A myoglobin antibody and its application and myoglobin detection product

Abstract

The present invention provides a myoglobin antibody, comprising protein a and / or protein b, wherein the protein a comprises a heavy chain complementary determining region whose amino acid sequence is sequentially shown in SEQ ID NO.1 to SEQ ID NO.3, and a light chain complementary determining region whose amino acid sequence is sequentially shown in SEQ ID NO.4 to SEQ ID NO.6; the protein b comprises a heavy chain complementary determining region whose amino acid sequence is sequentially shown in SEQ ID NO.7 to SEQ ID NO.9, and a light chain complementary determining region whose amino acid sequence is sequentially shown in SEQ ID NO.10 to SEQ ID NO.12. The myoglobin antibody provided by the present invention has good specificity, high biological activity, strong stability, high affinity with myoglobin, and can be used to prepare a product for detecting myoglobin.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a myoglobin antibody and an application thereof, as well as a myoglobin detection product. Background Art

[0002] Acute myocardial infarction (AMI) is an acute disease caused by acute blockage of the coronary artery, myocardial necrosis due to lack of blood supply, and damage to cardiac function. It has a high morbidity and mortality rate in clinical practice. The clinical manifestations of patients are squeezing pain or stuffiness in the precordial area lasting for more than 30 minutes, which may be accompanied by dyspnea. Severe cases often endanger life safety. Therefore, early diagnosis and early prediction of prognosis of such patients are crucial to improve patient prognosis in order to guide clinical individualized treatment measures. Traditional central laboratory testing (CLT) usually takes a long time (several hours) and requires large instruments and equipment and a large number of laboratory personnel. It is not suitable for the requirements of rapid emergency testing. In comparison, bedside testing technology (POCT) has the advantages of short detection cycle (TAT), portable instruments, simple operation, convenient and flexible use, and accurate results.

[0003] Myoglobin (Myo) is a low molecular weight heme protein present in myocardium and skeletal muscle. It increases 1-3 hours after the onset of myocardial infarction, reaches a peak value in 6-9 hours, and returns to normal in 18-30 hours. It is one of the earliest markers to increase in myocardial injury. Due to its short half-life, Myo negativity helps to exclude the diagnosis of AMI. Dynamic detection of secondary myoglobin levels can be used to diagnose the occurrence of acute myocardial infarction early, and can also observe whether there is re-infarction or re-expansion of infarction during the course of AMI. Myo is also a sensitive and accurate indicator for judging whether there is reperfusion during thrombolytic therapy. The biggest advantage is that it can greatly shorten the detection cycle (TAT). This is very important for the timely diagnosis and treatment of patients with acute myocardial infarction, because the diagnosis of myocardial infarction, monitoring of recurrent infarction, non-injury assessment of reperfusion and treatment are very time-sensitive.

[0004] In short, the bedside detection technology of myocardial markers is a diagnostic technology with broad prospects, especially in the field of emergency treatment. The correct application of POCT products has incomparable advantages over traditional methods for the timely diagnosis, disease monitoring, risk classification, prognosis assessment and treatment guidance of patients with acute myocardial infarction (AMI). Summary of the invention

[0005] The purpose of the present invention is to provide a myoglobin antibody, which specifically recognizes myoglobin; to provide the use of the myoglobin antibody in the preparation of a myoglobin detection product; to provide a myoglobin detection product, including a myoglobin detection test strip and a myoglobin detection kit, to solve the problems in the prior art.

[0006] In order to achieve the above object, the present invention provides a myoglobin antibody, comprising protein a and / or protein b:

[0007] The amino acid sequence of the heavy chain complementary determining region of the protein a is as follows: the amino acid sequence of CDR1-VH is shown in SEQ ID NO.1, the amino acid sequence of CDR2-VH is shown in SEQ ID NO.2, and the amino acid sequence of CDR3-VH is shown in SEQ ID NO.3;

[0008] The amino acid sequence of the light chain complementary determining region of the protein a is as follows: the amino acid sequence of CDR1-VL is shown in SEQ ID NO.4, the amino acid sequence of CDR2-VL is shown in SEQ ID NO.5, and the amino acid sequence of CDR3-VL is shown in SEQ ID NO.6;

[0009] The amino acid sequence of the heavy chain complementary determining region of the protein b is as follows: the amino acid sequence of CDR1-VH is shown in SEQ ID NO.7, the amino acid sequence of CDR2-VH is shown in SEQ ID NO.8, and the amino acid sequence of CDR3-VH is shown in SEQ ID NO.9;

[0010] The amino acid sequence of the light chain complementary determining region of the protein b is as follows: the amino acid sequence of CDR1-VL is shown in SEQ ID NO.10, the amino acid sequence of CDR2-VL is shown in SEQ ID NO.11, and the amino acid sequence of CDR3-VL is shown in SEQ ID NO.12.

[0011] Preferably, in any of the above items, the amino acid sequence of the heavy chain variable region of the protein a is as shown in SEQ ID NO.13.

[0012] Preferably, in any of the above items, the amino acid sequence of the light chain variable region of the protein a is as shown in SEQ ID NO.14.

[0013] Preferably, in any of the above items, the amino acid sequence of the heavy chain variable region of the protein b is as shown in SEQ ID NO.15.

[0014] Preferably, in any of the above items, the amino acid sequence of the light chain variable region of the protein b is as shown in SEQ ID NO.16.

[0015] Preferably, any of the above items is that the protein a or protein b is a mouse protein.

[0016] Preferably, in any of the above items, the amino acid sequence of the protein a light chain is as shown in SEQ NO ID.18.

[0017] Preferably, in any of the above items, the amino acid sequence of the heavy chain of protein a is as shown in SEQ NO ID.20.

[0018] Preferably, in any of the above items, the amino acid sequence of the protein b light chain is as shown in SEQ NO ID.22.

[0019] Preferably, in any of the above items, the amino acid sequence of the heavy chain of protein b is as shown in SEQ NO ID.23.

[0020] Preferably, in any of the above items, the amino acid sequence of the light chain constant region of protein a or protein b is as shown in SEQ NOID.19.

[0021] Preferably, in any of the above items, the amino acid sequence of the heavy chain constant region of the protein a or protein b is as shown in SEQ NOID.21.

[0022] The present invention also provides use of any of the above-mentioned myoglobin antibodies in the preparation of a myoglobin detection product.

[0023] The present invention also provides a myoglobin test strip, which comprises a base plate and a sample pad, a marking pad, a detection pad and a sample suction pad which are sequentially stacked on the base plate, wherein the marking pad contains any of the above-mentioned myoglobin antibodies, and the myoglobin antibodies are marked with a marker.

[0024] Preferably, the marker comprises at least one of colloidal gold, colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres.

[0025] Preferably, any of the above items has a particle size of colloidal gold of 40 to 100 nm; more preferably, 40, 50, 60, 80, 100 nm and ranges therebetween.

[0026] Preferably, any of the above items has a particle size of 100-300 nm; more preferably, 100, 200, 300 nm and ranges therebetween.

[0027] Preferably, any of the above items has a particle size of 100-300 nm of the time-resolved fluorescent microspheres; more preferably, 100, 200, 300 nm and ranges therebetween.

[0028] Preferably, any of the above items has a particle size of the quantum dot microspheres of 100-300 nm; more preferably, 100, 200, 300 nm and ranges therebetween.

[0029] Preferably, in any of the above items, the mass ratio of the myoglobin antibody to colloidal gold is: (0.04-0.32):1; more preferably, 0.04:1, 0.10:1, 0.15:1, 0.20:1, 0.25:1, 0.30:1, 0.32:1 and ranges therebetween.

[0030] Preferably, in any of the above items, the mass ratio of myoglobin antibody to colored microspheres is: (0.1-0.4):1; more preferably, it is 0.1:1, 0.2:1, 0.3:1, 0.4:1 and ranges therebetween.

[0031] Preferably, in any of the above items, the mass ratio of myoglobin antibody to time-resolved fluorescent microspheres is: (0.1-0.4):1; more preferably, it is 0.1:1, 0.2:1, 0.3:1, 0.4:1 and the range therebetween.

[0032] Preferably, in any of the above items, the mass ratio of myoglobin antibody to quantum dot microspheres is: (0.1-0.4):1; more preferably, it is 0.1:1, 0.2:1, 0.3:1, 0.4:1 and the range therebetween.

[0033] Preferably, any of the above items is provided with a detection line and a quality control line on the detection pad; the detection line is coated with the myoglobin antibody described in any of the above items; and the quality control line is coated with goat anti-mouse IgG polyclonal antibody.

[0034] Preferably, any of the above items is that the detection line is coated with 0.5-2 mg / mL of the myoglobin antibody; more preferably, 0.5, 1.0, 1.5, 2.0 mg / mL and ranges therebetween.

[0035] Preferably, any of the above items is coated with 0.5-2 mg / mL of goat anti-mouse IgG polyclonal antibody; more preferably, 0.5, 1.0, 1.5, 2.0 mg / mL and ranges therebetween.

[0036] Preferably, any of the above items is that the diluent for the myoglobin antibody and the goat anti-mouse IgG polyclonal antibody is a 10-50 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.1-1.0 g of trehalose; further preferably, the concentration of the PB buffer is 10, 20, 30, 40, 50 mM and ranges therebetween; further preferably, each 100 mL of the diluent contains 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 g and ranges therebetween of trehalose.

[0037] Any of the above items is preferably that a detection line and a quality control line are provided on the detection pad; the detection line is coated with the myoglobin antibody; the quality control line is coated with sheep anti-chicken IgY; the marking pad contains another myoglobin antibody, the myoglobin antibody on the marking pad is marked with a marker, and the marker is marked with chicken IgY.

[0038] Preferably, any of the above items is that the myoglobin antibody on the marking pad is different from the myoglobin antibody on the detection pad. Preferably, the myoglobin antibody on the marking pad is protein a, and the myoglobin antibody on the detection pad is protein b; preferably, the myoglobin antibody on the marking pad is protein b, and the myoglobin antibody on the detection pad is protein a.

[0039] The present invention provides a myoglobin detection kit, comprising any one of the above-mentioned myoglobin detection test strips and a shell, wherein the myoglobin detection test strip is arranged inside the shell.

[0040] Preferably, the shell includes an upper cover and a lower cover that are detachably connected.

[0041] Preferably, any one of the above items is that the upper cover is provided with an observation window and a sample addition hole.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The myoglobin antibody provided by the present invention has good specificity, high biological activity, strong stability, and high affinity with myoglobin, and can be used to prepare a product for detecting myoglobin.

[0044] The myoglobin test strip and kit provided by the present invention have the advantages of simple operation, rapid reaction, high sensitivity, strong specificity, suitability for rapid on-site detection, and economy and practicality. They can be used for rapid screening, and can quickly, accurately and safely indicate the risk of myocardial infarction in patients, so that patients can receive early diagnosis and early treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the electrophoresis diagram of the myoglobin monoclonal antibody in the preferred embodiment 1 of the present invention.

[0046] Figure 2 It is the upper cover of the myoglobin detection kit in the preferred embodiment 2 of the present invention.

[0047] Figure 3 It is the lower cover of the myoglobin detection kit in the preferred embodiment 2 of the present invention.

[0048] Figure 4 This is a structural diagram of the myoglobin detection test strip in preferred embodiment 2 of the present invention.

[0049] Figure 5 This is a display diagram of the detection results in preferred embodiment 3 of the present invention.

[0050] Icons: 1-Observation window; 2-Sample loading hole; 3-Test card strip loading area; 4-Bottom plate; 5-Test pad; 6-Sample suction pad; 7-Marking pad; 8-Sample pad; 9-Quality control line; 10-Test line. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below through the accompanying drawings and specific embodiments.

[0052] It should be noted that the "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy chain or light chain of an antibody. The variable domain of the heavy chain can be referred to as "VH". The variable domain of the light chain can be referred to as "VL". These domains are usually the most variable parts of the antibody and contain the antigen binding site. The light or heavy chain variable region is composed of a framework region interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The framework region of an antibody, that is, the combined framework region of the constituent light and heavy chains, plays a role in positioning and aligning the CDRs, which are primarily responsible for binding to the antigen.

[0053] "Framework" or "FR" regions mean the regions of an antibody variable domain excluding those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR1, FR2, FR3, and FR4) separated by CDRs.

[0054] Typically, the variable regions VL / VH of the heavy and light chains can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0055] In the present invention, CDR1-VH, CDR2-VH and CDR3-VH refer to the three hypervariable regions of the heavy chain variable region, respectively. Correspondingly, CDR1-VL, CDR2-VL and CDR3-VL refer to the three hypervariable regions of the light chain variable region, respectively.

[0056] In a first aspect, the present invention provides a myoglobin antibody selected from protein a and / or protein b;

[0057] The protein a includes heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH whose amino acid sequences are sequentially shown in SEQ ID NO.1 to SEQ ID NO.3, and light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL whose amino acid sequences are sequentially shown in SEQ ID NO.4 to SEQ ID NO.6;

[0058] The protein b includes heavy chain complementary determining regions CDR1-VH, CDR2-VH, and CDR3-VH whose amino acid sequences are shown in sequence as SEQ ID NO.7 to SEQ ID NO.9, and light chain complementary determining regions CDR1-VL, CDR2-VL, and CDR3-VL whose amino acid sequences are shown in sequence as SEQ ID NO.10 to SEQ ID NO.12.

[0059] The amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.12 are shown in Table 1.

[0060] Table 1

[0061]

[0062] The myoglobin antibody provided by the present invention has good specificity, high biological activity, strong stability, and high affinity with myoglobin, and can be used to prepare a product for detecting myoglobin.

[0063] In a preferred embodiment of the present invention, the protein a comprises a heavy chain variable region and a light chain variable region whose amino acid sequences are sequentially shown in SEQ ID NO.13 to SEQ ID NO.14;

[0064] The protein b includes a heavy chain variable region and a light chain variable region whose amino acid sequences are shown in SEQ ID NO.15 to SEQ ID NO.16;

[0065] Preferably, the myoglobin antibody is of murine origin.

[0066] The amino acid sequences shown in SEQ ID NO.13 to SEQ ID NO.14 are shown in Table 2.

[0067] Table 2

[0068]

[0069] Preferably, the amino acid sequences of the light chain, heavy chain and constant region of protein a (myoglobin monoclonal antibody 1), and the amino acid sequences of the light chain, heavy chain and constant region of protein b (myoglobin monoclonal antibody 2) are shown in Table 3.

[0070] Table 3

[0071]

[0072]

[0073] In a second aspect, the present invention provides the use of the myoglobin antibody in the preparation of a myoglobin detection product.

[0074] In a third aspect, the present invention provides a myoglobin test strip, the test strip comprising a base plate and a sample pad, a marking pad, a detection pad and a sample suction pad stacked in sequence on the base plate;

[0075] The label pad contains the myoglobin antibody, and the myoglobin antibody is labeled with a marker, and the marker can be detected to identify the location or concentration of the marker.

[0076] The myoglobin test strip provided by the present invention has the advantages of simple operation, rapid response, high sensitivity, strong specificity, suitability for rapid on-site detection, and economy and practicality. It can be used for rapid screening, and quickly, accurately and safely prompt patients of the risk of myocardial infarction, so that patients can receive early diagnosis and early treatment.

[0077] In a preferred embodiment of the present invention, the marker includes but is not limited to at least one of colloidal gold, colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres;

[0078] Preferably, the particle size of the colloidal gold may be, for example, but not limited to, 40 nm, 60 nm, 80 nm or 100 nm;

[0079] Preferably, the particle size of the colored microspheres, time-resolved fluorescent microspheres and quantum dot microspheres may be, for example, but not limited to, 100 nm, 150 nm, 200 nm, 250 nm or 300 nm;

[0080] Preferably, the mass ratio of myoglobin antibody coupled to colloidal gold is: (0.04-0.32):1; the mass ratio of myoglobin antibody to colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres is: (0.1-0.4):1.

[0081] By adjusting the particle size and dosage of the marker, the sensitivity of the test strip can be made higher.

[0082] In a preferred embodiment of the present invention, a detection line (T line) and a quality control line (C line) are provided on the detection pad;

[0083] The test line is coated with 0.5-2 mg / mL of the myoglobin antibody;

[0084] The quality control line was coated with 0.5~2mg / mL of goat anti-mouse IgG polyclonal antibody;

[0085] Preferably, the diluent for the myoglobin antibody and goat anti-mouse IgG polyclonal antibody is a 10-50 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.1-1.0 g of trehalose.

[0086] In a preferred embodiment of the present invention, the myoglobin antibody on the labeling pad is different from the myoglobin antibody on the detection pad.

[0087] For example, the protein on the labeling pad is myoglobin antibody a, where a is myoglobin monoclonal antibody 1, and the proteins on the detection pad are myoglobin antibody b, where b is myoglobin monoclonal antibody 2.

[0088] The test strip provided by the present invention is based on the principle of antigen-antibody reaction, and after labeling myoglobin monoclonal antibody with colloidal gold, colored microspheres, time-resolved fluorescent microspheres, and quantum dots, the marker is solidified on a glass cellulose membrane. Another monoclonal antibody coated with myoglobin on the detection pad (such as NC membrane) can be detected by the naked eye or a matching instrument within the detection time based on the principle of antigen-antibody reaction. If myoglobin is present in the sample, a double antibody sandwich structure is formed, forming a band visible to the naked eye or a light intensity signal in the instrument. If there is no myoglobin in the sample, no band appears on the NC membrane or there is no light intensity signal in the instrument. Negative or positive judgment is made based on the presence or absence of the signal, or the load of the test object is predicted based on the intensity of the light intensity signal.

[0089] By using the test strip provided by the present invention for testing, the test results can be obtained within 10-20 minutes of the entire process, which is fast and efficient, and helps medical personnel to obtain the test results in time, make comprehensive judgments based on the results, and take timely measures.

[0090] In a fourth aspect, the present invention provides a myoglobin detection kit, comprising the myoglobin detection test strip and a shell, wherein the myoglobin detection test strip is arranged inside the shell.

[0091] The myoglobin detection kit provided by the present invention contains a myoglobin detection test strip, and thus has all the beneficial effects of the myoglobin detection test strip.

[0092] In a preferred embodiment of the present invention, the housing comprises an upper cover and a lower cover that are detachably connected;

[0093] The upper cover is provided with an observation window and a sample adding hole.

[0094] The shapes of the observation window and the sample addition hole are not specifically limited in the present invention. The observation window can be, for example, square and located above the test line and the quality control line of the test strip for observing the test results. The sample addition hole can be, for example, a circular hole with a diameter of 0.5-1 cm and located above the sample pad.

[0095] Test kit sample loading:

[0096] It should be noted that the diluent for the myoglobin antibody and goat anti-mouse IgG polyclonal antibody of the present invention is a 10-50 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.1-1.0 g of trehalose; unless otherwise specified in the following embodiments, preferably, the diluent for the myoglobin antibody and goat anti-mouse IgG polyclonal antibody is a 20 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.5 g of trehalose.

[0097] The method for preparing the monoclonal antibody described in the present invention is the content of the prior art, and is only briefly described here. The specific operation method can be carried out according to the operation method recorded in the prior art:

[0098] The immunogen and Freund's adjuvant were mixed in equal volumes to a suitable volume and emulsified completely. The mice were immunized by intraperitoneal injection. Each mouse was injected with 50 μg of immunogen, the injection volume was 100 μL / mouse, and the injection was performed once a week.

[0099] After immunization for 4 times, the titer of antibodies in the serum was tested by the indirect ELISA method, and mice with an OD value greater than 1.0 detected by 16,000-fold serum dilution were screened;

[0100] The spleen cells of the screened mice were fused with myeloma cells, and the fused cells were plated and cultured using the limiting dilution method;

[0101] Screen the monoclonal cell wells and culture and expand them, and take the monoclonal cell well with the highest OD value detected in the cell culture supernatant as the target hybridoma cell;

[0102] The target hybridoma cells are amplified and injected into the mouse peritoneal cavity. The ascites is collected and the monoclonal antibodies in the ascites are purified using Protein A, which are the target monoclonal antibodies.

[0103] The amino acid sequence of the immunogen is the amino acid sequence shown in SEQ ID NO.17.

[0104] Example 1

[0105] Myoglobin antibodies include: myoglobin monoclonal antibody 1 (protein a), myoglobin monoclonal antibody 2 (protein b), and the variable region sequences of the above antibodies are shown in Table 2.

[0106] A 12% SDS-PAGE gel was prepared according to the conventional method, and 5 μg of the above antibodies were loaded and electrophoresed using a protein molecular weight standard as a reference. The results showed that the two myoglobin monoclonal antibodies showed two characteristic bands of about 25KD and 55KD, which were the light chain and heavy chain of IgG respectively ( Figure 1 ). After scanning and analysis, the antibody content of the bands was above 90%.

[0107] Figure 1 In the above, 1: Anti-1; 2: Anti-2; wherein, Anti-1 is myoglobin monoclonal antibody 1; Anti-2 is myoglobin monoclonal antibody 2.

[0108] The antibodies used in the following examples are the same as those in Example 1.

[0109] Example 2

[0110] A myoglobin detection kit, such as Figure 2~Figure 4 As shown, 1 is an observation window, 2 is a sample addition hole, 3 is a test card strip area, 4 is a bottom plate, 5 is a test pad, 6 is a sample suction pad, 7 is a marking pad, 8 is a sample pad, 9 is a quality control line, and 10 is a test line. The myoglobin detection kit includes a myoglobin detection test strip and a shell, and the myoglobin detection test strip is arranged inside the shell. The myoglobin detection test strip includes a bottom plate 4 and a sample pad 8, a marking pad 7, a test pad 5 and a sample suction pad 6 stacked on the bottom plate in sequence; the test pad 5 is provided with a test line 10 and a quality control line 9; the shell includes a detachably connected upper cover and a lower cover; the upper cover is provided with an observation window 1 and a sample addition hole 2, and the lower cover is provided with a test card strip area 3.

[0111] Example 3

[0112] Preparation of Myoglobin Antigen Detection Kit Using Colloidal Gold Labeled Antibody

[0113] 1 Main Materials

[0114] 1.1 Antibodies: Mouse anti-monoclonal antibodies are all myoglobin antibodies described in the present invention, which are mouse monoclonal antibodies, and are labeled and coated respectively; Sheep anti-mouse IgG antibodies: products of Shenzhen Feipeng Biological Co., Ltd., are used for coating nitrocellulose membrane quality control lines respectively.

[0115] 1.2 Nitrocellulose membrane: NC membrane is a product of Sartorius.

[0116] 1.3 Other consumables: PVC boards and other consumables are products of Beacon Labs; commonly used reagents are all analytical grade reagents.

[0117] 1.4 Acquisition of recombinant antigens: Based on the myoglobin data published by the NCBI database, protein recombinant antigens were commercially synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0118] 2 Methods

[0119] 2.1 Preparation of colloidal gold labeling pad:

[0120] The steps for preparing the colloidal gold labeling pad are as follows:

[0121] (1) Take 1 mL of colloidal gold solution with a particle size of 40 nm and adjust the pH to 8.5 with 0.2 M K2CO3;

[0122] (2) Add 25 μg of monoclonal antibody 1, the mass ratio of antibody to colloidal gold particles is 0.1:1, adjust the rotary shaker to a certain speed, rotate and label at room temperature for 1.5 hours, then add 20 μL of blocking solution;

[0123] (3) Centrifuge at 12000 rpm for 15 min and discard the supernatant;

[0124] (4) Add 100 μL of colloidal gold solution;

[0125] (5) The above concentrate was diluted in a ratio of 1:7, sprayed with gold, and placed in a drying oven at 37°C for 2 hours for later use;

[0126] 2.2 NC membrane coating:

[0127] Use 0.02M PB containing 0.5% trehalose to dilute monoclonal antibody 2 to 1.5mg / mL and goat anti-mouse IgG to 2mg / mL respectively; then use a film sprayer to draw the detection line T and the quality control line C on the nitrocellulose membrane respectively. After coating, dry the NC membrane in an oven at 37℃ for 24h for use.

[0128] 2.3 Assembly of the kit:

[0129] Place the coated nitrocellulose membrane in the middle of the plastic support plate in a drying room and stick it. Overlap the marker colloidal gold pad (1 / 3 of the colloidal gold pad) on one side of the T line of the nitrocellulose membrane and stick it. Overlap the sample pad (1 / 5 of the colloidal gold pad) on the other side of the colloidal gold pad. Overlap the sample pad (1 / 10 of the sample pad) on one side of the C line of the nitrocellulose membrane. Use a cutting machine to cut the pasted plastic plate into test strips of a certain width and then load them into the test card to form a myoglobin antigen test kit.

[0130] 2.4 Detection:

[0131] Step 1, take out the test kit and the sample to be tested and equilibrate to room temperature;

[0132] Step 2. Open the sealed aluminum foil bag, take out the test kit and place it flat on the table;

[0133] Step 3: Figure 2 Add 2 drops of sample (about 80-100 μL) to the sample well;

[0134] Step 4: Set the timer to count, and read the result after 10 minutes. Note that if the sample does not undergo lateral chromatography or diafiltration within 1 minute after adding the sample, it may be because the sample is too viscous and needs to be pretreated with saline.

[0135] 3 Results:

[0136] Under the action of lateral flow chromatography, when myoglobin is present in the sample, the test line will develop color, and the quality control line will also develop color ( Figure 5 a in the figure); when there is no myoglobin in the sample, the test line does not show color, but the quality control line shows color ( Figure 5 b); After loading, if the quality control line does not show color, the result is considered invalid regardless of whether the test line shows color or not ( Figure 5 c in Figure 5 d) in.

[0137] Example 4

[0138] The myoglobin antigen detection kit was prepared by time-resolved fluorescent microspheres labeled with antibodies.

[0139] The myoglobin monoclonal antibody of the present invention (protein a: myoglobin monoclonal antibody 1; protein b: myoglobin monoclonal antibody 2) is used to prepare a myoglobin antigen detection kit, which includes a detection card and a test strip. The detection card is divided into an upper cover and a lower bottom. The test strip is embedded with a monoclonal antibody 1 labeled with time-resolved fluorescent microspheres on a fluorescent pad, and the detection line is coated with monoclonal antibody 2. The double antibody sandwich method is used to quantitatively detect the myoglobin antigen in the sample.

[0140] 1 Kit preparation operation process:

[0141] The monoclonal antibody 1 prepared by the method of the present invention is labeled on the surface of time-resolved fluorescent microspheres. The specific embodiments are as follows:

[0142] Time-resolved fluorescent microsphere antibody labeling: 1 mL 1% carboxyl time-resolved fluorescent microspheres, add 9 mL MES buffer, then add 25 μL EDC solution (10 mg / mL) and 25 μL NHS solution (10 mg / mL), shake at room temperature for 30 minutes, and collect the precipitate by centrifugation. Add HEPES reconstitution solution, ultrasonically disperse evenly, add 1 mL 1 mg / mL monoclonal antibody 1, shake at room temperature for 120 minutes, and collect the precipitate by centrifugation. Then add 1 mL blocking solution and shake at room temperature for 120 minutes. Collect the microsphere precipitate by centrifugation and reconstitute with reconstitution solution.

[0143] Preparation of fluorescent pad: The labeled time-resolved fluorescent microspheres were diluted with the microsphere complex solution, and the fluorescent pads were sprayed with a gold spray film instrument at a spraying rate of 3 μL / cm and a spraying interval of 6 mm. After spraying, they were placed in a 37°C low humidity (<30%) oven for 2 h.

[0144] NC membrane coated CT line: T line uses monoclonal antibody 2 with a concentration of 1.5 mg / mL, C line uses goat anti-mouse IgG antibody with a concentration of 1 mg / mL, 1 μL / cm line is drawn, and then placed in 37℃ low humidity (<30%) for drying for 24 hours.

[0145] Sample pad treatment: The sample pad treatment solution is composed of buffer salt, sustained-release agent, cosolvent, blocking agent, etc. The specific formula is 20mM Tris buffer, and each 100mL Tris buffer contains 1g BSA, 0.5g Tween 20, and 2g sucrose. 2 Use 1mL of sample treatment solution to treat. After evenly treating, place in a 37℃ low humidity (<30%) oven to dry for 2h.

[0146] Test strip assembly: NC film, sample suction pad, fluorescent pad, and sample pad are sequentially pasted on the PVC board. The sample suction pad and fluorescent pad are pressed 1~2mm against the NC film, and the sample pad is pressed 1~2mm against the fluorescent pad. After assembly, the test strip is cut into a width of 4±0.4mm and loaded into a card shell. The card shell and desiccant are placed in an aluminum foil bag and sealed. After labeling and boxing, the finished test card is obtained.

[0147] Test sample preparation: The commercially synthesized protein recombinant antigen produced by Shanghai Bioengineering Co., Ltd. was tested after gradient dilution.

[0148] The amino acid sequence of the test recombinant antigen sample is shown in SEQ ID NO.17: MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASEDLKKHGATVLTALGGILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKHPGDFGADAQGAMNKALELFRKDMASNYKELGFQG

[0149] 2 Kit testing process

[0150] Place the test card on a clean, flat surface, draw 80-100 μL of the treated sample and drop it into the sample adding end of the test card, and set up a PBS control group at the same time.

[0151] The standard curve of the test strip was introduced. After 10 minutes, the fluorescent immunoassay analyzer was used to scan the detection area to obtain the fluorescent signal. After the test, the concentration value corresponding to the myoglobin antigen was displayed. The test results are shown in Tables 4 and 5 below.

[0152] Table 4 Test results of dilution samples

[0153]

[0154] Table 5 Recombinant antigen test results

[0155]

[0156] Example 5

[0157] Example 5 provides a myoglobin antigen detection kit prepared by using colored microspheres labeled with antibodies.

[0158] The myoglobin monoclonal antibody of the present invention (protein a: myoglobin monoclonal antibody 1; protein b: myoglobin monoclonal antibody 2) is used to prepare a myoglobin antigen detection test strip, wherein the test strip has monoclonal antibody 1 labeled with colored microspheres embedded on a marking pad, and the detection line is coated with monoclonal antibody 2, and the double antibody sandwich method is used to qualitatively detect the myoglobin antigen in the sample.

[0159] 1. Preparation of test strips:

[0160] 1.1 Colored microspheres labeled with antibodies

[0161] (1) Adjust the pH of 100 nm colored microspheres to 8.0 with 0.1 mol / L K2CO3;

[0162] (2) Take 1 mL of the above pH-adjusted solution and add 30 μg of monoclonal antibody 1 (the mass ratio of antibody to colored microspheres is 0.3:1). After reacting at room temperature for 1 hour, centrifuge and discard the supernatant; Take 1 mL of the above pH-adjusted solution and add 5 μg of chicken IgY (the mass ratio of antibody to colored microspheres is 0.05:1). After reacting at room temperature for 1 hour, centrifuge and discard the supernatant;

[0163] (3) Add 1 mL of 20% BSA and block for 2 h, then centrifuge and discard the supernatant;

[0164] (4) After re-dissolving the above microspheres with 100uL of pH 8.0 reconstitution solution, the two microspheres were mixed at a ratio of 5:1, and the mixture was diluted with the reconstitution solution at a ratio of 15%;

[0165] 1.2 Preparation of labeling pad

[0166] The dilution of the labeled colored microsphere antibody complex was sprayed on the marking pad using a gold spray film instrument at a spraying rate of 7.5 μL / cm and a spraying interval of 6 mm. After spraying, it was placed in a 37°C low humidity (<30%) drying room for 2 hours.

[0167] 1.3 Sample pad preparation

[0168] The sample pad treatment solution is composed of buffer salt, slow-release agent, cosolvent, blocking agent, etc. The specific formula is 20mM Tris, 1% BSA, 0.5% Tween 20, 2% sucrose. 2Use 1mL of sample treatment solution to treat. After evenly treating, place in a 37℃ low humidity (<30%) oven to dry for 2h.

[0169] 1.4 C / T line coating

[0170] The T line used monoclonal antibody 2 with a concentration of 1.5 mg / mL, and the C line used goat anti-chicken IgY antibody with a concentration of 2 mg / mL. The lines were drawn at 1 μL / cm and dried at 37°C with low humidity (<30%) for 24 hours.

[0171] 1.5 Test strip assembly

[0172] Paste the sample suction pad, NC film, and marking pad on the PVC board in sequence. Press the sample pad, sample suction pad, and marking pad with 1~2mm of NC film, and press the sample pad with 1~2mm of marking pad. After assembly, cut the test strip into a width of 4±0.4mm and install the card shell. Put the card shell and desiccant into an aluminum foil bag together. Put the label and box to get the finished test kit.

[0173] 1.6 Test sample preparation: The commercially synthesized protein recombinant antigen produced by Shanghai Sangon Biotechnology Co., Ltd. (the amino acid sequence of the test sample is shown in SEQ ID NO. 17) was diluted in series for testing.

[0174] 2. Detection

[0175] Place the test card on a clean and flat table, draw 80~100μL of the prepared recombinant antigen sample and drip it into the sample end of the test card, and set up a PBS control group. Observe the test card window after 10 minutes. There are 2 positive lines in the window and only one C line is negative. The test results are as follows: The results in Table 6 show that the antigen compliance rate of a pair of monoclonal antibody assembled test strips is 100%.

[0176] Table 6

[0177]

[0178] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A myoglobin antibody, comprising protein a or protein b, characterized in that: The amino acid sequence of the heavy chain complementary determining region of the protein a is as follows: the amino acid sequence of CDR1-VH is shown in SEQ ID NO.1, the amino acid sequence of CDR2-VH is shown in SEQ ID NO.2, and the amino acid sequence of CDR3-VH is shown in SEQ ID NO.3; the amino acid sequence of the light chain complementary determining region of the protein a is as follows: the amino acid sequence of CDR1-VL is shown in SEQ ID NO.4, the amino acid sequence of CDR2-VL is shown in SEQ ID NO.5, and the amino acid sequence of CDR3-VL is shown in SEQ ID NO.6; The amino acid sequence of the heavy chain complementary determining region of the protein b is: the amino acid sequence of CDR1-VH is shown in SEQ ID NO.7, the amino acid sequence of CDR2-VH is shown in SEQ ID NO.8, and the amino acid sequence of CDR3-VH is shown in SEQ ID NO.9; the amino acid sequence of the light chain complementary determining region of the protein b is: the amino acid sequence of CDR1-VL is shown in SEQ ID NO.10, the amino acid sequence of CDR2-VL is shown in SEQ ID NO.11, and the amino acid sequence of CDR3-VL is shown in SEQ ID NO.

12.

2. The myoglobin antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the protein a is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the protein a is shown in SEQ ID NO.

14.

3. The myoglobin antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the protein b is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the protein b is shown in SEQ ID NO.

16.

4. Use of the myoglobin antibody according to any one of claims 1 to 3 in the preparation of a myoglobin detection product.

5. A myoglobin test strip, comprising a base plate and a sample pad, a marking pad, a detection pad and a sample suction pad stacked on the base plate in sequence, characterized in that: The marking pad contains the myoglobin antibody according to any one of claims 1 to 3, and the myoglobin antibody is marked with a marker; the detection pad is provided with a detection line and a quality control line; the detection line is coated with the myoglobin antibody according to any one of claims 1-3; the quality control line is coated with a sheep anti-mouse IgG polyclonal antibody; the myoglobin antibody on the marking pad is different from the myoglobin antibody on the detection pad.

6. The test strip according to claim 5, characterized in that The marker includes at least one of colloidal gold, colored microspheres, time-resolved fluorescent microspheres or quantum dot microspheres.

7. The test strip according to claim 6, characterized in that: The diluent for the myoglobin antibody and goat anti-mouse IgG polyclonal antibody is a 10-50 mM PB buffer solution containing trehalose, and each 100 mL of the diluent contains 0.1-1.0 g of trehalose.

8. A myoglobin detection kit, characterized in that: It comprises the myoglobin detection test strip according to any one of claims 5 to 7 and a shell, wherein the myoglobin detection test strip is arranged inside the shell.

9. The myoglobin detection kit according to claim 8, characterized in that The housing comprises an upper cover and a lower cover which are detachably connected; the upper cover is provided with an observation window and a sample addition hole.

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