked immunofluorescence method protein detection kit of the present invention comprises the following steps:

[0048] 1) preparing a microplate pre-coated with a first mouse anti-protein monoclonal antibody or a first mouse anti-protein monoclonal antibody coupled to magnetic beads;

[0049] 2) Preparation of alkaline phosphatase-labeled mouse anti-protein monoclonal antibody;

[0050] 3) preparing protein calibrator preparations;

[0051] 4) Preparation of protein control preparations;

[0052] 5) preparing substrate solution;

[0053] 6) preparing detection buffer;

[0054] 7) Prepare concentrated washing solution.

[0055] Preferably, the preparation of the microplate coated with mouse anti-protein monoclonal antibody comprises the following steps:

[0056] 1) Take mouse anti-protein monoclonal antibody and dilute it with 10mmol / L CBS buffer (pH9.6);

[0057] 2) After dilution, the concentration of the obtained antibody solution is about 5.0 μg / mL. Then add 100 μL to each well of the microplate, store in a refrigerator at 2-8°C or at room temperature (18-25°C) overnight, then discard the solution in the wells, add 200 μL of 1% BSA solution to each well, let it stand at room temperature for 1-2 hours, then discard it, move the microplate to a drying room, drain it, and seal it in an aluminum foil bag.

[0058] Preferably, the preparation of the first mouse anti-protein monoclonal antibody coupled to magnetic beads comprises the following steps:

[0059] 1) Take 0.5 mg of evenly dispersed carboxyl magnetic bead solution into a 2.0 mL round-bottom centrifuge tube, retain the magnetic beads by magnetic force and discard the supernatant; wash with pre-cooled MES buffer 200 μL each time by vortexing and oscillating for 3 times;

[0060] 2) Discard the supernatant of the magnetic beads, quickly add 100 μL NHS solution, and immediately oscillate to disperse evenly; then add 100 μL EDC solution, and immediately oscillate to mix; continue mixing on a four-dimensional mixer at room temperature for 30 minutes, oscillating and mixing once every 2 to 5 minutes during this period;

[0061] 3) Magnetic retention of magnetic beads, discard the supernatant, add 200 μL pre-cooled MES buffer each time, shake and wash twice, and finally resuspend in 100 μL pre-cooled MES buffer.

[0062] 4) Take 10 μg (0.5 mg / mL) of pre-cooled antibody solution dissolved in MES buffer into a round-bottom centrifuge tube; transfer the activated magnetic bead suspension in 3) into the tube three times (maintain the suspension at about 4°C); after each addition of magnetic beads, immediately shake gently to mix; after completion, continue to keep at about 4°C for 10 to 15 minutes, and mix again every 3 minutes; finally, continue to shake gently to mix the suspension at room temperature for about 90 minutes.

[0063] 5) Retain the beads magnetically and discard the supernatant.

[0064] 6) Add 200 μL of blocking solution (1.0 M glycine), mix by gentle shaking, and react at room temperature for about 30 minutes.

[0065] 7) After magnetic separation, discard the supernatant, add 200 μL of washing solution (0.01 M PBS buffer) each time, and wash three times with a pipette.

[0066] 8) Magnetic separation: discard the supernatant, add preservation solution (20 mM HEPES buffer) to adjust the concentration of antibody-coupled magnetic beads to 2.5 mg / mL, gently shake and mix, and store at 2-8°C for later use.

[0067] The following Examples 1 to 6 are specifically described by using an enzyme-linked immunofluorescence assay kit for detecting ferritin, and Examples 7 to 9 are specifically described by using an enzyme-linked immunofluorescence assay kit for detecting carbohydrate antigen CA15-3.

[0068] Example 1

[0069] (a) The Fer detection kit described in this example (such as Figure 1 ) are as follows:

[0070] 1. Microplate (pre-coated with primary mouse anti-Fer monoclonal antibody)

[0071] The strip microplate (12 strips × 8 wells) pre-coated with mouse anti-Fer monoclonal antibody is placed in the frame, and the microplate material is PS (polystyrene), which has the advantages of high strength, fatigue resistance, dimensional stability, and small creep;

[0072] 2. Fer Calibrator

[0073] The Fer calibrator is a solution of extracted human natural Fer dissolved in phosphate buffer. Its six concentrations are 0, 40.0, 76.9, 120.0, 254.9, and 1000.0 ng / mL, numbered 1-6, and each concentration is packaged in one bottle, for a total of 6 bottles;

[0074] 3.Fer quality control products

[0075] Fer quality control product is a solution of extracted human natural Fer dissolved in phosphate buffer. There are 2 concentrations, about 50.0 ng / mL and 250.0 ng / mL, labeled 1 and 2 respectively. Each concentration is packaged in one bottle, for a total of 2 bottles;

[0076] 4. Detection Buffer

[0077] 10mmol / L PBS buffer that can be used directly, 1 bottle in total, each bottle of test buffer is at least 20.0mL;

[0078] 5. Alkaline phosphatase labeled secondary mouse anti-Fer monoclonal antibody

[0079] This enzyme-labeled antibody is dissolved in PBS buffer solution containing preservatives, a total of 1 tube, the volume is at least 0.2mL;

[0080] 6. Substrate solution

[0081] The directly usable Figure 5 ) Tris buffer solution, 1 bottle, volume at least 10.0 mL;

[0082] 7. Concentrated lotion

[0083] Concentrated washing buffer containing detergent and preservative, 1 bottle in total, diluted 10 times with purified water when used, the volume is at least 20.0mL;

[0084] 8.Sealing film

[0085] Adhesive film for sealing microplates during incubation, 2 pieces;

[0086] (II) The preparation process of the Fer detection kit described in this embodiment is as follows:

[0087] 1. Prepare microplate pre-coated with primary mouse anti-Fer monoclonal antibody

[0088] Mouse anti-Fer monoclonal antibody (IgG) was diluted with 10 mmol / L CBS buffer (pH 9.6), and the concentration of the obtained antibody solution was about 5.0 μg / mL; then 100 μL was added to each well of the microplate, and placed in a 2-8°C refrigerator or at room temperature (18-25°C) overnight, and then the solution in the well was discarded, and 200 μL of 1% BSA solution was added to each well, and the solution was discarded after standing at room temperature for 1-2 hours. The microplate was moved to a drying room for drying, and then sealed in an aluminum foil bag.

[0089] 2. Preparation of alkaline phosphatase labeled secondary mouse anti-Fer monoclonal antibody solution

[0090] A commercial labeling kit (such as the Alkaline Phosphatase Labeling Kit-NH2 from DOJINDO LABORATORISE) was used to label mouse anti-Fer monoclonal antibody (IgG) according to its instructions.

[0091] 3. Preparation of Fer Calibrator Preparation

[0092] Take the purchased extracted human natural Fer solution, calibrate the concentration with the national standard, and dilute it with 10mmol / L PBS buffer to the Fer concentration of 0, 40.0, 76.9, 120.0, 254.9, and 1000.0ng / mL, respectively. Take 0.4mL of each aliquot and seal it for storage.

[0093] 4. Preparation of Fer quality control preparation

[0094] Take the purchased extracted human natural Fer solution, calibrate the concentration with the national standard product, and dilute it with 10mmol / L PBS buffer. The Fer concentration in quality control product 1 is about 50.0ng / mL, and the Fer concentration in quality control product 2 is about 250.0ng / mL. 0.1% BSA and 0.01% Proclin 300 are added as stabilizers and preservatives, and 0.4mL of each is taken and packaged and sealed for storage.

[0095] 5. Prepare substrate solution

[0096] The TPE-phos solution dissolved in DMSO was diluted with 1.0 mol / L Tris buffer (pH 8.0) to a final concentration of 100 μM.

[0097] 6. Others

[0098] The detection buffer (10 mM PBS, pH 7.4) and concentrated washing solution (100 mM PBST, 10×) were routine laboratory test solutions, which were prepared by ourselves or purchased from other commercial companies.

[0099] Example 2

[0100] The Fer reference product was tested using the kit described in Example 1.

[0101] The kit of the present invention adopts double antibody sandwich magnetic enzyme-linked immunofluorescence method, which is based on a method in which Fer antigen is first combined with a monoclonal antibody coated on a microplate and then combined with an alkaline phosphatase-labeled Fer antibody.

[0102] First, the Fer in the added reference / quality control / calibrator is combined with the solid-phase antibody (first mouse anti-Fer monoclonal antibody IgG) in the microplate, and then the mouse anti-Fer monoclonal antibody labeled with alkaline phosphatase is added to selectively bind the target Fer to form a sandwich complex. After washing, the substrate solution is added. The substrate can be excited to emit fluorescence after enzyme catalysis. The intensity of the fluorescence is positively correlated with the Fer in the specimen. The relative fluorescence intensity is measured at 490nm on a multifunctional microplate reader.

[0103] 1. Inspection operation. Before use, balance all solutions to room temperature (18-25°C), determine the number of microplates required for the experiment, set two parallel wells for each sample, place an appropriate number of microplates on a plastic frame, seal the unused microplate strips together with the desiccant in an aluminum foil bag and return them to the original environment for storage. This kit can be used in either a one-step or two-step method;

[0104] A. One-step method The specific steps are as follows:

[0105] 1) Add 50 μL of Fer calibrator, quality control and reference to the appropriate wells, then add 50 μL of alkaline phosphatase-labeled mouse anti-Fer antibody solution, cover the microplate with a sealing film, and incubate at room temperature for 1 hour.

[0106] 2) Cleaning

[0107] Add 250 μL of diluted washing solution to each well and wash three times. After the last wash, pat dry on absorbent paper and discard the residual liquid.

[0108] 3) Add substrate solution and incubate

[0109] Add 100 μL of substrate solution to each well, cover with a sealing film, and incubate at room temperature with shaking at 200 rpm for 30 to 60 minutes.

[0110] 4) Determination of relative fluorescence intensity

[0111] RFU was measured at 490 nm (excitation wavelength 340 nm) on a multifunctional microplate reader.

[0112] B. The specific steps of the two-step method are as follows:

[0113] 1) One-time incubation

[0114] Add 100 μL of Fer calibrator, quality control and reference to appropriate wells, cover the microplate with a sealing film, and incubate at room temperature with shaking at 200 rpm for 1 hour.

[0115] 2) Cleaning

[0116] Add 250 μL of diluted washing solution to each well and wash three times. After the last wash, pat dry on absorbent paper and discard the residual liquid.

[0117] 3) Secondary incubation

[0118] 100 μL of alkaline phosphatase-labeled mouse anti-Fer antibody solution was added to each well, covered with a sealing film, and incubated at room temperature with shaking at 200 rpm for 1 hour.

[0119] 4) Cleaning

[0120] Same as 2).

[0121] 5) Add substrate solution and incubate

[0122] Add 100 μL of substrate solution to each well, cover with sealing film, and incubate at room temperature with shaking at 200 rpm for 30 to 60 minutes.

[0123] 6) Determination of relative fluorescence intensity

[0124] RFU was measured at 490 nm (excitation wavelength 340 nm) on a multifunctional microplate reader.

[0125] 2. Detection range

[0126] If the RFU of the sample to be tested is higher than 6 of the calibrator, it is recommended to dilute the sample 1:11 (1+10, such as 10μL sample + 100μL test buffer) and retest.

[0127] Example 3

[0128] Example 2 Test kit quality analysis (two-step method)

[0129] 1. Standard curve

[0130] The concentration and fluorescence intensity of the calibrators are shown in Table 1:

[0131] Table 1

[0132] Calibrator concentration (X value) RFU(Y value) 0 3664 40.0 24024 76.9 63302 120.0 103148 254.9 188994 1000.0 252771

[0133] Note: The multifunctional microplate reader used for the test is the American Berton H1MF, and the fluorescence test mode is the extended mode.

[0134] The test result RFU was used to make a four-parameter curve regression on the calibration sample concentration, and the obtained standard curve was as follows Figure 2As shown; where the four-parameter regression equation is: y = (AD) / [1 + (x / C) ^ B] + D

[0135] A=261139.2; B=-1.81098; C=152.5276; D=3675.461

[0136] r^2=0.9999.

[0137] 2. Sensitivity

[0138] The kit component detection buffer was measured 20 times, and the concentration corresponding to the mean value of the test result RFU plus two times the standard deviation was calculated. The results are shown in Table 2:

[0139] Table 2

[0140]

[0141] 3. Precision

[0142] The kit was used to test 2 Fer reference products 10 times each, and the results are shown in Table 3:

[0143] Table 3

[0144]

[0145] The sensitivity of ferritin detection in Table 2 and the precision in Table 3 were calculated by the calibration curve established with the data shown in Table 1, and the sensitivity was 3.1 ng / mL (less than the industry standard requirement of 5 ng / mL), and the precision was 6.7% and 5.7% (less than the industry standard requirement of CV < 15%), respectively. This can meet the requirements of clinical application.

[0146] Example 4

[0147] (I) The main components of the Fer detection kit described in this example are as follows:

[0148] 1. Magnetic bead solution (first mouse anti-Fer monoclonal antibody coupled to magnetic beads)

[0149] Mouse anti-Fer monoclonal antibody coupled to magnetic beads solution, 1 bottle, volume at least 10mL;

[0150] 2. Fer Calibrator

[0151] The Fer calibrator is a solution of extracted human natural Fer dissolved in phosphate buffer. Its six concentrations are 0, 15.6, 62.5, 125.0, 250.0 and 500 ng / mL, numbered 1-6, and each concentration is packaged in one bottle, for a total of 6 bottles;

[0152] 3.Fer quality control products

[0153] Fer quality control product is a solution of extracted human natural Fer dissolved in phosphate buffer. There are 2 concentrations, about 20.0ng / mL and 200.0ng / mL, labeled 1 and 2 respectively. Each concentration is packaged in one bottle, for a total of 2 bottles;

[0154] 4. Detection Buffer

[0155] 10mmol / L PBS buffer that can be used directly, 1 bottle in total, each bottle of test buffer is at least 20.0mL;

[0156] 5. Alkaline phosphatase labeled secondary mouse anti-Fer monoclonal antibody

[0157] This enzyme-labeled antibody is dissolved in PBS buffer solution containing preservatives, a total of 1 tube, the volume is at least 0.2mL;

[0158] 6. Substrate solution

[0159] The directly usable Figure 5 ) Tris buffer solution, 1 bottle, volume at least 10.0 mL;

[0160] 7. Concentrated lotion

[0161] Concentrated washing buffer containing detergent and preservative, 1 bottle in total, diluted 10 times with purified water when used, the volume is at least 20.0mL;

[0162] 10. Pre-treat the microplate

[0163] Microplate (12 strips × 8 wells), made of PS (polystyrene), blocked with 1% BSA solution;

[0164] 11.Sealing film

[0165] Adhesive film for sealing microplates during incubation, 2 pieces;

[0166] (II) The preparation method of the Fer detection kit described in this embodiment adopts the labeling method established by this laboratory, and the process is as follows:

[0167] 1. Prepare the first mouse anti-Fer monoclonal antibody coupled magnetic bead solution

[0168] 1) Take 0.5 mg of evenly dispersed carboxyl magnetic bead solution into a 2.0 mL round-bottom centrifuge tube, retain the magnetic beads by magnetic force and discard the supernatant; wash with pre-cooled MES buffer 200 μL each time by vortexing and oscillating for 3 times;

[0169] 2) Discard the supernatant of the magnetic beads, quickly add 100 μL NHS solution, and immediately oscillate to disperse evenly; then add 100 μL EDC solution, and immediately oscillate to mix; continue mixing on a four-dimensional mixer at room temperature for 30 minutes, oscillating and mixing once every 2 to 5 minutes during this period;

[0170] 3) Magnetic retention of magnetic beads, discard the supernatant, add 200 μL pre-cooled MES buffer each time, shake and wash twice, and finally resuspend in 100 μL pre-cooled MES buffer.

[0171] 4) Take 10 μg (0.5 mg / mL) of pre-cooled antibody solution dissolved in MES buffer into a round-bottom centrifuge tube; transfer the activated magnetic bead suspension in 3) into the tube three times (maintain the suspension at about 4°C); after each addition of magnetic beads, immediately shake gently to mix; after completion, continue to keep at about 4°C for 10 to 15 minutes, and mix again every 3 minutes; finally, continue to shake gently to mix the suspension at room temperature for about 90 minutes.

[0172] 5) Retain the beads magnetically and discard the supernatant.

[0173] 6) Add 200 μL of blocking solution (1.0 M glycine), mix by gentle shaking, and react at room temperature for about 30 minutes.

[0174] 7) After magnetic separation, discard the supernatant, add 200 μL of washing solution (0.01 M PBS buffer) each time, and wash three times with a pipette.

[0175] 8) Magnetic separation: discard the supernatant, add preservation solution (20 mM HEPES buffer) to adjust the concentration of antibody-coupled magnetic beads to 2.5 mg / mL, gently shake and mix, and store at 2-8°C for later use.

[0176] 2. Preparation of alkaline phosphatase labeled secondary mouse anti-Fer monoclonal antibody solution

[0177] As described in Example 1, labeling was performed using a commercial labeling kit according to its instructions.

[0178] 3. Preparation of Fer Calibrator Preparation

[0179] Take the purchased extracted human natural Fer solution, calibrate the concentration with the national standard, and dilute it with 10mmol / L PBS buffer to the Fer concentrations of 0, 15.6, 62.5, 125.0, 250.0 and 500ng / mL, respectively. Take 0.4mL of each aliquot and seal it for storage.

[0180] 4. Preparation of Fer quality control preparation

[0181] Take the purchased extracted human natural Fer solution, calibrate the concentration with the national standard, and dilute it with 10mmol / L PBS buffer. The Fer concentration in quality control product 1 is about 20.0ng / mL, and the Fer concentration in quality control product 2 is about 200.0ng / mL. Add 0.1% BSA and 0.01% Proclin300 as stabilizers and preservatives, take 0.4mL of each, and seal it for storage.

[0182] 5. Prepare substrate solution

[0183] The TPE-phos solution dissolved in DMSO was diluted with 10 mmol / L Tris buffer (pH 8.0) to a final concentration of 100 μM.

[0184] 6. Others

[0185] The detection buffer (10 mM PBS, pH 7.4) and concentrated washing solution (100 mM PBST, 10×) were routine laboratory test solutions, which were prepared by ourselves or purchased from other commercial companies.

[0186] Example 5

[0187] The Fer reference product was tested using the kit described in Example 4.

[0188] The kit of the present invention adopts a double antibody sandwich magnetic enzyme-linked immunofluorescence method, which is based on a method in which a Fer antigen is first combined with a monoclonal antibody coupled to magnetic beads and then combined with an alkaline phosphatase-labeled Fer antibody.

[0189] First, the Fer in the added reference / quality control / calibrator is combined with the solid-phase antibody (first mouse anti-Fer monoclonal antibody IgG) on the magnetic beads, and then the mouse anti-Fer monoclonal antibody labeled with alkaline phosphatase is added to selectively bind the target Fer to form a sandwich complex. After washing, the substrate solution is added. The substrate can be excited to emit fluorescence after enzyme catalysis. The intensity of the fluorescence is positively correlated with the Fer in the specimen. The relative fluorescence intensity is measured at 490nm on a multifunctional microplate reader.

[0190] 1. Inspection operation. Before use, balance all solutions to room temperature (18-25°C), determine the number of microporous strips required for the experiment, set two parallel holes for each sample, place an appropriate number of microporous strips on a plastic frame, seal the unused microporous strips together with the desiccant in an aluminum foil bag and return them to the original environment for storage. This kit can be used in either a one-step method or a two-step method. This embodiment selects a one-step method, and the specific steps are as follows:

[0191] 1) First, add 100 μL of magnetic bead solution to each well, then add 50 μL of Fer calibrator, quality control and reference to the appropriate wells, and then add 50 μL of alkaline phosphatase-labeled mouse anti-Fer antibody solution to each well. Cover the microplate with a sealing film and incubate at room temperature at 200 rpm for 1 hour.

[0192] 2) Cleaning

[0193] Add 250 μL of diluted washing solution to each well and wash three times.

[0194] 3) Add substrate solution and incubate

[0195] Add 100 μL of substrate solution to each well, cover with a sealing film, and incubate at room temperature with shaking at 200 rpm for 30 to 60 minutes.

[0196] 4) Determination of relative fluorescence intensity

[0197] RFU was measured at 490 nm (excitation wavelength 340 nm) on a multifunctional microplate reader.

[0198] Note: The magnetic plate must be used correctly during cleaning.

[0199] 2. Detection range

[0200] If the RFU of the sample to be tested is higher than 6 of the calibrator, it is recommended to dilute the sample 1:11 (1+10, such as 10μL sample + 100μL test buffer) and retest.

[0201] Example 6

[0202] Example 5: Quality analysis of the kit tested

[0203] 1. Standard curve

[0204] The concentration and fluorescence intensity of the calibrators are shown in Table 4:

[0205] Table 4

[0206] Calibrator concentration (X value) RFU(Y value) 0 212 15.6 406 62.5 943 125.0 1358 250.0 1794 500.0 1845

[0207] The test result RFU was used to make a four-parameter curve regression on the calibration sample concentration, and the obtained standard curve was as follows Figure 3 shown.

[0208] Among them, the four-parameter regression equation: y=(AD) / [1+(x / C)^B]+D

[0209] A=1997.46735; B=-1.48184; C=80.58513; D=232.85348

[0210] r^2=0.996.

[0211] 2. Sensitivity

[0212] Take the test buffer of the kit components, repeat the measurement 20 times, and calculate the concentration corresponding to the average value of the test result RFU plus two times the standard deviation. The results are shown in Table 5.

[0213] Table 5

[0214]

[0215] 3. Precision

[0216] The kit was used to test two Fer reference products 10 times each, and the coefficient of variation was calculated. The results are shown in Table 6.

[0217] Table 6

[0218]

[0219] The sensitivity of ferritin detection in Table 5 and the precision in Table 6 were calculated by the calibration curve established with the data shown in Table 4, and the sensitivity was 4.4 ng / mL (less than the industry standard requirement of 5 ng / mL), and the precision was 5.3% and 7.2% (less than the industry standard requirement of CV < 15%), respectively. This can meet the requirements of clinical application.

[0220] Example 7

[0221] (I) The main components of the carbohydrate antigen CA15-3 detection kit described in this example are as follows:

[0222] 1. Microplate (pre-coated with primary mouse anti-CA15-3 monoclonal antibody)

[0223] The strip microplate (12 strips × 8 wells) pre-coated with mouse anti-CA15-3 monoclonal antibody is placed in the frame, and the microplate material is PS (polystyrene), which has the advantages of high strength, fatigue resistance, dimensional stability, and small creep;

[0224] 2. CA15-3 Calibrator

[0225] The CA15-3 calibrator is a solution of extracted human natural CA15-3 dissolved in phosphate buffer. Its seven concentrations are 0, 7.8, 31.2, 62.5, 125.0, 250.0, and 500.0 U / mL, numbered 1-7, and each concentration is packaged in one bottle, for a total of seven bottles;

[0226] 3. CA15-3 quality control product

[0227] CA15-3 quality control product is a solution of extracted human natural CA15-3 dissolved in phosphate buffer. There are 2 concentrations, 50.0U / mL and 200.0U / mL, labeled 1 and 2 respectively. Each concentration is packaged in one bottle, for a total of 2 bottles;

[0228] 4. Detection Buffer

[0229] 10mmol / L PBS buffer that can be used directly, 1 bottle in total, each bottle of test buffer is at least 20.0mL;

[0230] 5. Alkaline phosphatase labeled secondary mouse anti-CA15-3 monoclonal antibody

[0231] This enzyme-labeled antibody is dissolved in PBS buffer solution containing preservatives, a total of 1 tube, the volume is at least 0.2mL;

[0232] 6. Substrate solution

[0233] The directly usable Figure 5 ) Tris buffer solution, 1 bottle, volume at least 10.0 mL;

[0234] 7. Concentrated lotion

[0235] Concentrated washing buffer containing detergent and preservative, 1 bottle in total, diluted 10 times with purified water when used, the volume is at least 20.0mL;

[0236] 8.Sealing film

[0237] Adhesive film for sealing microplates during incubation, 2 pieces;

[0238] (ii) The preparation process of the CA15-3 detection kit described in this embodiment is as follows:

[0239] 1. Prepare microplate pre-coated with primary mouse anti-CA15-3 monoclonal antibody

[0240] Mouse anti-CA15-3 monoclonal antibody (IgG) was diluted with 10 mmol / L CBS buffer (pH 9.6) to obtain an antibody solution with a concentration of about 5.0 μg / mL. 100 μL was then added to each well of the microplate and placed in a 2-8°C refrigerator or at room temperature (18-25°C) overnight. The solution in the wells was then discarded. 200 μL of 1% BSA solution was then added to each well. The solution was discarded after standing at room temperature for 1-2 hours. The microplate was moved to a drying room for drying and sealed in an aluminum foil bag.

[0241] 2. Preparation of alkaline phosphatase labeled secondary mouse anti-CA15-3 monoclonal antibody solution

[0242] A commercial labeling kit (such as the ALP labeling kit Alkaline Phosphatase Labeling Kit-NH2 from DOJINDO LABORATORISE) was used to label mouse anti-CA15-3 monoclonal antibody (IgG) according to its instructions.

[0243] 3. Preparation of CA15-3 Calibrator Preparation

[0244] Take the purchased extracted human natural CA15-3 solution, calibrate the concentration with the national standard, and dilute it with 10 mmol / L PBS buffer to CA15-3 concentrations of 0, 7.8, 31.2, 62.5, 125.0, 250.0, and 500.0 U / mL, respectively. Take 0.4 mL of each aliquot and seal it for storage.

[0245] 4. Preparation of CA15-3 quality control preparation

[0246] Take the purchased extracted human natural CA15-3 solution, calibrate the concentration with the national standard product, and dilute it with 10 mmol / L PBS buffer. The CA15-3 concentration in quality control product 1 is about 50.0 U / mL, and the CA15-3 concentration in quality control product 2 is about 200.0 U / mL. Add 0.1% BSA and 0.01% Proclin 300 as stabilizers and preservatives, take 0.4 mL of each aliquot, and seal it for storage.

[0247] 5. Prepare substrate solution

[0248] The TPE-phos solution dissolved in DMSO was diluted with 1.0 mol / L Tris buffer (pH 8.0) to a final concentration of 100 μM.

[0249] 6. Others

[0250] The detection buffer (10 mM PBS, pH 7.4) and concentrated washing solution (100 mM PBST, 10×) were routine laboratory test solutions, which were prepared by ourselves or purchased from other commercial companies.

[0251] Example 8

[0252] The CA15-3 reference substance was tested using the kit described in Example 7.

[0253] The kit of the present invention adopts a double antibody sandwich magnetic enzyme-linked immunofluorescence method, which is based on a method in which a CA15-3 antigen is first combined with a monoclonal antibody coated on a microplate and then combined with an alkaline phosphatase-labeled CA15-3 antibody.

[0254] First, the CA15-3 in the added reference / quality control / calibrator is combined with the solid-phase antibody (first mouse anti-CA15-3 monoclonal antibody IgG) in the microplate, and then the mouse anti-CA15-3 monoclonal antibody labeled with alkaline phosphatase is added to selectively bind the target CA15-3 to form a sandwich complex, and then the substrate solution is added after washing. The substrate can be excited to emit fluorescence after enzyme catalysis, and the intensity of the fluorescence is positively correlated with the CA15-3 in the specimen. The relative fluorescence intensity is measured at 490nm on a multifunctional microplate reader.

[0255] 1. Inspection operation. Before use, balance all solutions to room temperature (18-25°C), determine the number of microplates required for the experiment, set two parallel holes for each sample, place an appropriate number of microplates on a plastic frame, seal the unused microplate strips together with the desiccant in an aluminum foil bag and return them to the original environment for storage. This kit uses a two-step method, the steps are as follows:

[0256] 1) One-time incubation

[0257] Add 100 μL of the prepared CA15-3 calibrator, quality control and reference products to the appropriate wells, cover the microplate with a sealing film, and incubate at room temperature with shaking at 200 rpm for 1 hour.

[0258] 2) Cleaning

[0259] Add 250 μL of diluted washing solution to each well and wash three times. After the last wash, pat dry on absorbent paper and discard the residual liquid.

[0260] 3) Secondary incubation

[0261] 100 μL of alkaline phosphatase-labeled mouse anti-CA15-3 antibody solution was added to each well, covered with a sealing film, and incubated at room temperature with shaking at 200 rpm for 1 hour.

[0262] 4) Cleaning

[0263] Same as 2).

[0264] 5) Add substrate solution and incubate

[0265] Add 100 μL of substrate solution to each well, cover with sealing film, and incubate at room temperature with shaking at 200 rpm for 10 to 30 minutes.

[0266] 6) Determination of relative fluorescence intensity

[0267] RFU was measured at 490 nm (excitation wavelength 340 nm) on a multifunctional microplate reader.

[0268] 2. Detection range

[0269] If the RFU of the sample to be tested is higher than 7 of the calibrator, it is recommended to dilute the sample 1:11 (1+10, such as 10μL sample + 100μL test buffer) and retest.

[0270] Example 9

[0271] Example 8 Test kit quality analysis (two-step method)

[0272] 1. Standard curve

[0273] The concentration and fluorescence intensity of the calibrators are shown in Table 7:

[0274] Table 7

[0275] Calibrator concentration (X value) RFU(Y value) 0 998 7.8 1382 31.2 3702 62.5 7708 125.0 13525 250.0 20104 500.0 26239

[0276] Note: The multifunctional microplate reader used for the test is the American Berton H1MF.

[0277] The test result RFU was used to make a four-parameter curve regression on the calibration sample concentration, and the obtained standard curve was as follows Figure 4 As shown; where the four-parameter regression equation is: y = (AD) / [1 + (x / C) ^ B] + D

[0278] A=28277.99485; B=-1.43698; C=138.62195; D=961.13705r^2=0.9998.

[0279] 2. Sensitivity

[0280] The kit component detection buffer was measured 20 times, and the concentration corresponding to the mean value of the test result RFU plus two times the standard deviation was calculated. The results are shown in Table 8:

[0281] Table 8

[0282]

[0283] 3. Precision

[0284] The kit was used to test 2 CA15-3 reference products 10 times each, and the results are shown in Table 9:

[0285] Table 9

[0286]

[0287] The sensitivity of CA15-3 detection in Table 8 and the precision in Table 9 were calculated by using the calibration curve established with the data shown in Table 7, and the sensitivity was 3.53 U / mL, and the precision was 5.3% and 6.9% respectively (less than the general requirement of CV < 15%), which can meet the requirements of clinical application.

[0288] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An enzyme-linked immunofluorescence detection kit, characterized in that: There are two forms: (1) It includes a pre-coated microplate, a calibrator, a quality control product, an alkaline phosphatase-labeled antibody, an aggregation-induced luminescence fluorescent substrate solution, a detection buffer, and a washing solution; the microplate is pre-coated with a capture antibody for capturing the object to be detected in the sample; the calibrator and the quality control product are prepared from genetically engineered recombinant proteins of the object to be detected or extracted human natural proteins of different concentrations, the calibrator includes at least 5 concentrations, and the quality control product includes at least 1 concentration; or (2) It includes a pre-treated microplate / reaction tube / reaction cup, a magnetic bead solution, a calibrator, a quality control product, an alkaline phosphatase labeled antibody, an aggregation-induced luminescence fluorescent substrate solution, a detection buffer, and a washing solution; the magnetic beads are pre-coated with a capture antibody for capturing the object to be detected in the sample; the calibrator and the quality control product are prepared from genetically engineered recombinant proteins of the object to be detected or extracted human natural proteins of different concentrations, the calibrator includes at least 5 concentrations, and the quality control product includes at least 1 concentration; The aggregation-induced emission fluorescent substrate is The aggregation-induced luminescent fluorescent substrate is catalyzed by alkaline phosphatase to generate new product molecules, which then aggregate to form tiny aggregates and emit strong fluorescence under excitation light.

2. The enzyme-linked immunofluorescence assay kit according to claim 1, characterized in that: The pre-coated capture antibody and the alkaline phosphatase-labeled detection antibody should have different binding sites for the object to be detected.

3. The enzyme-linked immunofluorescence assay kit according to claim 1, characterized in that: The substrate solution is ready-to-use, or is prepared from the substrate stock solution via a substrate diluent; the capture antibody is a mouse monoclonal antibody of the protein.

4. The enzyme-linked immunofluorescence assay kit according to claim 1, characterized in that: The substrate solution The working concentration range is 80μmol / L~100μmol / L.

5. The enzyme-linked immunofluorescence detection kit according to claim 1, characterized in that: The concentration of the calibrator is 0-1000 ng / mL; the concentration of the quality control is 50-500 ng / mL.

6. The enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The preparation of the pre-coated microplate comprises the following steps: 1) Take the capture antibody and dilute it with CBS buffer; 2) Add the diluted antibody solution to each well of the microplate, let it stand in a 2-8℃ refrigerator or at room temperature of 18-25℃ for 8-16 hours, then discard the solution in the wells, add 200μL of 1% BSA solution to each well, let it stand at room temperature for 1-2 hours, then discard it, dry it, and seal it in an aluminum foil bag.

7. The enzyme-linked immunofluorescence detection kit according to claim 1, characterized in that: The preparation of the magnetic beads pre-coated with capture antibodies comprises the following steps: 1) Take the carboxyl magnetic bead solution, retain the beads by magnetic force and discard the supernatant; vortex and wash with pre-cooled MES buffer at least 3 times; 2) Discard the supernatant of the magnetic beads, add NHS solution, and oscillate to disperse evenly; then add EDC solution and oscillate to mix; continue mixing on a four-dimensional mixer at room temperature for 20-40 minutes, oscillating and mixing once every 2-5 minutes during this period; 3) Magnetic retention of magnetic beads, discard the supernatant, add pre-cooled MES buffer and shake to wash at least 2 times, and finally resuspend in pre-cooled MES buffer; 4) Transfer the magnetic bead suspension obtained in 3) to the pre-cooled capture antibody solution dissolved in MES buffer at least 3 times. After adding the magnetic bead suspension each time, oscillate and mix immediately. After completion, let it stand for 10-15 minutes, and mix it again every 2-4 minutes. Finally, continue to gently oscillate and mix the suspension at room temperature for 90-120 minutes. 5) Magnetic retention of magnetic beads and discarding of supernatant; 6) Add blocking solution, gently shake and mix, and react at room temperature for 20-40 minutes; 7) Discard the supernatant after magnetic separation, add washing solution, and wash at least 3 times with a pipette; 8) Discard the supernatant after magnetic separation, add preservation solution to adjust the concentration of antibody-coupled magnetic beads to 2-3 mg / mL, gently shake and mix, and store at 2-8°C for later use.

8. Use of the enzyme-linked immunofluorescence detection kit according to any one of claims 1 to 7 in detecting protein content.

9. Use of the enzyme-linked immunofluorescence assay kit according to claim 8 in detecting protein content, characterized in that: Used in non-disease diagnosis and treatment fields, the detection methods include one-step method and two-step method; the proteins are ferritin and glycoprotein CA15-3.

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