Recombinant dopamine rabbit monoclonal antibody and application

Abstract

The present application relates to the technical field of antibody, in particular to a recombinant dopamine rabbit monoclonal antibody and application. The present application utilizes phage display technology and alanine scanning mutagenesis technology, and screens to obtain a dopamine monoclonal antibody, compared with other antibodies and the antibody without alanine scanning mutagenesis, the affinity and specificity are strong, the detection sensitivity is high, and the consistency with the antigen detection concentration result in liquid quality is high. The antibody is used for the detection of dopamine in serum, and the test result shows that the detection sensitivity is high, the specificity is good, the repeatability is high, the stability is good, the precision is high, and the antibody has an important role for the clinical diagnosis of dopamine.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, specifically to recombinant dopamine rabbit monoclonal antibody and its applications. Background Technology

[0002] Dopamine is the most abundant catecholamine neurotransmitter in the brain. It's a chemical that helps cells transmit impulses. This brain secretion is related to human emotions and feelings; it transmits information about excitement and happiness. As a neurotransmitter, dopamine regulates various physiological functions of the central nervous system. Disorders of dopamine system regulation are associated with Parkinson's disease, schizophrenia, Tourette syndrome, attention deficit hyperactivity disorder (ADHD), and pituitary tumors.

[0003] However, the antibodies currently used to detect dopamine have weak affinity, low specificity, and limited selectivity. Obtaining monoclonal recombinant antibodies with improved affinity and specificity through expression in mammalian cells to solve the dopamine detection problem is an urgent issue to be addressed in this field. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a recombinant dopamine rabbit monoclonal antibody and its application.

[0005] This invention provides a dopamine monoclonal antibody.

[0006] The amino acid sequence of its heavy chain is GFSLSDYW in the CDR1 region, IRNKPYNYAT in the CDR2 region, and TGGFDF in the CDR3 region.

[0007] The amino acid sequence of its light chain is QSVNTD in the CDR1 region, SAS in the CDR2 region, and QHHYLPPYT in the CDR3 region.

[0008] Furthermore, the dopamine monoclonal antibody described in this invention,

[0009] Its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:3;

[0010] Its light chain variable region has an amino acid sequence as shown in SEQ ID NO:4.

[0011] Furthermore, the dopamine monoclonal antibody of the present invention has a constant region of heavy chain that is rabbit IgG1 subtype and a constant region of light chain that is rabbit κ1 type.

[0012] This invention utilizes phage display technology and alanine scanning mutation technology to screen and obtain the dopamine monoclonal antibody described in this invention. Compared with other antibodies and antibodies without alanine scanning mutation, this monoclonal antibody exhibits high specificity, high detection sensitivity, and high consistency with the antigen concentration detection results in liquid chromatography-mass spectrometry. When this antibody was used for the detection of dopamine in serum, the experimental results showed high detection sensitivity, good specificity, high repeatability, good stability, and high precision.

[0013] This invention provides a biological material comprising at least one of the following: (I) to (IV)

[0014] I) Nucleic acid encoding the dopamine monoclonal antibody of the present invention;

[0015] II) A recombinant vector comprising a vector backbone and nucleic acids as shown in I);

[0016] III) Transfect or transform host cells with the recombinant vector shown in II);

[0017] IV) Culturing the host cells as described in III) to obtain a culture containing the dopamine monoclonal antibody.

[0018] The present invention also provides an expression module comprising the nucleic acid, the expression module including a promoter, a terminator and the nucleic acid described in the present invention.

[0019] Furthermore, the expression module also includes an expression module formed by combining one or more nucleic acids described in this invention in tandem, fusion expression or other feasible ways, and this invention does not limit this.

[0020] The present invention also provides a transcription unit, which refers to a DNA sequence from the start of a promoter to the end of a terminator. Regulatory fragments may also be included flanking or between the promoter and terminator. These regulatory fragments may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, transmembrane signal peptides, and homologous recombination sites operatively linked to a nucleic acid sequence, such as enhancers of promoters, ITR sequences, polyA, MIS signal peptides, etc.

[0021] The recombinant vector of the present invention includes a vector backbone and the nucleic acid of the present invention.

[0022] Furthermore, the vector backbone of the present invention can be derived from plants, animals, bacteria, fungi, bacteriophages, or viruses, and the present invention does not limit this. The bacteriophage vector includes phage particles and helper vectors, and the phage particles include, but are not limited to, pBluescript II-KS(+), pcomb3XSS, pCANTAB5E, or pKK233.3. The mammalian vectors include, but are not limited to, pcDNA 3.1, pIRES, pTT3, pCEP4, pATX1, or pCHO1.0. The bacterial vectors include, but are not limited to, pET28a, pET16b, pET26b, pET28a, pET31b, pBAD, pBADHis, pTrc99a, pTrcHis, pACYCduet-1, pET duet-1, pCDFduet-1, pColdI, pColdII, etc. The fungal vectors include, but are not limited to, pYES2, pYES3, pYES6, pAUR23, etc.

[0023] In some embodiments of the present invention, the nucleic acid encoding the dopamine monoclonal antibody is integrated with the vector backbone pcomb3XSS for screening of phage libraries; in other embodiments of the present invention, the nucleic acid encoding the dopamine monoclonal antibody is integrated with the vector backbone pCHO 1.0 to construct a recombinant vector capable of replication and expression in host cells.

[0024] The recombinant vector described in this invention refers to a recombinant nucleic acid vector, a recombinant DNA molecule containing the desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of an operatively linked coding gene in a specific host organism. Nucleic acid sequences or elements essential for expression in viruses, microorganisms, or mammalian cells include promoters, ribosome binding sites, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In this specification, "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. However, this invention intends to include other forms of expression vectors that perform equivalent functions and are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors, and / or simply potential genomic inserts. In specific embodiments, the nucleic acid encoding the progesterone monoclonal antibody provided by this invention can be constructed in various expression vectors.

[0025] The host cells described in this invention can be derived from plants, animals, microorganisms, or viruses, and this invention does not limit the source. This invention uses vectors constructed using recombinant DNA technology to transform or transfect host cells, thereby enabling the transformed host cells to replicate the protein-encoding vector or express the desired protein.

[0026] In this invention, the transformation methods include chemical transformation and electrotransformation; the transfection methods include calcium phosphate coprecipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0027] Furthermore, in some specific embodiments, the host cell described in this invention is a mammalian cell, specifically selected from at least one of HEK-293, HEK293T, Hep G2, HELA, CHO-K1, COS-1, COS-7, NIH3T3, A204, A549, D-407, CHO, HCS-2, HT-29, U87, Sf9, or FD-CHOS. In some specific embodiments, the host cell described in this invention is a CHO cell, used for the expression of progesterone monoclonal antibody before mutation. Results show that the progesterone monoclonal antibody described in this invention has high expression level, high purity, and high concentration, meeting production requirements and showing good application prospects in clinical diagnostic reagents.

[0028] The present invention provides labeled antibodies, which include a marker and the dopamine monoclonal antibody described in the present invention.

[0029] Furthermore, the markers include chemical markers and biological markers; the biological markers include biotin, avidin, or enzymes, and the enzymes include horseradish peroxidase or alkaline phosphatase; the chemical markers include isotopes and / or chemical drugs.

[0030] The present invention provides a conjugate comprising a conjugate medium and the dopamine monoclonal antibody described in this invention.

[0031] Furthermore, in the coupling material described in this invention, the coupling medium includes a solid medium or a semi-solid medium.

[0032] Furthermore, the coupling medium is selected from colloidal gold, polystyrene sheets, or beads.

[0033] The application of any one of the following (a) to (g) in the preparation of a product for detecting dopamine levels:

[0034] a) The dopamine monoclonal antibody described in this invention;

[0035] b) The biomaterials described in this invention;

[0036] c) The labeled antibody described in this invention;

[0037] d) The coupling compound described in this invention.

[0038] This invention provides a product for detecting dopamine levels, the raw materials of which include any one of the following: A) to D).

[0039] A) The dopamine monoclonal antibody described in this invention;

[0040] B) The biomaterials of this invention;

[0041] C) The labeled antibody described in this invention;

[0042] D) The coupling compound described in this invention.

[0043] The samples mentioned in this invention are serum, blood, or plasma.

[0044] In a specific embodiment of the present invention, the product is a magnetic microparticle luminescence reagent kit.

[0045] This invention provides a method for detecting dopamine, which involves using the product described in this invention to detect the dopamine content in a sample.

[0046] Furthermore, the sample is serum, blood, or plasma.

[0047] This invention utilizes phage display technology and alanine scanning mutation technology to screen and obtain dopamine monoclonal antibodies. Compared with other antibodies and antibodies without alanine scanning mutation, this monoclonal antibody exhibits strong affinity and specificity, high detection sensitivity, and high consistency with antigen concentration detection results in liquid chromatography-mass spectrometry. When this antibody was used for the detection of dopamine in serum, experimental results showed high sensitivity, good specificity, high repeatability, good stability, and high precision, playing an important role in the clinical diagnosis of dopamine. Attached Figure Description

[0048] Figure 1 This is an agarose gel electrophoresis image of RNA extracted from the spleen and bone marrow;

[0049] Figure 2 This is an agarose gel electrophoresis image of the PCR products of the VL and VH genes. The marker sizes from top to bottom are: 100, 250, 750, and 2000.

[0050] Figure 3 This is an agarose gel electrophoresis image of the PCR products of the scFv gene. The sample wells from left to right are ScFv1, ScFv2, ScFv3, ScFv4 and the control.

[0051] Figure 4 This is an agarose gel electrophoresis image showing the antibody library recombination rate and bacterial culture PCR identification.

[0052] Figure 5 This is an SDS-PAGE electrophoresis image of the purified recombinant antibody;

[0053] Figure 6 The correlation between antibodies and positive controls in liquid chromatography before and after mutation is shown on the coordinate axis in pg / ml. Here, A represents the correlation between antibodies and positive controls after mutation, and B represents the correlation between antibodies and positive controls before mutation.

[0054] Figure 7 The correlation between antibody detection luminescence value and liquid chromatography-mass spectrometry antigen concentration is shown on the coordinate axis in pg / ml. A represents the mutated antibody AD3009#, B represents the positive control R601 autogenous antibody serum, and C represents the pre-mutation antibody. Detailed Implementation

[0055] This invention provides a recombinant dopamine rabbit monoclonal antibody and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0056] RbbScFVF-180521: ccgtggcccaggcggccgagctcg (SEQ ID NO: 1);

[0057] RbbScFVR-18-521:gtgctggccggcctggccactagtgactg (SEQ ID NO: 2);

[0058] The amino acid sequence of the variable region of the heavy chain of the mutated antibody:

[0059] QSLEESGGRLVTPGTPLTLTCTASGLSFSGYYMSWVRQAPGKGLEWIGLIEP

[0060] GGTIYYANWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARDGTGSVYY NIWGPGTLVTISS(SEQID NO:3);

[0061] The amino acid sequence of the variable region of the light chain of the mutated antibody:

[0062] DVVMTQTAASVSAAVGGTVTINCQSSPSVYKNNYLSWFQQKPGQPPKLLI

[0063] YEASRLASGVPQRFSGSGSGTQFTLTISGVQCGDAASYYCLGGYGLGNDL GAFGGGTKLEIK(SEQID NO:4);

[0064] The amino acid sequence of CDR1 in the heavy chain of the mutated antibody: GLSFSGYY (SEQ ID NO:5);

[0065] The amino acid sequence of CDR2 in the heavy chain of the mutated antibody: IEPGGTI (SEQ ID NO: 6);

[0066] The amino acid sequence of CDR3 in the heavy chain of the mutated antibody: ARDGTGSVYYNI (SEQ ID NO:7);

[0067] The amino acid sequence of CDR1 in the light chain of the mutated antibody: VYKNNY (SEQ ID NO:8);

[0068] The amino acid sequence of CDR2 in the light chain of the mutated antibody: EAS;

[0069] The amino acid sequence of CDR3 in the light chain of the mutated antibody: LGGYGLGNDLGA (SEQ ID NO: 9).

[0070] The reagents and consumables used in this invention are all commercially available products that can be purchased on the market.

[0071] The present invention will be further illustrated below with reference to the embodiments:

[0072] Example 1: Preparation for screening the phage platform provided by the present invention

[0073] 1. Immunizing animals with recombinant immunogen:

[0074] New Zealand white rabbits were immunized four times with the prepared antigen methyldopamine conjugated with KLH. The immunization cycle was 30 days, with multiple subcutaneous injections on the back. For the first immunization, 2 mg of the immunogen was emulsified with an equal volume of Freund's complete adjuvant and then immunized. For the subsequent three immunizations, 1 mg of the immunogen was emulsified with an equal volume of Freund's incomplete adjuvant and then immunized. On day 10 after the third immunization, blood was collected from the marginal ear vein, incubated at 37°C for 1 hour, and then centrifuged at 6000 rpm for 10 minutes. The supernatant (antiserum) was collected for ELISA to detect the immunization effect.

[0075] 2. Antiserum titer detection (reverse indirect method + competitive method)

[0076] Reverse indirect method: Rabbit serum was added to the anti-rabbit plate. The test serum was serially diluted 1:500, 50 μL / well, with a positive control included. The plate was incubated at 37°C for 30 min. After washing five times with PBST, the plate was dried, and biotinylated antigen was added, 50 μL / well. The plate was incubated at 37°C for 30 min. After washing five times with PBST, the plate was dried, and avidin was added. Luminescent substrates A and B were added, 50 μL each / well. The plate was incubated in the dark for 5 min, and the luminescence was detected using a luminescence analyzer. A competition experiment was conducted using a proportion of rabbits with signal values ​​consistent with the positive control. The positive control was R601 autologous antibody serum. 1# to 5# represent different rabbit numbers. The test results are shown in Table 1.

[0077] Table 1. Antiserum titer detection

[0078]

[0079] Conclusion: 1# was selected for competitive experiments at a dilution ratio of 1 / 8k, and 2# to 5# were selected at a dilution ratio of 1 / 16k.

[0080] Competitive method: Rabbit serum was added to the anti-rabbit plate at the dilution ratio described above. The plate was incubated at 37°C for 30 min; washed 5 times with PBST, dried, and calibrator and biotinylated antigen were added simultaneously, 50 μL / well, and incubated at 37°C for 30 min; washed 5 times with PBST, dried, and then avidin and luminescent substrates A and B were added, 50 μL / well each, and the reaction was carried out in the dark for 5 min. The plate was then subjected to luminescence analysis. The test results are shown in Table 2.

[0081] Table 2. Results of the competition method test

[0082]

[0083] In Table 2, S0, S1, S3, and S5 represent different concentrations of antigen added to the diluent matrix. The antigen concentration increases sequentially from S0 to S5. The antigen concentration in the diluent matrix is ​​0 pg / ml in S0; 113.8 pg / ml in S1; 891.5 pg / ml in S3; and 8023.2 pg / ml in S5. S0 / S5 represents the ratio of the signal value corresponding to S0 to the signal value corresponding to S5. Conclusion: The data shows that the serum gradient of rabbit #1 is better than that of the control, indicating the production of specific antibodies. Rabbit #1 can be boosted with immunization. Three days later, the animals are sacrificed, and spleen cells are extracted. Total RNA (e.g., 500g / ml) is routinely extracted from the spleen tissue using the Trizol method. Figure 1 (As shown), cDNA is synthesized by reverse transcription.

[0084] Example 2: scFv gene splicing and phage screening construction

[0085] 1. scFv gene splicing

[0086] PCR was used to amplify the heavy chain variable region and the light chain variable region of the antibody, respectively. The PCR reaction program was as follows:

[0087] Table 3. PCR reaction procedure

[0088]

[0089] PCR amplification primers:

[0090] RbbScFVF-180521: ccgtggcccaggcggccgagctcg (SEQ ID NO: 1);

[0091] RbbScFVR-18-521:gtgctggccggcctggccactagtgactg (SEQ ID NO: 2);

[0092] PCR products were recovered via 1% agarose gel electrophoresis, such as... Figure 2 and Figure 3 As shown. The amplified light chain variable region and heavy chain variable region were spliced ​​together using overlap-PCR to form scFv. The product was recovered by 1% agarose gel electrophoresis and stored at -20℃. Figure 3 As shown.

[0093] 2. Construction and screening of phage single-chain antibody libraries:

[0094] The phage vector pcomb3XSS and the purified ScFv fragment were digested with SfiI to construct a recombinant plasmid. The recombinant plasmid was electroporated into TG1 competent cells to construct a rabbit-derived immune single-chain antibody library (antibody library recombination rate was identified by bacterial PCR as follows). Figure 4 A primary phage single-chain antibody library was prepared. The primary phage single-chain antibody library was enriched and screened three times to screen for specific phage single-chain antibody libraries with high affinity and strong specificity. The screened antibodies are shown in Table 4. Based on the screening results, single clones were picked to prepare single-clone phage supernatant. Positive clones were identified by Phage-ELISA to obtain positive sequences.

[0095] Table 4. Screening of phage single-chain antibody libraries

[0096]

[0097]

[0098] Conclusion: After phage screening, antibody #1 was the optimal choice. Among them:

[0099] S1 and S3 represent calibrators, with S1 having a concentration of 113.8 pg / ml and S3 having a concentration of 891.5 pg / ml.

[0100] NE50 and NE500 represent cross-labeled products (concentrations of 50 pg / ml and 500 pg / ml, respectively);

[0101] S3 / S1 is the ratio of the signal value of S3 to the signal value of S1;

[0102] NE500 / 50 is the ratio of the signal value of NE500 to the signal value of NE50.

[0103] High / Low is the ratio of clinically high signal value to clinically low signal value;

[0104] Clinically representative real clinical samples; the difference between "clinically high" and "clinically low" refers to real clinical samples with different concentrations. Among these, higher S3 / S1, NE500 / 50, and high / low ratios indicate better antibody specificity. Example 31# Monoclonal Antibody Expression and Antibody Purification

[0105] 1. Construction of stable cell lines

[0106] Recombinant antibody genes were obtained by ligating the constant and variable regions of the heavy chain and the light chain separately using overlap-PCR. The heavy chain and light chain antibody genes were then digested with XmaJI / BstZ17I and EcoRV / PacI, respectively, and ligated into the expression vector pCHO 1.0. After transfecting the recombinant plasmids into competent DH5α cells, positive clones were selected for sequencing and plasmid extraction. The extracted plasmids were linearized with RruI and then transfected into CHO cells.

[0107] Forty-eight hours after transfection, ELISA was performed to detect antibody expression. 200 nM MTX and 20 μg / mL Puromycin were added to the culture medium for screening positive cell lines. After cell viability recovered, the screening concentrations were further increased (1000 MTX and 50 μg / mL Puromycin). Once cell viability recovered, the stable cell line pool was constructed. Subsequently, the stable cell line pool was screened using 96-well plates, 24-well plates, 6-well plates, and SF125 shake flasks to identify cell lines that stably and highly express the monoclonal antibody.

[0108] 2. Recombinant antibody expression

[0109] Resuscitate stable, high-expressing monoclonal cell lines, according to 5 × 10⁻⁶ 5 After passage two generations at a cell / mL density, when the cell viability is not lower than 95%, Fed-batch evaluation is started. Feed is added the next day, and the target protein supernatant is obtained when the cell viability drops to below 70%.

[0110] 3. Antibody SPA purification and SDS-PAGE column equilibration

[0111] Set the flow rate to 6.4 mL / min, rinse the column with 0.02 mol / L PBS pH 7.4 equilibration buffer, set the flow rate to 3.8 mL / min, and perform sample loading and purification. Add 10 μL of sample from the flow-through tube to 200 μL of G250 staining solution. When the solution turns blue, place the flow-through tube into a 250 mL Erlenmeyer flask to begin collecting the flow. Re-equilibrate: Set the flow rate to 6.4 mL / min, rinse the column again with 0.02 mol / L PBS pH 7.4 equilibration buffer until no protein flow-through occurs. Dissociation: Place 10 4 mL centrifuge tubes on the dissociation rack, add 200 μL of 1 mol / L Tris-HCl pH 8.5 to each tube, set the constant flow pump to 6.4 mL / min, and add 0.2 mol / L dissociation buffer. The target protein was dissociated using Gly + 0.15 mol / L NaCl at pH 2.7. A 10 μL sample was taken from an isoperfusion tube and added to 200 μL of G250 staining solution. Manual collection began when the solution turned blue, with 4 mL collected from each tube. Another 10 μL sample was taken from an isoperfusion tube and added to 200 μL of G250 staining solution. Collection stopped when the solution turned colorless. The collected proteins were pooled and analyzed by SDS-PAGE. Figure 5 As shown, the purified antibody has a purity of up to 98% and a concentration of 5.2 mg / mL.

[0112] Example 4: Comparison of antibody alanine scanning mutation and evaluation data before and after mutation

[0113] 1. Mutant library design: Alanine scanning of the CDR region was used to select amino acids with significant impact for soft mutation (i.e., the mutation method retains 70% of the original codon). Alanine scanning involves introducing one or more alanine residues onto charged residues and observing the effect of these changes on protein function. The mutated antibody was named AD 3009#, and its heavy chain amino acid sequence is shown in SEQ ID NO:3, while the light chain amino acid sequence is shown in SEQ ID NO:4. Compared with the unmutated sequence, only one amino acid in the heavy chain variable region changed. Specifically, leucine at position 21 of the heavy chain variable region was mutated to cysteine ​​to obtain the heavy chain variable region shown in SEQ ID NO:3.

[0114] 2. Evaluation of results before and after mutation, where the positive control here and thereafter is the self-produced superior rabbit serum R601 containing dopamine antibodies.

[0115] Table 5. Results of antibody evaluation before and after mutation.

[0116]

[0117]

[0118] In Table 5, 0 / 50, 50 / 200, 200 / 8000, and 0 / 8000 represent the ratio of the luminescence value before the slash to the luminescence value after the slash. The antibodies here are all purified naked antibodies and have not yet been labeled with magnetic microparticles.

[0119] Table 6. Clinical comparison of magnetic particle amplification before mutation.

[0120] sample Pre-mutation signal value Signal value after mutation Positive control signal value S0 39304 41404 48411 S5 3313 1986 3285 Clinical 1# 16146 24570 29360 Clinical 2# 16579 25527 31466 Clinical 3# 12822 20270 24797 Clinical 4# 17032 25313 30160 Clinical 5# 18996 28739 31710 Clinical 6# 17305 26443 29667 Clinical 7# 9744 18309 22693 Clinical 8# 3906 3248 6739 Clinical 9# 4889 10657 14399 Clinical 10# 15454 23611 29313 S0 / S5 11.86 20.85 14.74 Clinical 5 / 8 4.86 8.85 4.71

[0121] Clinical 5 / 8 represents the ratio of the clinical 5 signal value to the clinical 8 signal value. The larger the ratio, the better the antibody properties. The table shows the luminescence values ​​of clinical samples measured before and after antibody mutation.

[0122] Table 7. Results of Liquid Chromatography

[0123]

[0124]

[0125] The sample numbers mentioned above correspond to real clinical samples, and the dopamine content varies in each sample. Conclusion: Combining Tables 5-7 and... Figure 6 and Figure 7 The results showed that before antibody mutation, the gradient was small and the correlation was not high; after mutation, the gradient increased significantly and the correlation with liquid chromatography-mass spectrometry (LC-MS) was increased. 2 The value was 0.9735, indicating that the antibody performance was enhanced after mutation.

[0126] 3. Display of specific results

[0127] Table 8. Specificity of the mutated antibody

[0128] Sample number Dopamine mutation antibody luminescence value Cross rate 0 1529974 MN62 1509626 1％ NMN145 1452889 5％ 3MT28 1548033 -1％ E125 1471691 1％ NE600 1589808 -4％ LD100 1564611 -2％ MN620 1478489 -3％ NMN1450 1491674 3％ 3MT280 1505726 2％ E1250 1445857 5％ NE6000 1519349 1％ LD1000 1437650 -6％

[0129] The samples listed in this table are cross-samples, whose antigenic structures are similar to dopamine. The letter part of the sample number corresponds to the following analogues: dopamine (DA), epinephrine (E), norepinephrine (NE), methoxytyramine (3-MT), norepinephrine (NMN, also known as methoxynorepinephrine), and norepinephrine (MN, also known as methoxyadrenaline), and levodopamine (LD). The numerical part represents the concentration of each sample, in pg / ml.

[0130] Our antibodies should ideally detect dopamine but not its analogues, and the lower the cross-linking rate, the better.

[0131] Conclusion: The cross-cross rate was verified to be ≤5%, indicating that the antibody of the present invention has good specificity and is qualified.

[0132] 4. Precision assessment

[0133] Table 9. Precision Assessment of Mutated Antibodies

[0134]

[0135]

[0136] Among them, the serum matrix is prepared by adding high-value antigen to normal human serum, which is closer to real clinical samples. The calibration matrix is the matrix of the calibrator. 1 / 8K is the antigen dilution ratio. X8 and J8 are just identification symbols without special significance.

[0137] Conclusion: From the data, it can be seen that when the DA high-value antigen is added to the serum matrix and the calibration matrix, the mutation rate is within 5%, and there is no gap phenomenon, indicating good precision.

[0138] 5. Stability Assessment

[0139] Table 10. Stability of Mutated Antibodies

[0140]

[0141]

[0142] Among them, S0-S5 are calibrators (antigen content increases sequentially), PQ1-PQ5 and Q1, Q2 represent quality control products; P1-P12 are real clinical samples.

[0143] Conclusion: <所给内容中“

[0144] ”无对应中文,无法准确翻译此部分。若按照原文格式保留,翻译如下:

[0144] After the antibody is placed at 4°C and 37°C simultaneously for 7 days, the decrease is within 5%, which is qualified.

[0145] 6. Summary:

[0146] Combining all the above data, it can be proved that this antibody has good specificity, accuracy, sensitivity and stability, and can be used for clinical detection.

[0147] The above is only the preferred implementation mode of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. 需注意,原文中“

[0144] ”部分无对应中文内容,按照要求保留了原文格式。若这部分原文有误,请提供准确信息以便准确翻译。

Claims

1. A dopamine monoclonal antibody, characterized in that, The amino acid sequence of its heavy chain is GLSFSGYY in the CDR1 region, IEPGGTI in the CDR2 region, and ARDGTGSVYYNI in the CDR3 region. The amino acid sequence of its light chain is VYKNNY in the CDR1 region, EAS in the CDR2 region, and LGGYGLGNDLGA in the CDR3 region.

2. The dopamine monoclonal antibody according to claim 1, characterized in that, Its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:3; Its light chain variable region has an amino acid sequence as shown in SEQ ID NO:

4.

3. The dopamine monoclonal antibody according to claim 1 or 2, characterized in that, The constant region of the heavy chain is rabbit IgG1 subtype; the constant region of the light chain is rabbit κ1 type.

4. A biomaterial, characterized in that, Includes at least one of the following: (I) to (IV) I) The nucleic acid encoding the dopamine monoclonal antibody according to any one of claims 1 to 3; II) A recombinant vector comprising a vector backbone and nucleic acids as shown in I); III) Transfect or transform host cells with the recombinant vector shown in II); IV) Culturing host cells as described in III) to obtain a culture containing the dopamine monoclonal antibody.

5. A labeled antibody, characterized in that, Includes markers and dopamine monoclonal antibodies as described in any one of claims 1 to 3.

6. The labeled antibody according to claim 5, characterized in that, The markers include chemical markers and biological markers; the biological markers include biotin, avidin, or enzymes; the chemical markers include isotopes.

7. A coupling, characterized in that, It includes a coupling medium and the dopamine monoclonal antibody as described in any one of claims 1 to 3; the coupling medium is selected from solid media or semi-solid media.

8. Any one of the following applications (a) to (d) in the preparation of products for detecting dopamine levels: a) The dopamine monoclonal antibody according to any one of claims 1 to 3; b) The biomaterial as described in claim 4; c) The labeled antibody as described in claim 5 or 6; d) The coupling compound as described in claim 7.

9. A product for detecting dopamine levels, characterized in that, The raw materials include any one of the following: A) to D) A) The dopamine monoclonal antibody according to any one of claims 1 to 3; B) The biomaterial as described in claim 4; C) The labeled antibody as described in claim 5 or 6; D) The coupling compound according to claim 7.

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