3-MT antigen, 3-MT recombinant monoclonal antibody and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a 3-MT antigen, a 3-MT recombinant monoclonal antibody and application of the 3-MT antigen and the 3-MT recombinant monoclonal antibody. The preparation method comprises the following steps: coupling 3-MT with KLH through an intermediate arm to obtain an activated 3-methoxydopamine antigen, immunizing a rabbit with the antigen, constructing a phage library, and elutriating and screening to obtain a 4354 # antibody. Experiments show that the antibody is high in titer and good in gradient, the specificity, repeatability, stability and other properties meet the requirements of the magnetic particle chemiluminescence 3-MT kit, and the market blank is filled.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to 3-MT antigen, 3-MT recombinant monoclonal antibody and application thereof. Background Art

[0002] Catecholamine is a neurotransmitter containing catechol and amine groups, which are bound by enzymatic steps of L-tyrosine in the sympathetic nerves, adrenal medulla and chromophilic cells. Usually, catecholamine refers to dopamine, norepinephrine and epinephrine. These three catecholamines are converted from tyrosine as a precursor. The three metabolites of metabolites include 3-methylepinephrine (metanephrine, MN), 3-methylnorepinephrine (normetanephrine, NMN) and 3-methoxydopamine (3-methoxytyramine, 3-MT), which are 3-oxymethylated metabolites of epinephrine, norepinephrine and dopamine, respectively.

[0003] Pheochromocytoma and paraganglioma (PPGL) are a group of neuroendocrine tumors originating from the adrenal medulla or extra-adrenal sympathetic nerve chain. They are one of the causes of secondary hypertension, with a prevalence of 0.2% to 0.6% in general hypertension clinics.

[0004] Most PPGLs can occur in all age groups, with the peak incidence at 30 to 50 years old, and an annual incidence of 2 to 8 per million people. PPGLs usually secrete catecholamines (CA) paroxysmically or continuously, with typical clinical manifestations of paroxysmal or persistent hypertension and the triad of headache, palpitations, and sweating. Excessive release of CA can induce severe cardiovascular manifestations, including Takotsubo cardiomyopathy, hypertensive crisis, and acute myocardial infarction, and eventually develop into multi-system failure and death. Therefore, timely diagnosis is particularly important.

[0005] 3-MT is an intermediate metabolite of catecholamines, and its metabolic pathway is the conversion of dopamine to 3-methoxytyramine (3-MT) catalyzed by COMT enzyme. Early studies have shown that plasma 3-MT can be used to detect PPGL or head and neck paraganglioma (HNPGL) that secrete dopamine (DA). In addition, 3-MT is also considered a new biomarker for metastatic PPGL. Studies have shown that compared with those without metastasis, the plasma 3-MT level in patients with metastatic PPGL is significantly increased, and the results of the receiver operating characteristic (ROC) curve analysis show that the accuracy of plasma 3-MT level in predicting metastatic PPGL is better than that of methylnorepinephrine. High levels of plasma 3-MT are associated with poor disease-specific survival (DSS) in patients with metastatic PPGL and (or) HNPGL, suggesting that 3-MT may play a certain role in the individualized management and follow-up of patients with metastatic PPGL and (or) HNPGL. In the "Expert Consensus on the Diagnosis and Treatment of Pheochromocytoma and Paraganglioma 2020", it is mentioned that 3-MT is recommended as one of the biochemical indicators for PPGL detection.

[0006] In short, hypertension is one of the common chronic diseases. PPGL, as an important cause of secondary hypertension, may lead to uncontrollable malignant hypertension and cardiovascular and cerebrovascular diseases. Therefore, early detection and timely diagnosis of PPGL are of great significance. 3-MT can provide accurate judgment for the diagnosis and differentiation of benign and malignant PPGL.

[0007] At present, there are no relevant immunological products for the detection of 3-MT at home and abroad. The detection is mainly based on high-performance liquid chromatography (HPLC), but HPLC detection takes a long time and is costly. The conditions of different laboratories vary greatly, making it difficult to achieve batch determination. Therefore, screening 3-MT antibodies and applying them to magnetic particle luminescence platforms and other related catecholamine projects has become an important demand.

[0008] Dopamine analogs such as 3-MT, NMN and MN are all small molecules with very similar structures, so it is very difficult to develop antibodies and detection kits that meet the requirements of good gradient and high specificity. It is of great practical significance to provide a new 3-MT antigen for screening antibodies with strong specificity and high titer, and then develop a 3-MT kit with good specificity, high accuracy and high sensitivity. Summary of the invention

[0009] In view of this, the present invention provides 3-MT antigen, 3-MT recombinant monoclonal antibody and application thereof.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] 3-methoxydopamine hapten, characterized in that its structural formula is as shown in Formula I:

[0012]

[0013] Here, n is any integer selected from 0 to 8.

[0014] In some embodiments, n is equal to 8, and the structure of the antigen is shown in Formula II:

[0015]

[0016] The present invention also provides a 3-methoxydopamine complete antigen, which is a conjugate of the 3-methoxydopamine hapten and a carrier protein;

[0017] The carrier protein includes one or more of KLH, bovine serum albumin, human serum albumin, ovalbumin, etc.

[0018] In the present invention, the 3-methoxydopamine complete antigen is a conjugate obtained by coupling the amino group at the terminal of the 3-methoxydopamine hapten with the amino group of the carrier protein.

[0019] In some specific embodiments, the 3-methoxydopamine complete antigen, referred to as 3-MT-BS3-KLH, has a structure as shown in Formula III:

[0020]

[0021] The present invention also provides a method for preparing the complete 3-methoxydopamine antigen, comprising the following steps:

[0022] Synthesize a hapten of formula I;

[0023] The hapten is coupled to the carrier protein, and the coupled product is dialyzed to obtain the complete 3-methoxydopamine antigen.

[0024] In some embodiments, the coupling reaction conditions are: shaking reaction at 18-27° C. overnight, and the shaking reaction time is ≥16 h.

[0025] The present invention also provides the use of the hapten or the complete antigen in any of the following:

[0026] (1) preparing 3-MT recombinant monoclonal antibody;

[0027] (2) preparing products for detecting the levels of 3-MT and its derivatives;

[0028] (3) Preparation of diagnostic products for pheochromocytoma and / or paraganglioma.

[0029] The invention provides a 3-MT recombinant monoclonal antibody, which is a 3-MT recombinant monoclonal antibody prepared by immunizing a rabbit with the 3-methoxydopamine complete antigen.

[0030] The present invention provides a 3-MT recombinant monoclonal antibody, wherein the three CDR regions of the heavy chain respectively have:

[0031] A), the amino acid sequence shown in SEQ ID NO: 1 to 3; or

[0032] B) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in A); or

[0033] C) has at least 80% of the sequence shown in A) or B).

[0034] The three CDR regions of the light chain of the antibody respectively have:

[0035] D), the amino acid sequence shown in SEQ ID NO: 4 to 6; or

[0036] E) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in D); or

[0037] F) has at least 80% of the sequence shown in D) or E).

[0038] In some embodiments, the 3-MT antibody of the present invention comprises a heavy chain variable region and a light chain variable region;

[0039] The light chain variable region has:

[0040] G), the amino acid sequence shown in SEQ ID NO: 7; or

[0041] H) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in G); or

[0042] I) and the sequence shown in H) or I) have at least 80% of the sequence.

[0043] The heavy chain variable region has:

[0044] J), the amino acid sequence shown in SEQ ID NO: 8; or

[0045] K) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in J); or

[0046] L), and the sequence shown in J) or K) have at least 80% of the sequence.

[0047] In some embodiments, the constant region of the heavy chain is of IgG type; and the constant region of the light chain is of K type.

[0048] In some specific embodiments, the present invention uses the 3-MT-BS3-KLH antigen screening described above to obtain the 3-MT recombinant rabbit monoclonal antibody of the present invention, named 4354#. Experiments show that the monoclonal antibody has strong specificity, good repeatability, and high detection sensitivity, which are better than the control antibody 1605#.

[0049] The present invention also provides a method for preparing the 3-MT recombinant rabbit monoclonal antibody, comprising: immunizing the spleen of a rabbit with the 3-MT-BS3-KLH antigen, constructing a phage library, and obtaining the anti-TPO mouse monoclonal antibody through panning and screening.

[0050] The present invention also provides a biomaterial, characterized in that it comprises any of the following:

[0051] (I) a nucleic acid encoding the 3-MT antibody;

[0052] (II) an expression cassette or a recombinant vector comprising the nucleic acid described in (I);

[0053] (III) a host comprising the nucleic acid described in (I) or the recombinant vector described in (II).

[0054] The present invention also provides the use of any one of the following a) to b) in the preparation of a detection product:

[0055] a) the 3-MT recombinant monoclonal antibody of the present invention;

[0056] b) the biological material according to the present invention;

[0057] The detection products include: products for detecting the level of 6-carbonylated derivatives of 3-MT, or diagnostic products for pheochromocytoma and / or paraganglioma.

[0058] In the application of the present invention, it is characterized in that the 3-MT derivative is a compound of the following formula IV:

[0059]

[0060] The present invention also provides a kit for detecting the level of 3-MT derivatives, which comprises the complete antigen and / or the 3-MT recombinant monoclonal antibody of the present invention.

[0061] In some embodiments, the kit is a magnetic microparticle chemiluminescence kit, which includes: magnetic microparticles coated with the antibody of the present invention. Further, the magnetic microparticle chemiluminescence kit also includes a labeled antigen. The antigen in the labeled antigen is a compound shown in the above formula IV. The kit also includes other common components, such as buffer, washing solution, blocking solution, etc. The present invention does not specifically limit the specific types of these components, and any commonly used components in the art can be used.

[0062] The present invention also provides a method for detecting the level of 3-MT or its derivatives for non-diagnostic purposes, comprising: using the kit of the present invention to detect a sample to be tested. In some specific embodiments of the present invention, the sample to be tested is a 6C-acylated serum sample.

[0063] In the present invention, a plasma acylating agent is used to treat the sample to be tested to obtain a 6C-acylated serum sample. The plasma acylating agent includes a reagent that can make 3-MT generate a 3-MT derivative shown in Formula IV. In a specific embodiment of the present invention, the structure of the acylating agent and the acylation reaction process are as follows:

[0064]

[0065] In order to achieve specific detection of 3-MT, the applicant has tried to use 3-MT antigens of different structures for screening antibodies after years of research. Finally, the complete antigen prepared by the hapten shown in Formula II was screened to obtain the 4354# antibody with high titer and good gradient. The antibody titer and / or gradient obtained by the complete antigen prepared by other antigens in the structure of Formula I and other antigens outside the structure of Formula I were not ideal, and the effect was obviously not as good as the 4354# antibody. In addition, the applicant also performed different pretreatments on the serum samples (including 12C acylation, 6C acylation of the present application, PEGylation, etc.). It was found that the detection of serum samples after 6C acylation can significantly improve the sensitivity and specificity of the detection, which has obvious advantages for the detection of 3-MT.

[0066] The present invention uses 3-MT to couple KLH through an intermediate arm to obtain an activated form of 3-methoxydopamine antigen, uses the antigen to immunize rabbits, constructs a phage library, and obtains a 4354# antibody through panning and screening. Experiments show that the antibody has high titer, good gradient, and the specificity, repeatability and stability meet the requirements of the magnetic particle chemiluminescent 3-MT kit, filling the market gap. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The electrophoresis results of 3-MT4354# antibody are shown, the heavy chain is 53±3KD, and the light chain is 22±3KD;

[0068] Figure 2 3- HPLC results of MT4354# antibody are shown. DETAILED DESCRIPTION

[0069] The present invention provides 3-MT antigen, 3-MT recombinant monoclonal antibody and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0070] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0071] The present invention couples KLH at a specific position of 3-methoxydopamine through a connecting arm BS3 to obtain a new antigen 3-MT-BS3-KLH with immunogenicity.

[0072] The present invention uses the immunogen 3-MT-BS3-KLH to immunize rabbits and construct a phage library; after the panning antigen: 3-MT-8C-BSA panning, 3-MT-6C competitive panning is added; 3MT monoclonal screening strategy: preliminary screening of better clones: only using 2-point evaluation (extremely low, extremely high), using 0pg / ml value and 1500pg / ml calibration; simultaneous screening of major crossovers: major crossover NMN 2-point detection (low point, high point), 150 and 15000pg / ml detection, screening of low crossover rate clones and retesting of superior clones: selecting clones with better calibrators and crossover rates for retesting, increasing the calibrator gradient: 0, 40, 200, 1000pg / ml, selecting 0 / 40 point better clones; increasing crossover screening: NMN / MN repeated determination, determining that the crossover rate is normal; and finally screening out a strain of 3-MT antibody, named 3-MT8574#. Experiments show that the requirements of the kit are met.

[0073] The light chain variable region of the 3-MT8574# antibody has the amino acid sequence shown in SEQ ID NO:7, and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:8.

[0074] Wherein, the sequence of the light chain variable region SEQ ID NO.7 is as follows:

[0075] ALVMTQTPASVSVPVGDTVTIDC QASESVYNNNHLS WFQQKPGQPPK QLIY GASTLDSGVSSRFKGSGSGTQFTLTITDVVCDDAATYYC AGYKSLTS DGRA FAGGTEVVVK.

[0076] The light chain variable region includes three CDR regions, which are as follows:

[0077] CDR1: QASESVYNNNHLS(SEQ ID NO.1);

[0078] CDR2: GASTLDS (SEQ ID NO. 2);

[0079] CDR3: AGYKSLTSDGRA (SEQ ID NO. 3).

[0080] The sequence of the heavy chain variable region SEQ ID NO.8 is as follows:

[0081] QEQLKESGGRLVTPGTPLTLTCTASGFSLS NYYMS WVRQAPGKGLEYI G TIYGVGNTFYASWAKG RFTISKTATTVDLKVTSPTTEDTATYFCGR DAPS VGADL WGPGTLVTVSS.

[0082] The heavy chain variable region includes three CDR regions, which are as follows:

[0083] CDR1: NYYMS (SEQ ID NO. 4);

[0084] CDR2:TIYGVGNTFYASWAKG(SEQ ID NO.5);

[0085] CDR3: DAPSVGADL (SEQ ID NO. 6).

[0086] In the present invention, the 3-MT8574# antibody further comprises a heavy chain constant region and a light chain constant region. Further, the heavy chain constant region is of IgG type; the light chain constant region is of K type. In some specific embodiments, the sequence of the heavy chain constant region is shown in SEQ ID NO.9:

[0087] GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK。

[0088] The sequence of the light chain constant region is shown in SEQ ID NO.10:

[0089] RDPVAPSVLLFPPSKEELTTGTATIVCVANKFYPSDITVTWKVDGTTQQSGIENSKTPQSPEDNTYSLSSTLSLTSAQYNSHSVYTCEVVQGSASPIVQSFNRGDC。

[0090] The sequence of the Pcombxsss 283 plasmid is shown in SEQ ID NO.11:

[0091]

[0092] The present invention will be further described below in conjunction with embodiments:

[0093] Example 1 Preparation of 3-MT-BS3-KLH antigen and antibody

[0094] 1. Synthesis of Antigens:

[0095] The reaction process includes the following steps:

[0096] 1.1 Cross-linking: Take 0.932mg (186μL) of dissolved 3-MT antigen and slowly add it dropwise into 20mg (2ml) KLH solution, shaking while adding to ensure full mixing. Take 2.288mg BS3 (114μL) and slowly add it dropwise into the above 3-MT and KLH mixture, shaking while adding to ensure full mixing, and then shake and react at room temperature overnight (room temperature requirement, 18-27℃, reaction time ≥16h).

[0097] 1.2. Dialysis: Prepare a dialysis bag (14kd, MD25), cut the dialysis bag, place it in purified water, let it stand for 0.5h, and then replace the purified water. Use 0.01M PBS pH7.2 (single dialysis volume 2L), dialyze 4 times, change the liquid 3 times during the period, the first dialysis lasts 3h, the second dialysis lasts 3h, the third dialysis lasts 3h, and the fourth dialysis lasts overnight for 15h-16h.

[0098] 1.3. Sample processing: Undry the dialysis bag, measure the volume with a 5ml pipette and transfer it into a 10ml brown glass vial. Measure the concentration with A280, label it, and store it at -20℃.

[0099] 2. Rabbit immunization

[0100] The 3-MT-BS3-KLH antigen was diluted with PBS (0.01M pH 7.4) and fully emulsified with Freund's adjuvant at a volume ratio of 1:1. The immunization dose was 1 mg / rabbit for the first immunization and 0.5 mg / rabbit for the second immunization. The immunization route was systemic subcutaneous injection at multiple points. The interval between the first and second times was 30 days, and the interval between the second and third times was 30 days. The maximum immunization dose per rabbit did not exceed 0.8 ml.

[0101] The titer and gradient were determined by plate chemiluminescence; when the titer was 128K and the IC50 reached 400pg, a rabbit phage library was constructed.

[0102] 3. Nucleic Acid Extraction and Library Preparation

[0103] RNA was extracted from rabbit spleen, and cDNA was reverse transcribed. The variable regions of the light chain and heavy chain were amplified by PCR, and then ligated with the vector pCOMb3xss (sequence shown in SEQ ID NO.11).

[0104] 3.1 PCR reaction system (50 μl system):

[0105] VL (light chain variable region) mix: 50ng;

[0106] VH (heavy chain variable region) mix: 50ng;

[0107] 2933 (10 μM): 1.5 μl;

[0108] 2934 (10 μM): 1.5 μl;

[0109] 2× Primerstar Max: 25 μl;

[0110] High-pressure purified water: make up to 50 μl system.

[0111] PCR reaction parameter settings:

[0112]

[0113] 3.2 Enzyme digestion reaction system (50 μl system, SCFV: 50 μl / tube * 5 tubes; pcomb3xss: 50 μl / tube * 8 tubes):

[0114] scFv: 2 μg;

[0115] sfiI (5u / μl): 2.5μl;

[0116] 10×bufferG: 5μl;

[0117] High-pressure purified water: make up to 50 μl.

[0118] Pcomb3xss: 4 μg;

[0119] sfiI (5u / μl): 2.5μl;

[0120] 10× buffer G: 5 μl;

[0121] High-pressure purified water: make up to 50 μl.

[0122] 3.3 Ligation reaction:

[0123] System (20μl system), vector: 0.4μg scFv: 246ng ligase: 1μl 5×ligase buffer: 10μl high pressure purified water: make up to 50μl. Connection parameter setting: 37℃, overnight (14-18h), electroporation and efficient transformation of E. coli TG1. The next day, count the number of plate clones.

[0124] 4. Calculation of storage capacity and electrical conversion efficiency:

[0125] Reservoir capacity after electrotransformation = colony count * dilution gradient / coating volume * 30ml, in cfu;

[0126] Electroporation efficiency = colony number * dilution gradient / coating volume * 30 ml / input nucleic acid amount (μg) cfu / μg.

[0127] The electroporation efficiency is ≥10^9cfu / μg. The gene library capacity is ≥10^8cfu. It can meet the experimental requirements, otherwise the gene library needs to be reconstructed.

[0128] 5. Preparation of Phage Antibody Library

[0129] 5.1 Add 5*1012-4*1013 pfu helper phage M13KO7 (1:20 ratio) to the library after electroporation, incubate at 37℃ for 30 minutes. Then culture at 37℃, 250rpm for 1h. (The helper phage should be vortexed before use) Centrifuge at 4500rpm, 10min, discard the supernatant, add 120ml AK, culture at 250rpm, 30℃ overnight.

[0130] 6. Titer Determination

[0131] 6.1 Take the overnight cultured bacteria and centrifuge at 8000rpm for 10min, collect the supernatant. Transfer 40ml of the supernatant to 50ml conical bottom centrifuge tubes containing 10ml 5xPEG8000 / NaCl, divide into 3 tubes, mix thoroughly, place in an ice bath or 4℃ refrigerator for 20min. Centrifuge at 8000rpm for 10min at 4℃, discard the supernatant.

[0132] Centrifuge at 8000 rpm for 2 min and remove all excess liquid.

[0133] Add 2 ml 1xPBS (gibco) to each tube to resuspend the precipitate. Centrifuge at 8000 rpm for 5 min, take the supernatant (phage antibody library) to a new centrifuge tube, add 6% DMSO, mix well, dispense into 1.5 ml centrifuge tubes, and store at -20°C.

[0134] 6.2 Take 20 μl of phage and 180 μl of 2YT and make 10-fold serial dilutions: 10 1 -10 10 Then add 180 μl of TG1 (OD600 = 0.4-0.6) (prepared in 3.5.2.1) and incubate at 37℃ for 30 min. 9 -10 10100 μl of bacterial solution was coated on Amp+2YT plates. After the plates were dried, they were inverted and cultured overnight at 37°C. The statistics were collected the next day. The phage titer was also recorded. The statistical formula was titer = 2*Ax*10 x+1 pfu / ml; (X: dilution factor, Ax: number of clones at X-fold dilution ratio).

[0135] 7. Main steps of antibody screening

[0136] The day before, take the immunotube and coat the immune antigen 3-MT-BS3-BSA 1ml (20μg, 10μg in the second round, 5μg in the third round) according to the panning scheme. The next day, discard the coating solution, wash the immunotube 3 times with 0.05% PBST, add 5ml casein, and block at 37℃ for 1h. Take 1ml of the centrifuged phage and add it to the immunotube, incubate at 37℃, 70rpm, and incubate for 2h. Discard the unbound phage in the immunotube, add 4ml 0.05% PBST washing solution to each tube, wash the immunotube (6 times in the first round, 8 times in the second round, 10 times in the third round, increasing in sequence), tap for 30s each time, and then wash twice with 1xPBS. Add 1ml 3-MT-6C elution solution (5μg in the first round, 10μg in the second round, 20μg in the third round) to each tube, and incubate at 37℃, 70rpm for 60min. The dissociated phages were transferred into a new 1.5 ml centrifuge tube.

[0137] Quickly take 1ml of eluted phage and add it to 5mlTG1, then add 1mlTG1 to the corresponding immune tube, and incubate at 37℃ for 30min. (Mix the bacteria in the immune tube and the centrifuge tube, mix well, take 10μl for dilution and titer measurement (take 10μl of bacterial solution and make 10-fold serial dilutions, 101-105 20μl phage + 180μl 2YT), apply 100μl. Calculate the amount of storage) Add 6μl A+, incubate at 37℃, 250rpm for 1h. Add M13K07 (MOI=5), 37℃, infect for 30min. Add 10ml2%AG-2YT, incubate at 37℃, 250rpm for 1h. Centrifuge at room temperature 4500rpm for 10min, discard the supernatant, add 5mlAK-2YT, and culture overnight at 30℃, 250rpm. Count the storage data the next day. The amount of the stock = 6*Ax*10x+1pfu / ml (X: dilution factor, Ax: number of clones at X-fold dilution ratio.)

[0138] 8. Phage monoclonal antibody screening

[0139] In a 96-well medium-well plate, add 400 μl / well 2YT-AG (final concentration 50 μg / ml Amp+, 2% glucose). Pick a single clone in a deep-well plate and mark it with PC (positive control) and NC (negative control). Remove the tip of the pipette and cover it with a sealing film. Culture overnight at 37°C and 250 rpm. Prepare 2YT-0.1% AG liquid culture medium, dispense 100 μl / well in a 96-well deep-well plate, and add 5 μl of the single clone culture solution picked overnight. Incubate at 37°C and 250 rpm pm, activate and culture for 2-3h, (OD value is about 2). Prepare 2YT+helper phage (MOI=5) mixture, add 20μl / well to the deep-well plate. Incubate at 37℃ for 30 minutes. Incubate at 37℃, 220rpm for 1.5h, then add 300μl 2YT-AK (final concentration 100μg / mlAmp+, 50μg / mlKan+) to each well, 30℃250rpm, culture overnight for 16-18h. The next day, centrifuge at 4000rpm for 15 minutes at 4℃, and take the supernatant for ELISA detection.

[0140] 9. Phage monoclonal ELISA detection: (indirect method, competitive method)

[0141] Take the chemiluminescent plate, dilute 3-MT-DSS-BSA to 0.25 μg / mL with CB, and coat at 4°C overnight with 100 μl / well. The next day, wash the plate twice with PBST, pat dry, and add 150 μl / well of casein with a dispenser. Mix well with a micro-oscillator, block at room temperature for 2 hours, and shake off the liquid in the wells with absorbent paper and pat dry.

[0142] Indirect reaction: Take 50μl / well of the supernatant of phage centrifugation and add it to the antigen-coated plate. Mix by shaking on a micro-oscillator and react at 37℃ for 0.5h. Wash with PBST solution 5 times. Pat dry. Add enzyme conjugate anti-M13-HRP (1 / 5k), 100μl / well. React at 37℃ for 30min. Wash the plate: Wash the plate manually with PBST solution 5 times. Pat dry. : Add luminescent substrate A + substrate B and mix well, 100μl / well. Detect with LUMO luminometer. Detect immediately and after 5min. The result after 5min shall prevail.

[0143] Competition reaction—3MT monoclonal competition screening strategy:

[0144] (1) Preliminary screening of superior clones: using only 2-point evaluation (extremely low, extremely high), using 0 pg / ml value and 1500 pg / ml calibrator;

[0145] (2) Screening of major crossovers simultaneously: The major crossover NMN was tested at two points (low point and high point), 150 and 15000 pg / ml, and clones with low crossover rate were screened and retested for superior clones: clones with good calibrators and crossover rates were selected for retesting, and the calibrator gradient was increased: 0, 40, 200, 1000 pg / ml, and clones with good 0 / 40 points were selected;

[0146] (3) Add crossover screening: Repeat the NMN / MN measurement to confirm that the crossover rate is normal.

[0147] According to the luminescence value of the indirect method, use diluent to reduce the phage dilution to the luminescence value of about 5w, different calibrators 50μl / well, preliminary screening of better clones: only use 2-point evaluation (very low, very high), use 0pg / ml value and 1500pg / ml calibrators; simultaneous screening of major cross-objects: main cross-object NMN 2-point detection (low point, high point), 150 and 15000pg / ml detection, add diluted phage 50μl / well, add to the antigen coated plate. Oscillate and mix on a micro-oscillator, react at 37℃ for 0.5h. Manually wash the plate, PBST wash solution, 5 times. Pat dry. Add enzyme conjugate anti-M13-HRP (1 / 5k), 100μl / well. React at 37℃ for 30min. Wash the plate: Manually wash the plate, PBST wash solution, 5 times. Pat dry. Color development: Add luminescent substrate A + substrate B and mix well, 100μl / well. Detect by LUMO luminometer. Detect immediately and after 5min. The 5min result shall prevail. Select clones with good calibration and crossover rate for retest, increase calibration gradient: 0, 40, 200, 1000pg / ml, select clones with good 0 / 40 points; increase crossover screening: repeat the determination of NMN / MN to confirm that the crossover rate is normal, and the rest is the same as above.

[0148] Antibody sequencing: 20 dominant phage strains were screened for sequencing.

[0149] 10. Construction of full-length antibodies

[0150] According to the sequencing results, different primers for the variable region of rabbit monoclonal antibody were selected. The primer pairing scheme and the optimal annealing temperature were used to set the PCR reaction system (25 μl system): template DNA: 2.5 μl F (10 μM): 0.75 μl R (10 μM): 0.75 μl 2×primerstar Max: 12.5 μl high pressure purified water: 8.5 μl PCR reaction parameter settings:

[0151]

[0152] Nucleic acid gel recovery and purification.

[0153] 11. Homologous recombination reaction

[0154] Homologous recombination reaction system (10 μl system): Vector: 50 ng Fragment: 40 ng 2× homologous recombination enzyme: 5 μl High-pressure purified water: make up to 10 μl. React at 50℃ for 1 hour and place at 4℃. Transform into Top10 E. coli. Spread on a plate and invert and culture at 37℃ for more than 12 hours.

[0155] 12. Recombinant Screening

[0156] Pick a single colony and place it in 3 ml LB liquid medium (Amp+), and culture it at 37°C with shaking at 220 rpm for 3 hours. Prepare PCR reaction solution (50 μl system)

[0157] F: 1.5 μl

[0158] R: 1.5 μl

[0159] 2×Taq MixPLUS: 25 μl

[0160] High pressure purified water: 20 μl

[0161] Each tube is divided into 15 μl and 1 μl of bacterial solution is added

[0162] The PCR reaction parameters were set as follows:

[0163]

[0164] Agarose gel electrophoresis was performed, positive clones were sequenced, and plasmids were extracted from the clones with correct sequencing.

[0165] 13.3-MT 4354# IgG antibody expression

[0166] For plasmid extraction, select the MN kit to extract the plasmid (mid-extraction);

[0167] Plasmid transient transfection

[0168] On the third day of 293F cell culture, sterile samples were taken for cell counting, and the cell density reached (3.0-5.0)×10 6 cells / ml, viability ≥ 90%, discard appropriate amount of cell suspension and add fresh culture medium to 500ml, adjust the cell density to 2.0×10 6 cells / ml and cultured in a shaking incubator at 37°C, 5% CO2, and 130 rpm (amplitude 25 mm).

[0169] On the second day of cell culture, sterile samples were taken for cell counting, and the cell density reached (3.0-5.0)

[0170] ×10 6cells / ml, viability ≥ 90%, discard appropriate amount of cell suspension and add fresh culture medium to 500ml, adjust the cell density to 3.0×10 6 cells / ml and cultured in a shaking incubator at 37°C, 5% CO2, and 130 rpm (amplitude 25 mm).

[0171] Preparation for transfection of a single bottle of 200mL cell suspension: In the clean bench, take two sterile 50mL centrifuge tubes, mark A and B respectively, add the light chain and heavy chain plasmids to tube A according to the calculation results, shake gently to mix, add 2mg / mL PEI to tube B, shake gently to mix, incubate solution A and solution B at room temperature for 5min, then use a pipette to add solution B to solution A to prepare AB mixed solution (see Table 1), shake gently to mix, and incubate at room temperature for 10min. After incubation at room temperature, use a pipette to slowly add the AB mixed solution to a 1L shake flask, shake gently while adding the sample to mix evenly, and then place it in a shaker at 37°C, 5% CO2, and 130rpm (amplitude 25mm) for incubation.

[0172] Table 1

[0173]

[0174]

[0175] 14.3-Purification of MT4354# Antibody (SPA Method)

[0176] Pass 250ml of the supernatant sample to be purified through a 400-mesh nylon mesh, and use A280 to determine the IgG concentration (0.02M PBS pH 7.4 is the blank buffer). Take the culture supernatant and filter it through a 0.45μm filter membrane, load it and set it aside. Take 20μl of the filtered sample and dilute it to 2mg / ml with 0.02M PBS pH 7.4. The experiment uses AKTA purification equipment, SPA column, and goes through equilibration, loading, rinsing and elution steps. After elution, dialyze in pbs buffer, concentrate with an ultrafiltration tube, filter the antibody with a 0.22μm filter membrane, and dilute it with 0.01M PBS pH 7.2 to a final antibody concentration of 5.0±0.2mg / ml. The electrophoresis results are as follows Figure 1 As shown, the HPLC results are as Figure 2 shown.

[0177] HPLC showed that the purity of 3-MT was >98%.

[0178] 15.3-MT4354# Biotinylated Antibody Labeling

[0179] According to the ratio of Biotin / antibody = 2:1 (molar ratio), 270 μl of Biotin was added to the 3-MT4354# antibody solution, and the solution was added dropwise while being shaken. After the addition was completed, the solution was thoroughly mixed by vortexing and reacted at 25 degrees for 16 hours.

[0180] Example 2 Performance evaluation of the kit of the present invention

[0181] The biotinylated 3-MT4354# antibody of Example 1 was used to coat magnetic beads, and HRP-labeled 3-MT antigen and other components were used to prepare a magnetic particle 3-methoxytyramine detection kit (magnetic particle chemiluminescence method). The competition method was used for detection: the antibody was coated on magnetic particles, and the antigen was labeled with horseradish peroxidase. An antibody-enzyme-labeled antigen complex was formed through immune reaction. The complex catalyzed the luminescent substrate solution to emit photons, and the luminescence intensity was inversely proportional to the content of the substance to be detected.

[0182] The 1605# and 4354# selected in Example 1 were used as coating antibodies, respectively, and the plasma after liquid quality control was used as a sample to compare the performance difference between the two.

[0183] 1. Valence and gradient evaluation

[0184] Table 2

[0185]

[0186]

[0187] The results showed that the titer and gradient of 4354# rabbit monoclonal antibody were better than those of the control antibody 1605#, meeting the requirements of kit development.

[0188] 2. Repeatability

[0189] Table 3

[0190]

[0191]

[0192] The results showed that when the 4354# antibody was coated at 0.003μg / T, the Q1 concentration value variation was 6.24%, the Q2 concentration value variation was 6.53%, and the Q3 concentration value variation was 5.60%. The repeatability could meet the requirements of the 3-MT project.

[0193] 3. Stability

[0194] Table 4

[0195]

[0196] Results: After accelerated treatment at 37℃ for 7 days, the signal value of 4354# calibration matrix and plasma matrix decreased by about -2.37%, and the concentration value variation was less than 10%. The stability can meet the requirements of project 3-MT.

[0197] 4. Crossover rate.

[0198] Table 5

[0199]

[0200]

[0201] Note: Norepinephrine (NE), epinephrine (E), and dopamine (D) are all catechol analogs.

[0202] Conclusion: The crossover rate with low values ​​of MN, NMN, NE, D, and E is <5%, and the crossover rate with high values ​​of MN, NMN, NE, D, and E is <15%, which meets the requirements for project 3-MT detection.

[0203] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. 3-methoxydopamine hapten, characterized in that Its structural formula is shown in Formula I: Here, n is any integer selected from 0 to 12.

2. The 3-methoxydopamine hapten according to claim 1, characterized in that The structural formula of the 3-methoxydopamine hapten is shown in Formula II: 3.3-methoxydopamine complete antigen, characterized in that It is a conjugate of the 3-methoxydopamine hapten and a carrier protein as claimed in claim 1 or 2; The carrier protein includes one or more of KLH, bovine serum albumin, human serum albumin, and ovalbumin; The carboxyl group at the end of the 3-methoxydopamine hapten is coupled to the amino group of the carrier protein; Preferably, the structure of the 3-methoxydopamine complete antigen is as shown in Formula III:

4. The method for preparing the complete 3-methoxydopamine antigen according to claim 3, characterized in that: The steps include: Synthesize a hapten of formula I; coupling the hapten with the carrier protein, and dialyzing the coupling product to obtain the complete 3-methoxydopamine antigen; Preferably, the coupling reaction conditions are: shaking reaction at 18-27° C. overnight, and the shaking reaction time is ≥16 h.

5. Use of the hapten according to any one of claims 1 to 2 or the complete antigen according to any one of claims 3 to 4 in any of the following: (1) preparing 3-MT recombinant monoclonal antibody; (2) preparing products for detecting the levels of 3-MT and its derivatives; (3) Preparation of diagnostic products for pheochromocytoma and / or paraganglioma. 6.3-MT recombinant monoclonal antibody, characterized in that This includes any of the following: (1) a 3-MT recombinant monoclonal antibody prepared by immunizing an animal with the complete 3-methoxydopamine antigen according to claim 3 or 4; (2) 3-MT recombinant monoclonal antibody, the three CDR regions of its light chain respectively have: A), the amino acid sequence shown in SEQ ID NO: 1 to 3; or B), a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in A); or C) has at least 80% of the sequence shown in A) or B); The three CDR regions of its heavy chain have: D), the amino acid sequence shown in SEQ ID NO: 4 to 6; or E), a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in D); or F) has at least 80% of the sequence shown in D) or E).

7. The 3-MT recombinant monoclonal antibody according to claim 8, characterized in that: It includes a heavy chain variable region and a light chain variable region; The light chain variable region has: G), the amino acid sequence shown in SEQ ID NO: 7; or H) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in G); or I), and the sequence shown in H) or I) has at least 80% of the sequence; The heavy chain variable region has: J), the amino acid sequence shown in SEQ ID NO: 8; or K), a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in J); or L), and the sequence shown in J) or K) have at least 80% of the sequence.

8. Biomaterial, characterized in that Includes any of the following: (I) a nucleic acid encoding the 3-MT recombinant monoclonal antibody according to any one of claims 6 to 7; (II), an expression cassette or recombinant vector comprising the nucleic acid described in (I); (III) a host comprising the nucleic acid described in (I) or the recombinant vector described in (II).

9. Application of any one of the following a) to b) in the preparation of a detection product: a), the 3-MT recombinant monoclonal antibody according to any one of claims 6 to 7; b) the biomaterial according to claim 8; The test products include: Products for detecting the level of 3-MT or its derivatives, or diagnostic products for pheochromocytoma and / or paraganglioma.

10. A kit comprising the complete antigen according to claim 3 or 4 and / or the 3-MT recombinant monoclonal antibody according to any one of claims 6 to 7.

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