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

Abstract

This invention discloses an anti-human TH glycoprotein monoclonal antibody, a urine stain identification kit based on the TH glycoprotein / IgG ratio method, and a detection method. The kit includes fluorescently labeled anti-human TH glycoprotein-specific monoclonal antibody, fluorescently labeled anti-human IgG-specific monoclonal antibody, fluorescently labeled DNP-BSA, coated anti-human TH glycoprotein-specific monoclonal antibody, coated anti-human IgG-specific monoclonal antibody, rabbit anti-DNP polyclonal antibody, chromatographic test strips, and other related materials. This invention eliminates interference from other animal urine stains by specifically detecting human TH glycoprotein in urine stains; and eliminates interference from bloodstains, sweat stains, and other fading stains by determining the content of human TH glycoprotein in urine stains and its ratio to human IgG using a quantitative fluorescence method. The kit of this invention can be applied to the accurate and rapid identification of human urine stains in criminal investigations.

Description

Technical Field

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

[0002] Urine stain detection is an essential part of criminal investigation. People often experience urinary incontinence under extreme fear, thus leaving traces of urine at crime scenes. Extracting and analyzing urine samples from these scenes is crucial for determining the crime scene and the likelihood of a murder. Currently, the conventional method for detecting urine at crime scenes is to observe fluorescence under ultraviolet light. While sensitive, this method lacks specificity and cannot rule out interference from other animal urine or samples containing fluorescent substances. There are currently no reagents or instruments for the rapid detection and identification of specific components in human urine. Summary of the Invention

[0003] To address the aforementioned technical problems in existing technologies, we have developed a specific monoclonal antibody against human TH glycoprotein, targeting the TH glycoprotein, a specific protein found in urine. We have also invented a urine stain identification kit and detection method based on the TH glycoprotein / IgG ratio. TH glycoprotein, also known as Tamm-Horsfall glycoprotein, uromodulin, or uromucoprotein, is a kidney-specific protein primarily found in urine, with only trace amounts present in serum. TH glycoprotein is the most abundant specific protein in urine, with normal levels in human urine ranging from 20-40 mg / L and serum levels around 50 μg / L, making it an ideal target for urine stain identification.

[0004] The technical solution adopted in this invention is as follows:

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

[0006] 1) Based on the human TH glycoprotein sequence, the C-terminal 30 amino acids containing the GPI anchoring signal peptide were removed, and then the EK protease cleavage site Flag tag was attached to the C-terminus. Then, the Fc segment of mouse IgG was attached as a purification tag to form the THP(GPIfree)-Flag-mFc fusion protein gene. After adding the Kozak sequence GCCACC before the start codon ATG of its gene sequence, 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 labeled 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 pH1 plasmid and pH2 plasmid into lentiviral packaging cells 293V to prepare THP(GPI free)-mFc.GFP lentivirus. The lentivirus was then transfected into HEK293 cells, and clones were picked under a fluorescence microscope to establish a stable THP(GPI free)-mFc.GFP / 293 cell line.

[0008] 3) Expand and culture the THP (GPI free)-mFc.GFP / 293 cells prepared in step 2) using a cell factory, collect the culture supernatant, extract and purify the fusion protein using a protein A column, and then cleave the mFc tag with EK enzyme to obtain recombinant human TH glycoprotein, which is labeled as THP (GPI free) protein.

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

[0010] 2. The method for preparing an anti-human TH glycoprotein monoclonal antibody as described in 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 anchoring signal peptide, namely A611-Q640, are specifically ATSVRAFSSLGLLKVWLPLLLSATLTLTFQ;

[0011] The THP(GPI-free)-Flag-mFc fusion protein gene, after host cell codon optimization, has the following nucleic acid sequence as shown in SEQ ID NO: 1. The gene was synthesized and subcloned into the CMV promoter of the lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP. 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] Further, in step 4), when preparing anti-human TH glycoprotein monoclonal antibodies using mouse monoclonal antibody technology, human urine samples and urine samples from different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice are used for 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. These are the anti-human TH glycoprotein monoclonal antibodies.

[0013] This invention also discloses a urine stain identification kit based on the TH glycoprotein / IgG ratio method. The kit includes an immunochromatographic test strip, which comprises a backing, an NC membrane, a sample pad, a release pad, and an absorbent pad. The sample pad, release pad, NC membrane, and absorbent pad are sequentially overlapped on the backing. The release pad and absorbent pad are respectively stacked on both ends of the NC membrane, forming a detection area on the surface of the NC membrane. The sample pad is stacked on the release pad. The NC membrane in the detection area has detection lines T1 and T2 near the release pad and a control line C near the absorbent pad. DNP-BSA is coupled to carboxyl quantum dot microspheres and labeled as Q-DNP. Q-hTHP01, Q-hIgG01, and Q-DNP are mixed and sprayed onto the release pad. C-hTHP02 is coated on the T1 line area and C-hIgG02 is coated on the T2 line area of ​​the NC membrane. Rabbit anti-DNP polyclonal antibody is coated on the C line area.

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

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

[0016] Furthermore, the preparation process of the anti-human IgG specific monoclonal antibody is as follows: mice are immunized with human IgG as an antigen, and anti-human IgG antibodies are prepared using mouse monoclonal antibody technology. In antibody screening, human urine samples and urine samples from different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice are tested to obtain a pair of anti-human IgG specific monoclonal antibodies that do not react with urine samples from animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice.

[0017] The detection method of the urine stain identification kit based on the TH glycoprotein / IgG ratio includes 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 for human IgG protein is set at 50 ng / mL. Quantitative analysis is performed using a dry fluorescence chromatography analyzer. Quantitative standard curves for human TH glycoprotein and human IgG protein are preset in the detection instrument, and the corresponding protein content in the actual sample is calculated based on the corresponding standard curves.

[0019] 2) Dissolve the suspected urine sample in PBS buffer (pH 7.4) and then test it using the kit described above. After the chromatography has stood for 10-15 minutes, perform quantitative analysis using a dry fluorescence chromatography analyzer. If TH glycoprotein is not detected (TH glycoprotein value <100 ng / mL), the sample is negative and can be excluded as human urine stain. If the TH glycoprotein value is ≥100 ng / mL and human IgG is not detected, or if the human IgG value is ≥50 ng / mL but the TH glycoprotein / IgG ratio is ≥0.1, the sample is positive for human urine stain. If the TH glycoprotein value is ≥100 ng / mL and the human IgG value is ≥50 ng / mL but the TH glycoprotein / IgG ratio is <0.1, the sample is contaminated with human blood or other substances containing human IgG.

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

[0021] Step 1: Constructing Standard Curve 1: Using recombinant human TH glycoprotein as a standard, a series of standard solutions with concentrations ranging from 100 ng / mL to 3200 ng / mL were prepared using PBS buffer at pH 7.4. PBS buffer at pH 7.4 was used as a blank control. The solutions were detected using the kit described above. After chromatography, the fluorescence values ​​of the T1 line and C line were measured using a dry fluorescence chromatography analyzer. The ratio of the T1 line fluorescence value to the C line fluorescence value (T1 / C fluorescence value ratio) was plotted on the ordinate, and the concentration of recombinant human TH glycoprotein was plotted on the abscissa.

[0022] Step 2: Constructing Standard Curve 2: Using human IgG protein as the standard, prepare a series of standard solutions with different concentrations ranging from 25 ng / mL to 800 ng / mL using PBS buffer at pH 7.4. Use PBS buffer at pH 7.4 as a blank control. Detect the solutions using the kit described above. After chromatography, measure the fluorescence values ​​of the T2 line and C line using a dry fluorescence chromatography analyzer. Plot the standard curve 2 with the ratio of the T2 line fluorescence value to the C line fluorescence value (T2 / C fluorescence value ratio) as the ordinate and the concentration of human IgG protein as the abscissa.

[0023] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0024] (1) This invention develops a specific monoclonal antibody against human TH glycoprotein and prepares an immunochromatographic rapid test strip that can be used for accurate and rapid identification of human urine stains. Furthermore, IgG is a plasma abundance protein with a normal value of 7.5-15.6 g / L, while the normal value of IgG in human urine is 0.0-14.0 mg / L. Although the urinary IgG level may be elevated in patients with kidney damage, the difference from serum IgG levels is still more than two orders of magnitude. This invention uses a quantitative analysis method to simultaneously measure the content of human TH glycoprotein and human IgG in the sample and calculate the TH glycoprotein / IgG ratio, which can more accurately determine whether the sample is human urine stains, excluding interference from other stains such as bloodstains and sweat stains.

[0025] (2) This invention uses quantum dot fluorescence chromatography quantitative analysis technology to quickly determine the content of human TH glycoprotein and human IgG in suspected human urine stains, and uses the ratio of TH glycoprotein content to IgG content to exclude interfering substances and accurately and quickly identify human urine stains.

[0026] (3) The kit of the present invention can be used in criminal investigation to accurately and quickly identify human urine stains. Attached Figure Description

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

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

[0029] Figure 3 Standard curve for hIgG content determination. Detailed Implementation

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

[0031] This 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 document and appropriately modify the process parameters to achieve the same result. 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 preparation method and application of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the preparation method and application described herein without departing from the content and scope of this invention to realize and apply the technology of this invention.

[0032] Explanation of some terms and abbreviations:

[0033] THP: Tamm-Horsfall glycoprotein, also known as uromodulin or uromucoprotein.

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

[0035] hIgG: Human Immunoglobulin G.

[0036] The lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP is a commercially available product; see http: / / www.youbio.cn / product / vt1479 for more information.

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

[0038] The carboxyl quantum dot microspheres used in this embodiment of the invention are commercially available products from Hangzhou Boyin Biotechnology Co., Ltd., with product number QM605-300C. (See website for more information.)

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

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

[0041] The human TH glycoprotein sequence, namely the gene and protein sequence numbered M15881.1 in the GenBank gene database, has the C-terminal 30 amino acids, A611-Q640, containing the GPI anchoring signal peptide removed. The 30 amino acids A611-Q640 are specifically ATSVRAFSSLGLLKVWLPLLLSATLTLTFQ. Then, an EK protease cleavage site Flag tag was attached to the C-terminus, and a mouse IgG Fc fragment was attached as a purification tag to form the THP (GPI free)-Flag-mFc fusion protein. The Kozak sequence GCCACC was added before the ATG start codon in its gene sequence. The nucleic acid sequence after host (human) codon optimization is shown in SEQ ID NO: 1. EcoRI and NotI restriction endonucleases were designed at both ends, and the amino acid sequence after expression is shown in SEQ ID NO: 2. After gene synthesis, the EcoRI and NotI restriction sites were ligated and cloned into the CMV promoter of the lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP to construct the eukaryotic expression plasmid pMLD-THP(GPI free)-mFc.GFP.

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

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

[0044] 1) The day before the experiment, cells were plated in 5 15cm dishes to ensure that the lentiviral packaging line cells 293V reached 70%-80% confluence before transfection.

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

[0046] 3) Prepare a 15mL centrifuge tube, add 5mL of 1×HBS, then add 100μg of pMLD-THP(GPIfree)-mFc.GFP plasmid and 100μg of PH1 / PH2 (mixed at a mass ratio of 3:1) mixed plasmid, 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 5 equal portions and add them evenly into 5 15cm dishes to be transfected. Gently shake to distribute the complex evenly.

[0049] 6) After 5.5 h of transfection, prepare a 15 mL centrifuge tube, add 5 mL of 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 portions and add them evenly into 5 15cm dishes to be transfected. Gently shake to distribute the complex evenly.

[0053] 10) Change the medium 6 hours after transfection, replacing it with DMEM complete medium (+10% FBS+1% penicillin / streptomycin solution).

[0054] 11) Collect the supernatant 48 h after transfection, centrifuge at 8000g for 15 min, and freeze at -80℃.

[0055] 12) Collect the supernatant 72 h after transfection, centrifuge at 8000g for 15 min, combine the supernatant collected 48 h after transfection, filter through a 0.45 μm filter membrane, and then centrifuge at 85000g for 2 h.

[0056] 13) Discard the supernatant, resuspend the precipitate in 1 mL of DMEM complete medium (+10% FBS+1% penicillin / streptomycin solution), and infect HEK293 cells (1 well in a 6-well plate, cells at 60%-70% confluence).

[0057] 14) After culturing infected HEK293 cells for 2 days, they were passaged into 12 wells in two 6-well plates. Once the dispersed single cells formed clonal cell clusters (about 1 week), single clonal cell clusters that highly expressed green fluorescent protein were picked under a fluorescence microscope for amplification culture to establish a stable THP (GPI free)-mFc.GFP / 293 cell line.

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

[0059] THP(GPI-free)-mFc.GFP / 293 stable transgenic cells were amplified and cultured using protein-free 293 cell culture medium. The supernatant culture medium was collected, and the THP(GPI-free)-mFc fusion protein was purified using protein A / G. The THP(GPI-free) was then cleaved from the column using EK enzyme to obtain recombinant human TH glycoprotein. The specific steps are as follows:

[0060] 1) Expand THP (GPI free)-mFc.GFP / 293 stable transgenic cells to a five-layer cell factory and culture them in Hektor HEK293 cell culture medium that does not contain protein or peptides;

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

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

[0063] 4) Prepare EK enzyme buffer (250mM Tris-HCl, 50mM NaCl, pH 7.4, 2.5mM CaCl2). Add EK enzyme to the EK enzyme buffer at a concentration of 0.5 U / mL. After desalting the protein solution, load it onto a Protein A / G column to adsorb the target protein. Wash with washing buffer and add the prepared EK enzyme buffer. Incubate at 4°C for 16 hours. Use EK enzyme to cleave the THP (GPI-free) protein portion from the column 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 protein molecular weight cutoff of 10KD, and the PBS buffer (pH 7.4) is replaced. The solution is then aliquoted and frozen for later use. The resulting THP (GPI free) protein is the recombinant human TH glycoprotein.

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

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

[0067] Mice were immunized with human IgG as the antigen, and anti-human IgG antibodies were prepared using mouse monoclonal antibody technology. In antibody screening, human urine samples, as well as urine samples from different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice, were tested to obtain a pair of anti-human IgG specific monoclonal antibodies that did not react with urine samples from these animals. One antibody was labeled hIgG01, and hIgG01 antibody was labeled with quantum dot fluorescent microspheres, designated Q-hIgG01. The other antibody was used as a coating antibody and designated C-hIgG02.

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

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

[0070] 2) Microsphere activation: Add 7.5 μl of crosslinking agent EDC (76.7 mg / ml) and 7.5 μl of NHS (69.1 mg / ml) sequentially, mix quickly, and react at 37℃ for 15 min. After the reaction is complete, centrifuge at 15000g for 15 min; resuspend the precipitate in 10 mmol / L MES pH 6.0 solution to a volume of 3 ml (to make the microsphere concentration approximately 1 μmol / L), mix well, and disperse ultrasonically if necessary. If aggregation occurs during the reaction, it can be dispersed and mixed by ultrasonication.

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

[0072] 4) Antibody-conjugated 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 aggregation occurs during the reaction, try sonicating to disperse and mix.

[0073] 5) Washing and blocking: Add blocking agent (100mM glycine solution containing 1% BSA) to tubes A, B, and C respectively; react at 37℃ for 0.5h; centrifuge at 13000g for 40min and discard the supernatant; add 200μL of PB buffer (pH7.0, 1% BSA, 8% sucrose, 0.05% NaN3) to each tube for resuspension, dispersion, and ultrasonic dispersion to obtain quantum dot fluorescently labeled protein Q-hTHP01 solution, Q-hIgG01 solution, and Q-DNP solution, and store at 4℃ for later use.

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

[0075] Quantum dot fluorescent TH glycoprotein / IgG ratio chromatographic test strips were prepared using the traditional immunochromatographic test strip preparation process.

[0076] 1) Take 5 μL each of the Q-hTHP01 solution and Q-hIgG01 solution prepared in Example 4, and 1 μL of the Q-DNP solution, add them to 49 μL of gold sputtering diluent (0.1M PB solution + 1% BSA + 4% sucrose + 0.05% NaN3), mix well, and then use a gold sputtering instrument to spray onto the fluorescent release pad at a spraying rate of 2 μL / cm. Dry at 37°C for 12 h, and seal in a bag for later use.

[0077] 2) Using coating dilution buffer (150mM PB, pH 7.4), anti-human TH glycoprotein monoclonal antibody C-hTHPO2 and anti-human IgG monoclonal antibody C-hIgG02 were coated on the T1 and T2 detection lines of the NC membrane, respectively. Rabbit anti-DNP polyclonal antibody (i.e., Figure 1 The anti-DNP (anti-DNP) was coated in the control line C region. The coating concentration was 1 mg / mL, the splash volume was 30 μL / 30 cm, and the membrane was dried at 37℃ for 12 h before being sealed and stored.

[0078] 3) Assemble the sample pad, release pad, NC membrane, and absorbent pad on the substrate, overlapping them. The release pad and absorbent pad are respectively stacked on both ends of the NC membrane, forming a detection area (T1 line area) on the surface of the NC membrane near the release pad (i.e., Figure 1 The sample pad (with the fluorescent pad in the middle, the T2 line area in the center, and the C line area near the absorbent pad) is stacked on top of the release pad. After assembly, a test strip is formed, which is then cut into strips with a width of 3.8 mm to produce the human urine stain identification test strip based on the human TH glycoprotein-human IgG ratio method. Figure 1 As shown. See also Figure 1 The sample pad has a sample application hole.

[0079] Example 6: Application of the Urine Stain Identification Kit Based on the 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 this invention can be applied to the accurate and rapid identification of human urine stains in criminal investigations.

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

[0082] 2) Standard Curve 2: Using human IgG protein as the standard, concentrations of 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL, and 800 ng / mL were prepared in PBS at pH 7.4. The blank was PBS at pH 7.4. 100 μl of the test strips prepared in Example 5 of this invention was spotted onto each well, and the chromatography time was 10 minutes. The fluorescence values ​​at the T2 and C lines were measured using a dry fluorescence chromatography analyzer. Five replicates were performed for each concentration. Standard Curve 2 was plotted with the average T2 / C fluorescence ratio as the ordinate and the human IgG protein concentration as the abscissa. The results are shown in [Figure 2]. Figure 3 The equation of the curve is Y = 0.0024X + 0.1736, R. 2 =0.9074.

[0083] 3) Ten volunteers were recruited, half male and half female. Each volunteer collected 200 μL of urine and dripped it onto a piece of cotton cloth approximately 1 cm × 1 cm in size. At the same time, 200 μL of blood from a fingertip was dripped onto another piece of cotton cloth approximately 1 cm × 1 cm in size. After natural drying, 0.5 cm × 0.5 cm samples were cut from each piece of cotton cloth and placed in 0.5 ml of concentrated PBS solution. After shaking and soaking for 3 minutes, 100 μL of the sample solution was dripped into the sample well of the test strip prepared in Example 5 of this invention. After standing for 10 minutes, the fluorescence values ​​of the T1 line, T2 line, and C line were measured using a dry fluorescence chromatography analyzer. The T1 / C fluorescence value ratio was substituted into standard curve 1 to calculate the hTH glycoprotein content, and the T2 / C fluorescence value ratio was substituted into standard curve 1 to calculate the hIgG content. The hTH glycoprotein / hIgG content ratio was also 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 from 10 volunteers.

[0085]

[0086]

[0087] Table 2. Detection results of the reagent kit of the present invention on blood samples from 10 volunteers.

[0088]

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

Claims

1. A urine stain identification kit based on the TH glycoprotein / IgG ratio method, characterized in that, The kit includes an immunochromatographic test strip, which comprises a backing, an NC membrane, a sample pad, a release pad, and an absorbent pad. The sample pad, release pad, NC membrane, and absorbent pad are sequentially overlapped and assembled on the backing. The release pad and absorbent pad are respectively stacked on both ends of the NC membrane, forming a detection area on the surface of the NC membrane. The sample pad is stacked on the release pad. On the NC membrane of the detection area, there are detection lines T1 and T2 near the release pad and control line C near the absorbent pad. DNP-BSA is coupled to carboxyl quantum dot microspheres and labeled as Q-DNP. Q-hTHP01, Q-hIgG01, and Q-DNP are mixed and sprayed onto the release pad. C-hTHP02 is coated on the T1 line area, C-hIgG02 is coated on the T2 line area, and rabbit anti-DNP polyclonal antibody is coated on the C line area of ​​the NC membrane. Two anti-human TH glycoprotein monoclonal antibodies were selected. One antibody was labeled with quantum dot fluorescent microspheres and named Q-hTHP01, while the other was used as a coating antibody and named C-hTHP02. One of the two anti-human IgG specific monoclonal antibodies was labeled with quantum dot fluorescent microspheres and named Q-hIgG01, while the other was used as a coating antibody and named C-hIgG02.

2. The urine stain identification kit based on the TH glycoprotein / IgG ratio method as described in claim 1, characterized in that, The method for preparing the anti-human TH glycoprotein monoclonal antibody includes the following steps: 1) Based on the human TH glycoprotein sequence, the C-terminal 30 amino acids containing the GPI anchoring signal peptide were removed, and then the EK protease cleavage site Flag tag was attached to the C-terminus. Then, the Fc segment of mouse IgG was attached 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 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 labeled as pMLD-THP(GPI free)-mFc.GFP. 2) The plasmid pMLD-THP(GPI free)-mFc.GFP constructed in step 1) was co-transfected with pH1 plasmid and pH2 plasmid into lentiviral packaging cells 293V to prepare THP(GPI free)-mFc.GFP lentivirus. The lentivirus was then transfected into HEK293 cells, and clones were picked under a fluorescence microscope to establish a stable THP(GPI free)-mFc.GFP / 293 cell line. 3) Expand and culture the THP (GPI free)-mFc.GFP / 293 cells prepared in step 2) using a cell factory, collect the culture supernatant, extract and purify the fusion protein using a protein A column, and then cleave the mFc tag with EK enzyme to obtain recombinant human TH glycoprotein, which is labeled as THP (GPI free) protein. 4) Immunize mice with the recombinant human TH glycoprotein antigen prepared in step 3), and prepare anti-human TH glycoprotein monoclonal antibody using mouse monoclonal antibody technology; 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 anchoring signal peptide, namely A611-Q640, are specifically ATSVRAFSSLGLLKVWLPLLLSATLTLTFQ. The THP(GPI-free)-Flag-mFc fusion protein gene, after host cell codon optimization, has the following nucleic acid sequence as shown in SEQ ID NO:

1. The gene was synthesized and subcloned into the CMV promoter of the lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP. 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 urine stain identification kit based on the TH glycoprotein / IgG ratio method as described in claim 2, characterized in that, Step 4) When preparing anti-human TH glycoprotein monoclonal antibodies using mouse monoclonal antibody technology, in the antibody screening process, human urine stains and urine stain samples from different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice are used for testing to obtain a pair of anti-human urine TH glycoprotein-specific monoclonal antibodies that do not react with urine stains from different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice. This is the anti-human TH glycoprotein monoclonal antibody.

4. The urine stain identification kit based on the TH glycoprotein / IgG ratio method as described in claim 1, characterized in that... The preparation process of the anti-human IgG specific monoclonal antibody is as follows: mice are immunized with human IgG as antigen, and anti-human IgG antibodies are prepared using mouse monoclonal antibody technology. In antibody screening, human urine samples and urine samples from different animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice are tested to obtain a pair of anti-human IgG specific monoclonal antibodies that do not react with urine samples from animals such as pigs, cattle, sheep, dogs, cats, rabbits, and mice.

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