Monoclonal antibody recognizing N-acetyl-β-D-glucosaminidase and its application

Abstract

The present invention belongs to the field of immunoassay detection technology, and relates to a monoclonal antibody for identifying N-acetyl-β-D-glucosaminidase, a hybridoma cell line secreting the antibody, and an antigen-binding fragment derived from the antibody, and a detection reagent containing the antibody or antigen fragment. The antibody can be used to detect N-acetyl-β-D-glucosaminidase, and has the effects of strong specificity, strong anti-interference ability, high sensitivity and high stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay detection, and relates to antibodies and antigen-binding fragments of antibodies for identifying N-acetyl-β-D-glucosaminidase (NAG), a kit containing the antibodies and antibody fragments, and analytical tests using the antibodies and antibody fragments. Background Art

[0002] N-acetyl-β-D-glucosaminidase (NAG, β-2-acetamido-2-deoxy-D-glucoside acetamidodeoxyglucohydrolase, EC3.2.1.30) is an acid hydrolase located in lysosomes, with a molecular weight of about 112500±19000. It is present in all tissues, and the highest content is in the prostate and renal proximal tubular lysosomes. Studies over the years have shown that NAG can be used as an early diagnostic indicator for renal tubular damage. NAG in urine is mainly derived from renal proximal tubular epithelial cells. When proximal tubular epithelial cells are damaged, the activity of NAG in urine will increase significantly and earlier than other urinary enzymes, and the degree of NAG increase is proportional to the degree of tubular damage. Therefore, it can be used as a urine marker protein, which has great value for the early diagnosis of renal damage, especially tubular damage. Summary of the invention

[0003] The present invention is based at least in part on the development of NAG protein-specific antibodies, which can be used to qualitatively or quantitatively analyze NAG proteins in biological samples (e.g., clinical samples). These antibodies and antigen-binding fragments thereof, kits containing these antibodies and antibody fragments, and various methods of using these antibodies and antibody fragments are provided herein.

[0004] The amino acid sequence of NAG protein is shown in SEQ ID NO.1.

[0005] SEQ ID NO.1:

[0006] .

[0007] The present invention provides antibodies and antigen-binding fragments thereof that bind to NAG protein. The antibodies and antigen-binding fragments thereof are produced by hybridoma cells or compete with antibodies produced by the hybridoma cells for binding. The hybridoma cells are deposited in China Center for Type Culture Collection (CCTCC), and the deposit numbers are CCTCC No: C202381, CCTCC No: C202398, CCTCC No: C202399, and CCTCC No: C2023100, respectively.

[0008] The term "competitive binding" means that the binding of one antibody or antibody fragment to an antigen reduces the binding of a second antibody or antibody fragment to the same antigen. When the two antibodies or antibody fragments bind to the same sequence on a given antigen (e.g., NAG protein), one antibody or antibody fragment will compete with another antibody or fragment for binding.

[0009] The present invention first discovered that there is a specific amino acid sequence in NAG protein that can be recognized. The amino acid sequence is one or more of the following:

[0010] SEQ ID NO.2: GDEVEFKCWESNPKIQDFMRQKGFC;

[0011] SEQ ID NO.3: SKDVRDMDDAYDC;

[0012] SEQ ID NO.4: GQDWRKYYKVEPLDFGGGTQKQKQC;

[0013] SEQ ID NO. 5: KDLLTPCYSRQNKLDSFGPINPC.

[0014] The present invention also provides a method for measuring the level of NAG in a human sample. The method includes binding of NAG protein in the sample to at least one antibody or fragment thereof described herein.

[0015] The present invention also provides diagnostic reagents and kits, which contain at least one (eg, two, three or four) antibodies or antigen-binding fragments thereof described herein.

[0016] The term "antibody" as used herein refers to a protein containing heavy chain polypeptides and light chain polypeptides. Antigen recognition and binding occur in the variable regions of the heavy and light chains, and the antibody contains one of five different types of heavy chains, α, δ, ε, γ and μ, which are classified based on the amino acid sequence of the heavy chain constant region. These different types of heavy chains form five types of antibodies, namely IgA (IgA1 and IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3 and IgG4) and IgM. The antibody also contains one of two types of light chains, called κ and λ, which are classified based on the amino acid sequence of the light chain constant region. IgG, IgD and IgE antibodies usually contain two identical heavy chains and two identical light chains, and the variable region (VH) of each heavy chain and the variable region (VL) of the light chain form an antigen binding fragment, forming a total of two antigen binding fragments. IgA is composed of two monomers, each of which is composed of two heavy chains and two light chains, so the IgA molecule has four antigen binding fragments, each of which is also composed of VH and VL. IgM is generally composed of five monomers, each of which is also composed of two heavy chains and two light chains. Therefore, the IgM molecule has an antigen-binding fragment composed of VH and VL.

[0017] The term "chimeric antibody" as used herein refers to an antibody that has been modified to include at least one human antibody constant region. For example, one or all light chain variable regions and one or all heavy chain variable regions of a mouse monoclonal antibody can each be linked to a human antibody constant region.

[0018] The term "antibody fragment" as used herein refers to a polypeptide derived from an antibody heavy chain and / or light chain. Antibody fragments include Fab fragments, F(ab')2 fragments, scFV (single-chain FV) fragments, linear antibodies, monospecific or multispecific antibody fragments, such as bispecific, trispecific and multispecific antibodies, miniantibodies, nanoantibodies, camelized antibodies, and antibodies containing VHH, etc.

[0019] The term "humanized antibody" as used herein refers to an antibody that is modified to include one or more human framework regions in the variable region together with non-human (e.g., mouse, rat, hamster or rabbit) heavy and / or light chain complementary determining regions (CDRs). In some embodiments, the humanized antibody is derived from human antibody sequences except for the CDR region.

[0020] The "complementarity determining region" or "CDR" refers to short polypeptide sequences within the variable regions of heavy and light chain polypeptides that are primarily responsible for mediating specific antigen recognition.

[0021] The "framework region" refers to the amino acid sequence in the variable region of the heavy chain and light chain polypeptides that is not a CDR sequence, and is mainly responsible for maintaining the correct positioning of the CDR sequence for antigen binding.

[0022] Specifically, this application provides the following solutions:

[0023] (1) The present application first provides a hybridoma cell capable of secreting N-acetyl-β-D-glucosaminidase monoclonal antibody, which is one of the following cells:

[0024] Deposited in China Center for Type Culture Collection, the deposit numbers are CCTCC No: C202381, CCTCC No: C202398, CCTCC No: C202399 or CCTCC No: C2023100.

[0025] The above cell lines may be paired hybridoma cell lines, specifically:

[0026] Cell lines deposited in China Center for Type Culture Collection with the deposit numbers CCTCC No: C202381 and CCTCC No: C202398; or,

[0027] The cell lines are deposited in China Center for Type Culture Collection with the deposit numbers CCTCC No: C202399 and CCTCC No: C2023100.

[0028] (2) The present invention further provides a monoclonal antibody to N-acetyl-β-D-glucosaminidase, wherein the antibody is produced by any one of the aforementioned paired hybridoma cell lines.

[0029] The N-acetyl-β-D-glucosaminidase monoclonal antibody of the present invention can recognize the amino acid sequence shown in SEQ ID NO.2-5.

[0030] (3) The present invention also provides an isolated peptide, which can be recognized by the aforementioned antibody, and the amino acid sequence of the peptide is shown in any one of SEQ ID NOs. 2-5.

[0031] (4) The present invention also provides an antigen-binding fragment of a monoclonal antibody to N-acetyl-β-D-glucosaminidase, wherein the antigen-binding fragment is a Fab fragment or a F(ab')2 fragment of any of the aforementioned antibodies.

[0032] (5) The present invention also provides a chimeric or humanized antibody, wherein the chimeric or humanized antibody comprises the aforementioned antigen-binding fragment.

[0033] (6) The present invention also provides a detection reagent, which comprises any one or more of the aforementioned N-acetyl-β-D-glucosaminidase monoclonal antibody, the antigen-binding fragment of the N-acetyl-β-D-glucosaminidase monoclonal antibody, or the aforementioned chimeric or humanized antibody.

[0034] Preferably, the detection reagent can be any one of a colloidal gold detection reagent, an ELISA detection reagent, an immunochromatography detection reagent, an immunoturbidimetric detection reagent, a magnetic particle detection reagent, a chemiluminescence detection reagent, an immunofluorescence detection reagent or a radioimmunoassay detection reagent.

[0035] Further preferably, the reagent comprises a monoclonal antibody against N-acetyl-β-D-glucosaminidase secreted by the following cell lines or a Fab fragment or F(ab')2 fragment thereof:

[0036] The cell lines deposited in China Center for Type Culture Collection with the deposit numbers CCTCC No: C202381 and CCTCC No: C202398;

[0037] Or, cell lines deposited in China Center for Type Culture Collection with deposit numbers of CCTCC No: C202399 and CCTCC No: C2023100.

[0038] (7) Use of the aforementioned N-acetyl-β-D-glucosaminidase monoclonal antibody, antigen-binding fragment of N-acetyl-β-D-glucosaminidase monoclonal antibody, or chimeric or humanized antibody, wherein the use is for preparing a reagent for detecting N-acetyl-β-D-glucosaminidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1The specificity evaluation results of the NAG colloidal gold test strip of the present invention are shown below;

[0040] Figure 2 The anti-interference ability evaluation results of the NAG colloidal gold test strip of the present invention are shown;

[0041] Figure 3 The results of sensitivity evaluation using the NAG colloidal gold test strip of the present invention are shown below;

[0042] Figure 4 The figure shows the stability evaluation result of the NAG colloidal gold test strip of the present invention. DETAILED DESCRIPTION

[0043] The technical scheme of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work conditions belong to the scope of protection of the present invention. In the following examples, if specific conditions are not specified, they are all carried out according to the conditions recommended by normal conditions or manufacturers. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained by commercial purchase. Those skilled in the art can make appropriate improvements to realize and apply the technology of the present invention with reference to the contents herein, but similar replacements and changes should be deemed to be included in the scope of the content disclosed by the present invention.

[0044] Example 1: Extraction of N-acetyl-β-D-glucosaminidase (NAG) protein

[0045] After thawing, the human placenta was removed from the umbilical cord, washed with pre-cooled pure water, the blood vessels and membranes were removed, and the placenta was chopped; it was rinsed three times with pre-cooled pure water containing 0.5mM EDTA and 1% NaN3. 2L pre-cooled buffer (containing 0.5mM EDTA, 1% NaN3, 0.01M phosphate buffer (Na2HPO4 / KH2PO4), pH=6.0) was added to the washed and chopped placenta for homogenization; after sufficient homogenization, centrifuged at 4℃ and 10000×g for 10min, and the supernatant was collected. 1L of pre-cooled buffer (containing 0.5mM EDTA, 1% NaN3, 0.01M phosphate buffer (Na2HPO4 / KH2PO4), pH=6.0) was added to the precipitate again for homogenization; after sufficient homogenization, centrifuge at 4℃ and 10000×g for 10min, take the supernatant, and dialyze into buffer (0.5M NaCl, 1% NaN3, 0.125M sodium succinate, pH=6.0). After the sample is completely dialyzed, centrifuge at 4℃ and 12000×g for 1h, and the supernatant is filtered through a 0.45μM membrane and waits for loading. Equilibrate 30 mL of 5 mL Con A Beads 4FF chromatography column with equilibration solution (0.5 M NaCl, 0.1% NaN3, 12.5 mM sodium succinate, pH = 6.0); load the filtered sample at a flow rate of 0.5 mL / min. After loading, wash with 10 mM phosphate buffer (Na2HPO4 / KH2PO4), 0.5 M NaCl, pH = 7.0 until the baseline is flat; elute with 10 mM phosphate buffer (Na2HPO4 / KH2PO4), 0.2 M α-D-methyl glucoside, pH = 7.0. The eluted sample is dialyzed into 5 mM phosphate buffer (Na2HPO4 / KH2PO4), pH = 7.5. After complete dialysis, centrifuge the sample at 12000 × g at 4 ° C for 10 min, and filter the supernatant through a 0.45 μM membrane for loading. Equilibrate 1 mL Capto with 5 mM phosphate buffer (Na2HPO4 / KH2PO4), pH = 7.5 TMMMC ImpRes chromatography column 20mL; filter the sample at a flow rate of 0.5mL / min, after loading, wash with 5mM phosphate buffer (Na2HPO4 / KH2PO4), pH = 7.5 balance solution until the baseline is flat; 5mM phosphate buffer (Na2HPO4 / KH2PO4), 1M NaCl, pH = 7.5 elution. The eluted sample is dialyzed into 20mM phosphate buffer (Na2HPO4 / NaH2PO4), 0.15M NaCl, pH = 7.4 buffer; the dialyzed sample is measured with BCA Protein Quantification Kit. The protein concentration is 1.1mg / mL, a total of 13.5mg, and 1% NaN3 is added and stored at -20℃.

[0046] The purified NAG protein was sequenced and analyzed to obtain the protein sequence, which was then compared with the NAG sequence in the UniProt protein database (Uniprot: P07686). The results were completely consistent, confirming that the extracted target protein was NAG.

[0047] Example 2: Preparation of NAG mouse monoclonal antibody

[0048] (1) Animal immunization

[0049] After the obtained NAG protein was fully emulsified with Freund's complete adjuvant, female Balb / c mice aged 6-8 weeks were subcutaneously immunized at multiple points, with an initial immunization dose of 50 μg / mouse. The second and third immunizations were performed 14 and 28 days after the first immunization, respectively, with an immunization dose of 50 μg / mouse. Blood was collected from the tail of the mice about 14 days after the third immunization, and the serum titer was detected indirectly using a 96-well plate coated with NAG.

[0050] (2) Construction of B cells

[0051] Select the SP / 20 myeloma cell line with fast growth and high fusion efficiency for fusion. Select cells in logarithmic growth phase, good cell morphology and activity during fusion. Before fusion, use an elbow dropper to gently blow the myeloma cells off the bottle wall, collect them in a centrifuge tube, centrifuge at 1000r / min for 5min, discard the supernatant, resuspend the cell pellet with 30mL culture medium, centrifuge at 1000r / min for 5min, discard the supernatant, and mix the pellet with 20mL culture medium for later use.

[0052] Take BALB / c mice that have completed animal immunization, remove the eyeballs to collect blood, and use a centrifuge to separate the serum as the positive control serum for antibody detection. Kill the mice by cervical dislocation, soak the mice in 75% alcohol for 5 minutes, and remove the spleen with sterile in a clean bench. Remove the spleen and place it in a plate containing 10mL of culture medium, wash it gently, and peel off the surrounding connective tissue. Use a sterile syringe to pierce the spleen with liquid until the spleen turns white, collect the spleen cells in the plate in a 50mL centrifuge tube, centrifuge at 1000r / min for 10 minutes, discard the supernatant, add 10mL of culture medium, and take a small amount of 10 dilution counts.

[0053] Three days before fusion, positive mice were boosted with immunization, mouse spleen cells were mixed with myeloma cells sp2 / 0 at a ratio of 10:1, PEG fused, and then cultured in HAT selection medium. Ten days later, hybridoma cell supernatants were screened by ELISA, and the screened positive hybridoma cells were cloned by limiting dilution. After five screenings, four strains were finally determined to be positive cells.

[0054] According to the NAG protein sequence, four specific site sequences were selected to synthesize polypeptides, which were coupled with KLH and used for ELISA screening of hybridoma cells.

[0055] Weigh 3 mg of KLH and dissolve it in 600 μl of PBS; weigh 0.5 mg of sulf-SMCC and dissolve it in 100 μl of PBS; then slowly drop the dissolved SMCC solution into the KLH solution, gently mix while dropping, and then react at room temperature for 2 hours; add the solution to a dialysis bag, dialyze with PBS to remove excess SMCC, and dialyze overnight; on the next day, after changing 1-2 dialysate, slowly drop 4 mg of the dissolved polypeptide into the semi-conjugate, drop and mix while dropping, and react at room temperature for 4 hours; after the reaction is complete, directly package and store.

[0056] The KLH-coupled polypeptide was coated on a 96-well plate at 10 μg / mL, 50 μL / well, and adsorbed at 37 degrees for 1 hour. The liquid in the well was discarded, and the well was washed with washing solution 3 times, patted dry, and 100 μL of blocking solution was added to each well for blocking at 37°C for 1 hour, washed twice, and patted dry. 100 μL of the culture supernatant of the 4 hybridoma cells to be tested was added to each well, and positive, negative and blank control standards were set up at the same time. The plates were incubated at 37°C for 0.5 hours, washed 4 times, and patted dry. Horseradish enzyme-labeled goat anti-mouse IgG 1:10000 was taken, 100 μL per well was added to the ELISA plate, incubated at 37°C for 30 minutes, washed 4 times, and patted dry. Finally, 100 μL / well of TMB substrate colorimetric solution was added, and the reaction was terminated with 50 μL / well of 2 mol / L dilute hydrochloric acid after 15 minutes of color development. The OD value of each well was measured at dual wavelengths of 450nm and 630nm. The blank well value was below 0.02, and the negative well value was below 0.1. The positive and negative were clearly distinguished, and it could be judged that the anti-NAG hybridoma cells secreted antibodies that could specifically recognize the specific sites of the NAG protein.

[0057] Through the screening and analysis of peptides with different sequences, it was determined that 4 cell lines can secrete antibodies that specifically recognize NAG protein, and each has a corresponding recognition site sequence, as shown in Table 1:

[0058] Table 1 Recognition site sequences of specific antibodies secreted by four cell lines

[0059]

[0060] (3) Comparative example:

[0061] Different immunization methods and B cell construction methods were used to prepare the B cells of the control group, and the antibodies secreted by the obtained B cells were different from the aforementioned antibodies. The details are as follows:

[0062] Five female Balb / c mice aged 6-8 weeks were selected for immunization. The antigen was a mixture of purified NAG protein with Freund's complete adjuvant, Freund's incomplete adjuvant, and PBS. After complete emulsification, 200 μL was injected into each mouse at multiple points on the back, ventral groove, and axilla. Immunization steps: the first immunization, 50 μg of antigen / mouse, Freund's complete adjuvant was added for subcutaneous injection and multiple points on the back; the second immunization was performed two weeks later, the dosage route was the same as the first, and Freund's incomplete adjuvant was added; the third immunization was performed 15 days later, the dosage was the same as above, and Freund's incomplete adjuvant was added, and the intraperitoneal injection was performed; the last immunization was one month apart from the previous immunization, and NAG antigen 50 μg / mouse was used for muscle booster immunization (without adjuvant), and tail blood was collected 3 days later, and the serum antibody titer was detected by ELISA.

[0063] Select SP / 20 myeloma cell line, and select cells in logarithmic growth phase, good cell morphology and activity for fusion. On the day of fusion, use an elbow dropper to gently blow myeloma cells off the bottle wall, collect them in a 50mL centrifuge tube or fusion tube, centrifuge at 1000r / min for 5-10min, discard the supernatant, add 30mL culture medium to the precipitate, centrifuge and wash once, centrifuge for 5-10min, discard the supernatant, and mix the precipitate with 20mL culture medium for later use.

[0064] Take BALB / c mice that have completed animal immunization, remove the eyeballs to collect blood, and use a centrifuge to separate the serum as the positive control serum for antibody detection.

[0065] Mice were killed by cervical dislocation and soaked in 75% alcohol for 5 min. The spleen was removed aseptically in a clean bench and placed in a plate containing 10 mL of culture medium. The spleen was gently washed and the surrounding connective tissue was removed. The spleen was pierced with a sterile syringe until the spleen turned white. The spleen cells in the plate were collected in a 50 mL centrifuge tube and centrifuged at 1000 r / min for 10 min. The supernatant was discarded, 10 mL of culture medium was added, and a small amount of 10-fold dilution was taken for counting.

[0066] Three days before fusion, positive mice were boosted with immunization, mouse spleen cells were mixed with myeloma cells sp2 / 0 at a ratio of 10:1, PEG fused, and then cultured in HAT selection medium. Ten days later, hybridoma cell supernatants were screened by ELISA, and the screened positive hybridoma cells were cloned by limiting dilution. After five screenings, six cell lines were finally determined to be positive cells.

[0067] ELISA method: NAG antigen was coated on a 96-well plate at 10 μg / mL, 50 μL / well, and adsorbed at 37 degrees for 1 hour. The liquid in the well was discarded, and the well was washed with washing solution for 3 times, patted dry, and 100 μL of blocking solution was added to each well for blocking at 37°C for 1 hour, washed twice, and patted dry. 100 μL of hybridoma cell culture supernatant to be tested was added to each well, incubated at 37°C for 0.5h, washed 4 times, and patted dry. Horseradish enzyme-labeled goat anti-mouse IgG 1:10000 was taken, 100 μL per well was added to the ELISA plate, incubated at 37°C for 30min, washed 4 times, and patted dry. The above ELISA method determined that the anti-NAG B hybridoma cells secreted monoclonal antibodies A and B that could specifically recognize NAG protein.

[0068] (4) Testing: Testing the cell-secreted antibodies obtained by the above different methods.

[0069] NAG antigen, XH1852, XH1853, XH1855, and XH1856 polypeptides coupled to KLH were coated on 96-well plates at 10 μg / mL, 50 μL / well, and adsorbed at 37 degrees for 1 hour. The liquid in the wells was discarded, and the wells were washed 3 times with washing solution, patted dry, and 100 μL of blocking solution was added to each well at 37 degrees for 1 hour, washed twice, and patted dry. 100 μL of the culture supernatant of the hybridoma cells to be tested was added to the 5 wells of the plates, a total of 6 hybridoma cell culture supernatants, and physiological saline (0.85% sodium chloride solution) was used as a negative control group. Take HRP-labeled goat anti-mouse IgG and dilute it at a ratio of 1:2000, add 100 μL per well to the plate, incubate at 37°C for 30 minutes, wash 4 times, pat dry, add 100 μL substrate solution to each well, incubate at 37°C for 15 minutes, add 50 μL stop solution to terminate the reaction, and read with an ELISA reader. According to the test data, it can be determined that 15D12, 6E8, 4A3, and 7G9 can all recognize specific epitope peptides and whole molecule NAG antigens, while monoclonal antibodies A and B can only recognize whole molecule NAG antigens.

[0070] The results are shown in Table 2.

[0071] Table 2 NAG antibody recognition specific epitope test data

[0072]

[0073] Example 3: ELISA test of monoclonal antibody pairing

[0074] (1) ELISA functional sensitivity

[0075] The functional sensitivity of the test refers to the minimum concentration that satisfies CV≤15% by performing 20 repeated tests on a certain concentration of NAG standard. The concentration gradient of NAG standard test is 0, 2.5, 5, 10, 20 U / L, and all test concentrations meet the standard, so the functional sensitivity of the two pairs of 15D12 and 6E8, 4A3 and 7G9 is 2.5 U / L. The results are shown in Tables 3 and 4.

[0076] Table 3 Functional sensitivity of antibody 15D12 and 6E8 pairing

[0077] NAG(U / L) Average OD450 Standard Deviation CV 0 0.058 0.0083 14.3％ 2.5 0.207 0.0250 12.1％ 5 0.388 0.0345 8.9％ 10 0.493 0.0449 9.1％ 20 0.812 0.0512 6.3％

[0078] Table 4 Functional sensitivity of antibody 4A3 and 7G9 pairing

[0079] NAG(U / L) Average OD450 Standard Deviation CV 0 0.021 0.0083 14.6％ 2.5 0.083 0.0250 13.9％ 5 0.171 0.0345 9.1％ 10 0.356 0.0449 9.7％ 20 0.718 0.0512 5.8％

[0080] (2) ELISA precision

[0081] The precision evaluation of the test was performed according to EP5-A2 of the Clinical and Laboratory Standards Institute (CLSI) guide. Clinical urine samples at three concentration levels of high, medium and low were collected, and each concentration sample was divided into 20 portions, each 1 mL and frozen at -80°C. The divided samples were tested once a day, two tests were repeated at a time, one group every 10 days, a total of 20 days of testing, and completed within 2 months. The results are shown in Tables 5 and 6.

[0082] Table 5 Pairing precision of 15D12 and 6E8

[0083] sample Concentration value (U / L) Within group CV Total CV Low concentration 12 8.6％ 9.3％ Final concentration 58 7.9％ 8.2％ High concentration 189 7.5％ 8.1％

[0084] Table 6 Pairing precision of 4A3 and 7G9

[0085] sample Concentration value (U / L) Within group CV Total CV Low concentration 12 9.6％ 9.8％ Final concentration 58 8.8％ 8.5％ High concentration 189 8.5％ 8.6％

[0086] Example 4: Preparation of colloidal gold rapid test strips using monoclonal antibodies 15D12 and 6E8, and evaluation of specificity, anti-interference ability, sensitivity and stability of NAG protein detection

[0087] (1) Preparation of colloidal gold rapid test strips

[0088] The structure of the colloidal gold rapid test strip is as follows: ① colloidal gold pad, containing colloidal gold-labeled NAG monoclonal antibody 15D12; ② nitrocellulose membrane, with NAG monoclonal antibody 6E8 fixed on the T line and goat anti-mouse IgG polyclonal antibody fixed on the C line; ③ other test strip supports.

[0089] The urine sample is dripped vertically onto the sample pad of the test strip. Under the action of chromatography, the N-acetyl-β-D-glucosaminidase in the sample first combines with the colloidal gold-labeled NAG monoclonal antibody 15D12 on the colloidal gold pad to form a reaction complex. The reaction complex moves forward along the nitrocellulose membrane and is captured by the NAG monoclonal antibody 6E8 fixed on the detection line T of the nitrocellulose membrane and presents a color band. The more N-acetyl-β-D-glucosaminidase in the sample, the more complexes accumulate on the detection line (T) and the darker the color of the color band.

[0090] If the N-acetyl-β-D glucosaminidase in the sample is negative, no binding occurs, that is, no color is developed. Regardless of whether N-acetyl-β-D glucosaminidase is present in the sample, the quality control line (C) should show a color band. If not, the test is invalid.

[0091] Test result explanation:

[0092] Positive: Clear color bands appear at both the test line (T) and the quality control line (C), indicating that the activity of N-acetyl-β-D-glucosaminidase in the sample is high, suggesting that the patient has renal tubular damage or early diabetic kidney damage.

[0093] Negative: Only a clear color band appears at the quality control line (C), indicating that the N-acetyl-β-D-glucosaminidase activity in the sample is normal.

[0094] Invalid: There is no color band at the control line (C). Regardless of whether a color band appears at the test line (T), the test is invalid.

[0095] (2) Evaluation of specificity and anti-interference ability

[0096] Natural urine fibronectin and lysozyme were diluted 100 times, 1000 times, 10000 times and 100000 times respectively as samples for specificity evaluation. In addition, 50 urine samples from healthy people were selected as samples for anti-interference ability evaluation. The colloidal gold rapid test strips were prepared by the above method. The evaluation results were as follows: Figure 1 , Figure 2 shown.

[0097] (3) Sensitivity evaluation

[0098] Natural N-acetyl-β-D-glucosaminidase (1.1 mg / mL) (prepared according to the method of Experimental Example 1) was diluted 1000 times, 10000 times, 100000 times, and 200000 times as a sensitivity evaluation sample, and colloidal gold rapid test strips were prepared using the above method for testing. The evaluation results are as follows: Figure 3 shown.

[0099] (4) Stability evaluation

[0100] The colloidal gold rapid test strip prepared according to the above method was evaluated according to the above sensitivity evaluation method, and then placed at 55°C for accelerated destruction for 7 days to evaluate the stability. The evaluation results are as follows: Figure 4 shown.

[0101] Example 5: Preparation of colloidal gold rapid test strips using monoclonal antibodies 4A3 and 7G9

[0102] The structure of the colloidal gold rapid test strip is as follows: ① colloidal gold pad, containing colloidal gold-labeled NAG monoclonal antibody 4A3; ② nitrocellulose membrane, with NAG monoclonal antibody 7G9 fixed on the T line and goat anti-mouse IgG polyclonal antibody fixed on the C line; ③ other test strip supports.

[0103] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A paired hybridoma cell line capable of secreting N-acetyl-β-D-glucosaminidase monoclonal antibody, characterized in that: The paired hybridoma cell strains are the following cell strains: A cell line deposited in China Center for Type Culture Collection with a deposit number of CCTCC No: C202381, and a cell line deposited in China Center for Type Culture Collection with a deposit number of CCTCC No: C202398; or, The cell line deposited in China Center for Type Culture Collection with a deposit number of CCTCC No: C202399, and the cell line deposited in China Center for Type Culture Collection with a deposit number of CCTCC No: C2023100.

2. N-acetyl-β-D-glucosaminidase monoclonal antibody, characterized in that The N-acetyl-β-D-glucosaminidase monoclonal antibody is produced by any of the following hybridoma cell lines: The cell line is deposited in China Center for Type Culture Collection with the deposit number CCTCC No: C202381; The cell line is deposited in China Center for Type Culture Collection with the deposit number CCTCC No: C202398; The cell line is deposited in China Center for Type Culture Collection with the deposit number CCTCC No: C202399; The cell line is deposited in China Center for Type Culture Collection with the deposit number CCTCC No: C2023100.

3. The N-acetyl-β-D-glucosaminidase monoclonal antibody according to claim 2, further characterized in that: The N-acetyl-β-D-glucosaminidase monoclonal antibody can recognize the amino acid sequence shown in SEQ ID NO.2-5; wherein: The amino acid sequence shown in SEQ ID NO.2 is recognized by the monoclonal antibody against N-acetyl-β-D-glucosaminidase produced by the cell line with the deposit number CCTCC No: C202381; The amino acid sequence shown in SEQ ID NO.3 is recognized by the monoclonal antibody against N-acetyl-β-D-glucosaminidase produced by the cell line with the deposit number CCTCC No: C202398; The amino acid sequence shown in SEQ ID NO.4 is recognized by the monoclonal antibody against N-acetyl-β-D-glucosaminidase produced by the cell line with the deposit number CCTCC No: C202399; The amino acid sequence shown in SEQ ID NO.5 is recognized by the N-acetyl-β-D-glucosaminidase monoclonal antibody produced by the cell line with the deposit number of CCTCC No: C2023100.

4. An antigen-binding fragment of a monoclonal antibody to N-acetyl-β-D-glucosaminidase, characterized in that The antigen-binding fragment is the Fab fragment or F(ab')2 fragment of the N-acetyl-β-D-glucosaminidase monoclonal antibody according to claim 2.

5. A chimeric antibody, characterized in that The chimeric antibody comprises the antigen-binding fragment of claim 4.

6. A humanized antibody, characterized in that: The humanized antibody comprises the antigen-binding fragment of claim 4.

7. A detection reagent, characterized in that: The detection reagent comprises any one or more of the N-acetyl-β-D-glucosaminidase monoclonal antibody according to claim 2, the antigen-binding fragment of the N-acetyl-β-D-glucosaminidase monoclonal antibody according to claim 4, or the chimeric antibody according to claim 5.

8. A detection reagent, characterized in that The detection reagent comprises any one or more of the N-acetyl-β-D-glucosaminidase monoclonal antibody according to claim 2, the antigen-binding fragment of the N-acetyl-β-D-glucosaminidase monoclonal antibody according to claim 4, or the humanized antibody according to claim 6.

9. The detection reagent according to claim 7 or 8, further characterized in that: The detection reagent is any one of an ELISA detection reagent, an immunochromatographic detection reagent, an immunoturbidimetric detection reagent, a magnetic particle detection reagent, a chemiluminescent detection reagent, an immunofluorescent detection reagent or a radioimmunoassay detection reagent.

10. The detection reagent according to claim 7 or 8, wherein the detection reagent is a colloidal gold detection reagent.

11. The detection reagent according to claim 7 or 8, further characterized in that: The detection reagent comprises a monoclonal antibody to N-acetyl-β-D-glucosaminidase secreted by the following paired hybridoma cell lines or a Fab fragment or F(ab')2 fragment thereof: A cell line deposited in China Center for Type Culture Collection with a deposit number of CCTCC No: C202381, and a cell line deposited in China Center for Type Culture Collection with a deposit number of CCTCC No: C202398; Alternatively, the cell line deposited in China Center for Type Culture Collection with a deposit number of CCTCC No: C202399 and the cell line deposited in China Center for Type Culture Collection with a deposit number of CCTCC No: C2023100.

12. Use of the N-acetyl-β-D-glucosaminidase monoclonal antibody according to claim 2, the antigen-binding fragment of the N-acetyl-β-D-glucosaminidase monoclonal antibody according to claim 4, or the chimeric antibody according to claim 5, characterized in that: The application is to prepare N-acetyl-β-D-glucosaminidase detection reagent.

13. The use of the humanized antibody according to claim 6, characterized in that: The application is to prepare N-acetyl-β-D-glucosaminidase detection reagent.

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