Anti-human TH glycoprotein monoclonal antibody, urine stain identification kit based on TH glycoprotein / IgG ratio method and detection method

Abstract

The invention discloses an anti-human TH glycoprotein monoclonal antibody, a urine stain identification kit based on a TH glycoprotein / IgG ratio method and a detection method. The kit comprises a fluorescently-labeled anti-human TH glycoprotein specific monoclonal antibody, the kit is prepared from related materials such as a fluorescence-labeled anti-human IgG specific monoclonal antibody, a fluorescence-labeled DNP-BSA, an anti-human TH glycoprotein specific monoclonal antibody for coating, an anti-human IgG specific monoclonal antibody for coating, a rabbit anti-DNP polyclonal antibody, a chromatographic test strip and the like. According to the invention, human TH glycoprotein in urine traces is specifically detected, so that interference of urine traces of other animals is eliminated; the content of human-derived TH glycoprotein in urine stains and the ratio of the human-derived TH glycoprotein to human IgG are measured through a fluorescent quantitative method, so that interference of other retrogression such as blood stains and sweat stains is eliminated. The kit provided by the invention can be applied to accurate and rapid identification of human urine stains in criminal reconnaissance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an anti-human TH glycoprotein monoclonal antibody, a urine trace identification kit based on a TH glycoprotein / IgG ratio method, and a detection method. Background Art

[0002] Urine stain detection is one of the must-do items in criminal investigation. People often have urinary incontinence when they are extremely frightened, so urine stains of the victim are often left at the crime scene. Extracting and identifying urine stain samples at the scene is one of the important bases for judging the crime scene and the possibility of murder. The conventional method for discovering urine stains at the crime scene is to observe fluorescence under ultraviolet light. Although this method is sensitive, it has poor specificity and cannot rule out interference from urine stains of other animals and other samples containing fluorescent substances. At present, there are no reagents and instruments for rapid detection and identification of specific components in human urine stains. Summary of the invention

[0003] In response to the above-mentioned technical problems existing in the prior art, we have developed anti-human TH glycoprotein-specific monoclonal antibodies targeting the specific protein TH glycoprotein in urine, and invented a urine stain identification kit and detection method based on the TH glycoprotein / IgG ratio method. TH glycoprotein, namely Tamm-Horsfall glycoprotein, is also known as uromodulin and urinary mucin (Uromucoid). It is a specific protein of the kidney, mainly present in urine, and only in trace amounts in serum. TH glycoprotein is the specific protein with the highest content in urine. The normal value in human urine is 20-40 mg / L, and the content in serum is about 50 μg / L. Therefore, it can be used as the most ideal target for urine stain identification.

[0004] The technical solution adopted by the present invention is as follows:

[0005] A method for preparing an anti-human TH glycoprotein monoclonal antibody comprises the following steps:

[0006] 1) According to the human TH glycoprotein sequence, the C-terminal 30 amino acids containing the GPI anchor signal peptide were removed, and then the C-terminal was connected to the EK protease cleavage site Flag tag, and then connected to the Fc segment of mouse IgG as a purification tag to form the THP (GPI free) -Flag-mFc fusion protein gene. After adding the Kozak sequence GCCACC before the start codon ATG of its gene sequence, the host cell codon was optimized, the gene was synthesized and subcloned into the CMV promoter of the lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP to obtain a plasmid that can express the THP (GPI free) -Flag-mFc fusion protein, which was marked as pMLD-THP (GPI free) -mFc.GFP;

[0007] 2) The plasmid pMLD-THP(GPI free)-mFc.GFP constructed in step 1) was co-transfected with the pH1 plasmid and the pH2 plasmid into the lentiviral packaging cell 293V to prepare the THP(GPI free)-mFc.GFP lentivirus, and transfected into HEK293 cells. The clones were picked under a fluorescence microscope to establish a THP(GPI free)-mFc.GFP / 293 stable cell line;

[0008] 3) using a cell factory to amplify and culture the THP (GPI free)-mFc.GFP / 293 cells prepared in step 2), and collecting the culture supernatant, extracting and purifying the fusion protein therein using a protein A column, and then using EK enzyme to cut off the mFc tag to obtain a recombinant human TH glycoprotein, which is labeled as THP (GPI free) protein;

[0009] 4) Immunizing mice with the recombinant human TH glycoprotein antigen prepared in step 3), and preparing anti-human TH glycoprotein monoclonal antibody using mouse monoclonal antibody technology.

[0010] 2. The method for preparing an anti-human TH glycoprotein monoclonal antibody according to claim 1, characterized in that in step 1), the human TH glycoprotein sequence is the gene and protein sequence of GenBank: M15881.1; the removed C-terminal 30 amino acids containing the GPI anchor signal peptide, namely A611-Q640, are specifically ATVSRAFSSLGLLKVWLPLLLSATLTLTFQ;

[0011] Among them, the nucleic acid sequence of the THP (GPI free) -Flag-mFc fusion protein gene after host cell codon optimization is shown in SEQ ID NO: 1, the gene is synthesized and subcloned into the CMV promoter of the lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP, and the amino acid sequence of the THP (GPI free) -Flag-mFc fusion protein expressed by the constructed plasmid is shown in SEQ ID NO: 2.

[0012] Furthermore, in step 4), when the anti-human TH glycoprotein monoclonal antibody is prepared using mouse monoclonal antibody technology, human urine and urine samples of different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice are used for testing in antibody screening to obtain a pair of anti-human urine TH glycoprotein specific monoclonal antibodies that do not react with urine samples of different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice, which are the anti-human TH glycoprotein monoclonal antibodies.

[0013] The present invention also discloses a urine stain identification kit based on the TH glycoprotein / IgG ratio method, the kit comprises an immunochromatographic test strip, the immunochromatographic test strip comprises a bottom liner, a NC membrane, a sample pad, a release pad and a water absorbent pad; the sample pad, the release pad, the NC membrane and the water absorbent pad are sequentially overlapped and assembled on the bottom liner, the release pad and the water absorbent pad are respectively superimposed on the two ends of the NC, and a detection area is formed on the surface of the NC, and the sample pad is superimposed on the release pad; a detection line area T1 line and a T2 line close to the release pad and a control line area C line close to the water absorbent pad are arranged on the NC of the detection area; DNP-BSA is coupled to a carboxyl quantum dot microsphere and marked as Q-DNP; Q-hTHP01, Q-hIgG01 and Q-DNP are mixed and sprayed on the release pad, and the T1 line area on the NC membrane is coated with C-hTHP02, the T2 line area is coated with C-hIgG02, and the C line area is coated with rabbit anti-DNP polyclonal antibody;

[0014] Among them, one of the antibodies of the pair of anti-human urine TH glycoprotein specific monoclonal antibodies of the present invention is labeled with quantum dot fluorescent microspheres and is labeled as Q-hTHP01, and the other is used as a coating antibody and is labeled as C-hTHP02;

[0015] One of the anti-human IgG specific monoclonal antibodies was labeled with quantum dot fluorescent microspheres and labeled as Q-hIgG01, and the other was used as a coating antibody and labeled as C-hIgG02.

[0016] Furthermore, the preparation process of the anti-human IgG specific monoclonal antibody is: using human IgG as an antigen to immunize mice, using mouse monoclonal antibody technology to prepare anti-human IgG antibodies, and in the antibody screening, using human urine and urine samples of different animals such as pigs, cattle, sheep, dogs, cats, rabbits, mice, etc. for testing to obtain a pair of anti-human IgG specific monoclonal antibodies that do not react with urine samples of pigs, cattle, sheep, dogs, cats, rabbits, mice, etc.

[0017] The detection method of the urine stain identification kit based on the TH glycoprotein / IgG ratio method comprises the following steps:

[0018] 1) The detection limit of the kit for human TH glycoprotein is set at 100 ng / mL, and the detection limit of human IgG protein is set at 50 ng / mL, and a dry fluorescence chromatography analyzer is used for quantitative analysis, and quantitative standard curves of human TH glycoprotein and human IgG protein are respectively preset in the detection instrument, and then the corresponding protein content in the actual sample detection is deduced according to the corresponding standard curves;

[0019] 2) The suspected urine sample is dissolved with PBS buffer at pH 7.4, and then tested with the kit. After the detection chromatography is left for 10-15 minutes, a dry fluorescence chromatography analyzer is used for quantitative analysis. If TH glycoprotein is not detected, that is, the TH glycoprotein value is <100ng / mL, it is a negative sample and can be ruled out as human urine; if the TH glycoprotein value is ≧100ng / mL and human IgG is not detected or the human IgG value is ≧50ng / mL but the TH glycoprotein value / IgG value ratio is ≧0.1, it is a positive sample for human urine; if the TH glycoprotein value is ≧100ng / mL and the human IgG value is ≧50ng / mL but the TH glycoprotein value / IgG value ratio is <0.1, it is a sample mixed with human blood or other samples containing human IgG.

[0020] Furthermore, the preparation of the standard curve in step 1) includes the following process:

[0021] Step 1, preparing standard curve 1: using recombinant human TH glycoprotein as a standard, using pH 7.4 PBS buffer to prepare a series of standard solutions with different concentrations in the concentration range of 100 ng / mL-3200 ng / mL, using pH 7.4 PBS buffer as a blank control, respectively using the kit for detection, after chromatography, using a dry fluorescence chromatography analyzer to measure the T1 line fluorescence value and the C line fluorescence value, using the ratio of the T1 line fluorescence value to the C line fluorescence value, i.e., the T1 / C fluorescence value ratio, as the ordinate, and the recombinant human TH glycoprotein concentration as the abscissa to draw standard curve 1;

[0022] Step 2, preparing standard curve 2: using human IgG protein as the standard, using pH 7.4 PBS buffer to prepare a series of standard solutions with different concentrations in the concentration range of 25 ng / mL-800 ng / mL, using pH 7.4 PBS buffer as the blank control, and using the kit for detection, after chromatography, using a dry fluorescence chromatography analyzer to determine the T2 line fluorescence value and the C line fluorescence value, and using the ratio of the T2 line fluorescence value to the C line fluorescence value, i.e., the T2 / C fluorescence value ratio, as the ordinate and the human IgG protein concentration as the abscissa to draw standard curve 2.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention has developed a specific monoclonal antibody against human TH glycoprotein, and prepared an immunochromatographic rapid test strip that can be used to accurately and quickly identify human urine stains. In addition, IgG is an abundant protein in human plasma, with a normal value of 7.5-15.6 g / L, while the normal value of IgG content in human urine is 0.0-14.0 mg / L. Although the urine IgG content of patients with renal damage will increase, it is more than 2 orders of magnitude different from the serum IgG content. The present invention adopts a quantitative analysis method to simultaneously determine the human TH glycoprotein content and human IgG content in the sample, and calculate the TH glycoprotein content / IgG content ratio, so that it can more accurately determine whether it is a human urine sample, eliminating interference from other stains such as blood stains and sweat stains.

[0025] (2) The present invention uses quantum dot fluorescence chromatography quantitative analysis technology to quickly determine the human TH glycoprotein content and human IgG content in suspected human urine, and eliminates interfering substances through the TH glycoprotein content / IgG content ratio to accurately and quickly identify human urine.

[0026] (3) The kit of the present invention can be used for accurate and rapid identification of human urine traces in criminal investigation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the urine stain identification kit based on the TH glycoprotein / IgG ratio method;

[0028] Figure 2 Standard curve for determination of TH glycoprotein content;

[0029] Figure 3 hIgG content determination standard curve. DETAILED DESCRIPTION

[0030] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments:

[0031] The present invention discloses a urine stain identification kit based on the TH glycoprotein / IgG ratio method. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The preparation method and application of the present invention have been described through preferred embodiments. Relevant personnel can obviously modify or appropriately change and combine the preparation method and application described herein without departing from the content and scope of the present invention to realize and apply the technology of the present invention.

[0032] Explanation of some terms and abbreviations:

[0033] THP: Tamm-Horsfall glycoprotein, TH glycoprotein, also known as uromodulin and urinary mucin (Uromucoid).

[0034] GPI: Glycosylphosphatidylinositol, glycosylphosphatidylinositol; THP is a glycosylphosphatidylinositol-anchored protein (glycosylphosphatidylinositol-anchored proteins, GPI-AP).

[0035] hIgG: Human Immunoglobulin G.

[0036] The lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP is a commercially available product, see the website http: / / www.youbio.cn / product / vt1479.

[0037] Penicillin / streptomycin dual antibody solution: The concentrations of the two are penicillin 10000U / mL and streptomycin 10000μg / mL respectively.

[0038] The carboxyl quantum dot microspheres used in the embodiments of the present invention are commercially available products from Hangzhou Boyin Biotechnology Co., Ltd., with the product number QM605-300C.

[0039] https: / / www.biogenome.cn / 343.

[0040] Example 1 Construction of pMLD-THP (GPI free)-mFc.GFP plasmid

[0041] The human TH glycoprotein sequence, that is, the gene and protein sequence numbered M15881.1 in the gene database GenBank, has the C-terminal 30 amino acids A611-Q640 containing the GPI anchor signal peptide removed. The 30 amino acids A611-Q640 are specifically ATVSRAFSSLGLLKVWLPLLLSATLTLTFQ. Then, the C-terminus was connected to the Flag tag of the EK protease cleavage site, and then the Fc segment of mouse IgG was connected as a purification tag to form a THP (GPI free)-Flag-mFc fusion protein, and the Kozak sequence GCCACC was added before the start codon ATG of its gene sequence. The nucleic acid sequence after host (human) codon optimization is shown in SEQ ID NO: 1, and EcoR I and Not I restriction endonucleases are designed at both ends, respectively. The amino acid sequence after expression is shown in SEQ ID NO: 2; after the gene sequence was synthesized, it was cloned into the CMV promoter of the lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP by connecting the restriction sites EcoR I and NotI to construct the eukaryotic expression plasmid pMLD-THP (GPI free) -mFc.GFP.

[0042] Example 2 Establishment of THP (GPI free)-mFc.GFP / 293 stable cell line

[0043] The pMLD-THP(GPI free)-mFc.GFP plasmid, pH1 plasmid, and pH2 plasmid were co-transfected into the lentiviral packaging cell line 293V to prepare THP(GPI free)-mFc.GFP lentivirus, and transfected into HEK293 cells. The clones were picked under a fluorescence microscope to establish the THP(GPI free)-mFc.GFP / 293 stable cell line. The specific steps are as follows:

[0044] 1) Plate cells on five 15 cm dishes one day before the experiment to ensure that the lentiviral packaging cell line 293V reaches 70%-80% confluence before transfection.

[0045] 2) 1 to 2 hours before transfection, replace the culture medium in the dish with serum-free and antibiotic-free DMEM medium.

[0046] 3) Prepare a 15 mL centrifuge tube, add 5 mL 1×HBS, then add 100 μg of pMLD-THP(GPIfree)-mFc.GFP plasmid and 100 μg of PH1 / PH2 (mixed at a mass ratio of 3:1) mixed plasmids in sequence, and mix gently.

[0047] 4) Add 4 mL of PEI working solution (10 μM), mix gently, and incubate at 37°C for 20 min.

[0048] 5) Divide the transfection complex solution into five equal parts and add them evenly into five 15 cm dishes to be transfected. Shake gently to ensure that the complex is evenly distributed.

[0049] 6) 5.5 hours after transfection, prepare another 15 mL centrifuge tube, add 5 mL 1×HBS, then add 100 μg of pMLD-THP (GPI free) -mFc.GFP plasmid and 100 μg of PH1 / PH2 mixed plasmid (PH1: PH2 = 3:1) in sequence, and mix gently.

[0050] 7) Add 4 mL of PEI working solution (10 μM), mix gently, and incubate at 37°C for 20 min.

[0051] 8) Replace the culture medium in the dish with fresh serum-free and antibiotic-free DMEM medium.

[0052] 9) Divide the transfection complex solution into 5 equal parts and add them evenly into 5 15cm dishes to be transfected. Shake gently to ensure even distribution of the complex.

[0053] 10) 6 hours after transfection, the medium was replaced with DMEM complete medium (+10% FBS + 1% penicillin / streptomycin double antibody solution).

[0054] 11) After 48 hours of transfection, the supernatant was collected, centrifuged at 8000 g for 15 min, and then frozen in a -80°C refrigerator.

[0055] 12) The supernatant was collected 72 hours after transfection, centrifuged at 8000 g for 15 min, and then the supernatant collected 48 hours after transfection was combined, filtered through a 0.45 μm filter, and centrifuged again at 85000 g for 2 h.

[0056] 13) The supernatant was discarded, and the pellet was resuspended in 1 mL of DMEM complete medium (+10% FBS + 1% penicillin / streptomycin double antibody solution), and HEK293 cells were infected (1 well of a 6-well plate, with a cell confluence of 60%-70%).

[0057] 14) After culturing the infected HEK293 cells for 2 days, the cells were subcultured into 2 6-well plates with a total of 12 wells. The dispersed single cells formed cloned cell clusters (about 1 week), and the monoclonal cell clusters with high expression of green fluorescent protein were selected under a fluorescence microscope for expansion culture to establish the THP (GPI free)-mFc.GFP / 293 stable cell line.

[0058] Example 3 Preparation of THP (GPI free) protein

[0059] Use protein-free 293 cell culture medium to expand and culture THP (GPI free)-mFc.GFP / 293 stable cells, collect the supernatant culture fluid, and purify the THP (GPI free)-mFc fusion protein with protein A / G, and cut THP (GPI free) from the column with EK enzyme to obtain recombinant human TH glycoprotein. The specific steps are as follows:

[0060] 1) Expand THP (GPI free)-mFc.GFP / 293 stably transfected cells to a five-layer cell factory and culture them in Hektor HEK293 cell culture medium without proteins and peptides;

[0061] 2) Collect the culture medium, centrifuge at 1200g and 4°C for 20 min, take the supernatant, and precipitate the protein using the saturated ammonium sulfate method;

[0062] 3) The precipitated protein was re-dissolved with PBS (pH 7.4) of 1 / 10 to 1 / 100 of the original solution volume, desalted and concentrated using an ultrafiltration tube with a 30KD protein molecular weight cutoff, and replaced with a binding / washing buffer (0.5M NaCl, 20mM Na2HPO4, pH 8.0, the solvent is water) to obtain a desalted protein solution dissolved in the binding / washing buffer;

[0063] 4) Prepare EK enzyme buffer (250mM Tris-HCl, 50mM NaCl, pH7.4, 2.5mM CaCl2), add EK enzyme to the EK enzyme buffer, the EK enzyme concentration is 0.5U / mL. Step 3) Load the desalted protein solution onto a Protein A / G column to adsorb the target protein, wash with a washing buffer, add the above-prepared EK enzyme buffer containing EK enzyme, and act at 4°C for 16 hours. Cut the THP (GPI free) protein part from the column with EK enzyme to obtain a THP (GPI free) protein solution;

[0064] 5) The THP (GPI free) protein solution obtained in step 4) is concentrated using an ultrafiltration tube with a 10KD protein cutoff molecular weight, and replaced with PBS buffer (pH 7.4), and then aliquoted and frozen for later use. The obtained THP (GPI free) protein is the recombinant human TH glycoprotein.

[0065] Example 4 Preparation of quantum dot fluorescent labeled proteins Q-hTHP01, Q-hIgG01 and Q-DNP

[0066] The recombinant human TH glycoprotein antigen obtained in Example 3 was used to immunize mice, and anti-human TH glycoprotein monoclonal antibodies were prepared using mouse monoclonal antibody technology. In the antibody screening, human urine and urine samples of different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice were used for testing to obtain a pair of anti-human urine TH glycoprotein specific monoclonal antibodies that did not react with urine of animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice, one of which was labeled as hTHP01, and the hTHP01 antibody was labeled with quantum dot fluorescent microspheres and labeled as Q-hTHP01, and the other was used as a coating antibody and labeled as C-hTHP02.

[0067] Human IgG was used as an antigen to immunize mice, and anti-human IgG antibodies were prepared using mouse monoclonal antibody technology. In the antibody screening, human urine and urine samples from different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice were used for testing to obtain a pair of anti-human IgG specific monoclonal antibodies that did not react with urine from pigs, cattle, sheep, dogs, cats, rabbits, mice, and other animals; one of the antibodies was labeled as hIgG01, and the hIgG01 antibody was labeled with quantum dot fluorescent microspheres and marked as Q-hIgG01, and the other was used as a coating antibody and marked as C-hIgG02.

[0068] Anti-human TH glycoprotein monoclonal antibody hTHP01, anti-human IgG monoclonal antibody hIgG01, and DNP-BSA were coupled to carboxyl quantum dot microspheres respectively. The specific steps are as follows:

[0069] 1) Adjustment of the buffer system of carboxyl quantum dot microspheres: dilute 3 ml of 1 μmol / L carboxyl quantum dot microspheres with 3 ml of 20 mmol / L pH6 MES to a concentration of about 0.5 μmol / L, and the final buffer system is 10 mmol / L pH6 MES.

[0070] 2) Microsphere activation: Add 7.5μl of crosslinking agent EDC (76.7mg / ml) and 7.5μl of NHS (69.1mg / ml) in sequence, mix quickly and react at 37℃ for 15min. After the reaction is over, centrifuge at 15000g for 15min; resuspend the precipitate in 10mmol / L MES pH6.0 solution to 3ml volume solution (so that the microsphere concentration is about 1μmol / L), mix well, and disperse by ultrasound if necessary. If there is flocculation during the reaction, it can be dispersed and mixed by ultrasound.

[0071] 3) Dispense the activated microspheres into tube A (1 mL), tube B (1 mL), and tube C (1 mL).

[0072] 4) Antibody-coupled microspheres: Add 600 μg hTHP01, 600 μg hIgG01, and 600 μg DNP-BSA to tubes A, B, and C respectively; react at 37°C for 1 hour. If there is coagulation during the reaction, try to disperse and mix by ultrasonication.

[0073] 5) Washing and blocking: Add blocking agent (100 mM glycine solution containing 1% BSA) to tubes A, B, and C respectively; react at 37°C for 0.5 h; centrifuge at 13000 g for 40 min and remove the supernatant; add 200 μL of PB buffer (pH 7.0, 1% BSA, 8% sucrose, 0.05% NaN3) to each tube, resuspend, disperse, and ultrasonically disperse to obtain quantum dot fluorescent labeled protein Q-hTHP01 solution, Q-hIgG01 solution, and Q-DNP solution, which are stored at 4°C for later use.

[0074] Example 5 Preparation of urine stain identification kit based on TH glycoprotein / IgG ratio method

[0075] The quantum dot fluorescent TH glycoprotein / IgG ratio chromatography test paper was prepared by referring to the traditional immunochromatographic test paper preparation process.

[0076] 1) Take 5 μL of the Q-hTHP01 solution and Q-hIgG01 solution prepared in Example 4, take 1 μL of the Q-DNP solution, add them to 49 μL of gold spray diluent (0.1M PB solution + 1% BSA + 4% sucrose + 0.05% NaN3), mix well, and apply them on the fluorescent release pad with a gold spraying instrument at a spray volume of 2 μL / cm, dry them at 37°C with air blowing for 12 hours, and seal them for later use;

[0077] 2) Anti-human TH glycoprotein monoclonal antibody C-hTHP02 and anti-human IgG monoclonal antibody C-hIgG02 were coated on the NC membrane detection line area T1 line area and T2 line area respectively with coating diluent (150mM PB, pH 7.4), and rabbit anti-DNP polyclonal antibody (i.e. Figure 1 The anti-DNP in the control line area C was coated. The coating concentration was 1 mg / mL, the film volume was 30 μL / 30 cm, and the film was dried at 37℃ for 12 hours and sealed for later use;

[0078] 3) The sample pad, release pad, NC membrane and absorbent pad are overlapped and assembled on the substrate, and the release pad and the absorbent pad are respectively overlapped on the two ends of the NC membrane, and a detection area (T1 line area close to the release pad (i.e. Figure 1 The fluorescent pad in the middle), the T2 line area is located in the middle, and the C line area is close to the absorbent pad), the sample pad is superimposed on the release pad, and after assembly, a test paper is formed, which is cut into strips with a width of 3.8 mm to make the human urine stain identification test paper based on the human TH glycoprotein-human IgG ratio method, such as Figure 1 See Figure 1 , a sample adding hole is provided on the sample pad.

[0079] Example 6 Application of urine stain identification kit based on TH glycoprotein / IgG ratio method

[0080] The urine stain identification kit based on the TH glycoprotein / IgG ratio method developed based on the technical solution of the present invention can be used for accurate and rapid identification of human urine stains in criminal investigation.

[0081] 1) Standard curve 1: The recombinant human TH glycoprotein antigen obtained in Example 3 was used as the standard, and the concentrations were prepared into 100ng / mL, 200ng / mL, 400ng / mL, 800ng / mL, 1600ng / mL, and 3200ng / mL respectively with PBS at pH 7.4, and the blank was PBS at pH 7.4. The test strips prepared in Example 5 of the present invention were spotted separately, 100μl per well, the chromatography time was 10 minutes, and the T1 line fluorescence value and C line fluorescence value were measured with a dry fluorescence chromatography analyzer; 5 replicates were set for each concentration, and the standard curve 1 was drawn with the average T1 / C fluorescence value ratio as the ordinate and the recombinant human TH glycoprotein concentration as the abscissa. The results are shown in Figure 2 ; The curve equation is Y = 0.0008X + 0.0515, R 2 =0.9729.

[0082] 2) Standard curve 2: Human IgG protein was used as the standard, and the concentrations were prepared into 25ng / mL, 50ng / mL, 100ng / mL, 200ng / mL, 400ng / mL, and 800ng / mL respectively with PBS at pH 7.4, and the blank was PBS at pH 7.4. The test strips prepared in Example 5 of the present invention were spotted separately, 100μl per well, the chromatography time was 10 minutes, and the T2 line fluorescence value and C line fluorescence value were measured with a dry fluorescence chromatography analyzer; 5 replicates were set for each concentration, and the standard curve 2 was drawn with the average T2 / C fluorescence value ratio as the ordinate and the human IgG protein concentration as the abscissa. The results are shown in Figure 3 ; The curve equation is Y = 0.0024X + 0.1736, R 2 =0.9074.

[0083] 3) Recruit 10 volunteers, half male and half female, and each person takes 200 μL of urine and drips it on a cotton cloth of about 1 cm×1 cm in size, and takes 200 μL of fingertip blood and drips it on another cotton cloth of about 1 cm×1 cm in size. After natural drying, each piece of 0.5 cm×0.5 cm cotton cloth sample is cut and placed in 0.5 ml PBS concentrated solution, shaken and soaked for 3 minutes, and 100 μL of sample solution is taken and dripped into the sample addition hole of the test strip prepared in Example 5 of the present invention, and the chromatography time is left to stand for 10 minutes. The fluorescence value of the T1 line, the fluorescence value of the T2 line and the fluorescence value of the C line are measured by a dry fluorescence chromatography analyzer, and the hTH glycoprotein content is calculated by substituting the T1 / C fluorescence value ratio into the standard curve 1, and the hIgG content is calculated by substituting the T2 / C fluorescence value ratio into the standard curve 1, and the hTH glycoprotein / hIgG content ratio is calculated. The results are shown in Table 1. The results showed that all 10 urine samples were positive and all 10 blood samples were negative, with an accuracy rate of 100%.

[0084] Table 1 Detection results of the kit of the present invention on urine samples of 10 volunteers

[0085]

[0086]

[0087] Table 2 Test results of the kit of the present invention on 10 volunteers' blood samples

[0088]

[0089] The hTHP content in Table 1-2 is the content of hTH glycoprotein.

Claims

1. A method for preparing an anti-human TH glycoprotein monoclonal antibody, characterized in that The following steps are involved: 1) According to the human TH glycoprotein sequence, the C-terminal 30 amino acids containing the GPI anchor signal peptide were removed, and then the C-terminal was connected to the EK protease cleavage site Flag tag, and then connected to the Fc segment of mouse IgG as a purification tag to form the THP (GPI free) -Flag-mFc fusion protein gene. After adding the Kozak sequence GCCACC before the start codon ATG of its gene sequence, the host cell codon was optimized, the gene was synthesized and subcloned into the CMV promoter of the lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP to obtain a plasmid that can express the THP (GPI free) -Flag-mFc fusion protein, which was marked as pMLD-THP (GPIfree) -mFc.GFP; 2) The plasmid pMLD-THP(GPI free)-mFc.GFP constructed in step 1) was co-transfected with the pH1 plasmid and the pH2 plasmid into the lentiviral packaging cell 293V to prepare the THP(GPI free)-mFc.GFP lentivirus, and transfected into HEK293 cells. The clones were picked under a fluorescence microscope to establish a THP(GPI free)-mFc.GFP / 293 stable cell line; 3) using a cell factory to amplify and culture the THP (GPI free)-mFc.GFP / 293 cells prepared in step 2), and collecting the culture supernatant, extracting and purifying the fusion protein therein using a protein A column, and then using EK enzyme to cut off the mFc tag to obtain a recombinant human TH glycoprotein, which is labeled as THP (GPI free) protein; 4) Immunizing mice with the recombinant human TH glycoprotein antigen prepared in step 3), and preparing anti-human TH glycoprotein monoclonal antibody using mouse monoclonal antibody technology.

2. The method for preparing an anti-human TH glycoprotein monoclonal antibody according to claim 1, characterized in that In step 1), the human TH glycoprotein sequence is the gene and protein sequence of GenBank: M15881.1; the removed C-terminal 30 amino acids containing the GPI anchor signal peptide, namely A611-Q640, are specifically ATVSRAFSSLGLLKVWLPLLLSATLTLTFQ; Among them, the nucleic acid sequence of the THP (GPI free) -Flag-mFc fusion protein gene after host cell codon optimization is shown in SEQ ID NO: 1, the gene is synthesized and subcloned into the CMV promoter of the lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP, and the amino acid sequence of the THP (GPI free) -Flag-mFc fusion protein expressed by the constructed plasmid is shown in SEQ ID NO:

2.

3. The method for preparing an anti-human TH glycoprotein monoclonal antibody according to claim 1, characterized in that Step 4) When the anti-human TH glycoprotein monoclonal antibody is prepared using mouse monoclonal antibody technology, human urine and urine samples from different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice are used for testing in antibody screening to obtain a pair of anti-human urine TH glycoprotein specific monoclonal antibodies that do not react with urine samples from different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice, which are the anti-human TH glycoprotein monoclonal antibodies.

4. An anti-human TH glycoprotein monoclonal antibody prepared by the method according to any one of claim 3.

5. A urine stain identification kit based on the TH glycoprotein / IgG ratio method, characterized in that The kit comprises an immunochromatographic test strip, which comprises a bottom liner, a NC membrane, a sample pad, a release pad and a water-absorbing pad; the sample pad, the release pad, the NC membrane and the water-absorbing pad are sequentially overlapped and assembled on the bottom liner, the release pad and the water-absorbing pad are respectively overlapped on the two ends of the NC, and a detection area is formed on the surface of the NC, and the sample pad is overlapped on the release pad; a detection line area T1 line and a T2 line close to the release pad and a control line area C line close to the water-absorbing pad are arranged on the NC in the detection area; DNP-BSA is coupled to carboxyl quantum dot microspheres and marked as Q-DNP; Q-hTHP01, Q-hIgG01 and Q-DNP are mixed and sprayed on the release pad, and the T1 line area on the NC membrane is coated with C-hTHP02, the T2 line area is coated with C-hIgG02, and the C line area is coated with rabbit anti-DNP polyclonal antibody; Among them, one of the antibodies of a pair of anti-human urine TH glycoprotein specific monoclonal antibodies in claim 4 is labeled with quantum dot fluorescent microspheres and marked as Q-hTHP01, and the other is used as a coating antibody and marked as C-hTHP02; One of the anti-human IgG specific monoclonal antibodies was labeled with quantum dot fluorescent microspheres and labeled as Q-hIgG01, and the other was used as a coating antibody and labeled as C-hIgG02.

6. A urine stain identification kit based on the TH glycoprotein / IgG ratio method as claimed in claim 5, characterized in that The preparation process of the anti-human IgG specific monoclonal antibody is as follows: using human IgG as an antigen to immunize mice, using mouse monoclonal antibody technology to prepare anti-human IgG antibodies, and in antibody screening, using human urine and urine samples of different animals such as pigs, cattle, sheep, dogs, cats, rabbits, mice, etc. for testing to obtain a pair of anti-human IgG specific monoclonal antibodies that do not react with urine samples of animals such as pigs, cattle, sheep, dogs, cats, rabbits, mice, etc.

7. The detection method of the urine stain identification kit based on the TH glycoprotein / IgG ratio method according to claim 5, characterized in that The following steps are involved: 1) The detection limit of the kit for human TH glycoprotein is set at 100 ng / mL, and the detection limit of human IgG protein is set at 50 ng / mL, and a dry fluorescence chromatography analyzer is used for quantitative analysis, and quantitative standard curves of human TH glycoprotein and human IgG protein are respectively preset in the detection instrument, and then the corresponding protein content in the actual sample detection is deduced according to the corresponding standard curves; 2) The suspected urine sample is dissolved with PBS buffer at pH 7.4, and then tested with the kit. After the detection chromatography is left for 10-15 minutes, a dry fluorescence chromatography analyzer is used for quantitative analysis. If TH glycoprotein is not detected, that is, the TH glycoprotein value is <100ng / mL, it is a negative sample and can be ruled out as human urine; if the TH glycoprotein value is ≧100ng / mL and human IgG is not detected or the human IgG value is ≧50ng / mL but the TH glycoprotein value / IgG value ratio is ≧0.1, it is a positive sample for human urine; if the TH glycoprotein value is ≧100ng / mL and the human IgG value is ≧50ng / mL but the TH glycoprotein value / IgG value ratio is <0.1, it is a sample mixed with human blood or other samples containing human IgG.

8. The detection method according to claim 7, characterized in that The preparation of the standard curve in step 1) includes the following process: Step 1, preparing standard curve 1: using recombinant human TH glycoprotein as a standard, using pH 7.4 PBS buffer to prepare a series of standard solutions with different concentrations in the concentration range of 100 ng / mL-3200 ng / mL, using pH 7.4 PBS buffer as a blank control, respectively using the kit for detection, after chromatography, using a dry fluorescence chromatography analyzer to measure the T1 line fluorescence value and the C line fluorescence value, using the ratio of the T1 line fluorescence value to the C line fluorescence value, i.e., the T1 / C fluorescence value ratio, as the ordinate, and the recombinant human TH glycoprotein concentration as the abscissa to draw standard curve 1; Step 2, preparing standard curve 2: using human IgG protein as the standard, using pH 7.4 PBS buffer to prepare a series of standard solutions with different concentrations in the concentration range of 25 ng / mL-800 ng / mL, using pH 7.4 PBS buffer as the blank control, and using the kit for detection, after chromatography, using a dry fluorescence chromatography analyzer to determine the T2 line fluorescence value and the C line fluorescence value, and using the ratio of the T2 line fluorescence value to the C line fluorescence value, i.e., the T2 / C fluorescence value ratio, as the ordinate and the human IgG protein concentration as the abscissa to draw standard curve 2.

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