Preparation of Monoclonal Antibodies for IV Collagen and Application of Detection Kits

Abstract

The present invention discloses the preparation of a monoclonal antibody for IV collagen and its application in a detection kit, belonging to the field of immunological assays. The present invention expresses a highly specific conserved fragment of the IV collagen protein and prepares a polyclonal antibody against this fragment. Simultaneously, a pair of IV monoclonal antibodies with high specificity, high affinity, and high sensitivity are screened and obtained. Combined with dual-size liposome particle coating technology, a highly sensitive and specific IV collagen detection kit and method are developed. The kit has a minimum detection limit of approximately 3 ng / ml, a clinical false-positive rate of less than 5‰, and a detection range exceeding 600.00 ng / ml, helping to improve the accuracy of clinical diagnosis.

Description

Technical Field

[0001] The present invention belongs to the field of immunological determination, and relates to the preparation of a monoclonal antibody for IV collagen and the application of a detection kit, and particularly to the preparation of a highly specific and highly sensitive IV collagen monoclonal antibody and the development of a latex-enhanced immunoturbidimetric kit for the quantitative detection of IV collagen. Background Art

[0002] Type IV collagen (IV-C) is a member of the collagen family and a non-fibrillar collagen. It also possesses a unique heterotrimeric helical chain structure, composed of six α-peptide chains: α1, α2, α3, α4, α5, and α6. The genes COL4A1 and COL4A2, which synthesize the α1 and α2 chains, are present as a pair on human chromosome 13. The genes COL4A3 and COL4A4, which synthesize the α3 and α4 chains, are present as a pair on human chromosome 2. The genes COL4A5 and COL4A6, which synthesize the α5 and α6 chains, are present as a pair on human chromosome X. Type V collagen is the most abundant structural basement membrane component. It consists of a predominant triple helical structure flanked by an N-terminal 7S domain and a C-terminal non-collagenous domain, NC1. There are six distinct type IV collagen chains, α1-α6 (IV), which exhibit tissue-specific distribution patterns. The three α chains fold to form a heterotrimeric molecule known as the protocollagen. Six genetically distinct α chains assemble together to form only three prototypes: α1α1α2(IV), α3α4α5(IV), and α5α5α6(IV). The predominant form of type IV collagen, α1α1α2(IV), is present in the basement membranes of all tissues, while the other two forms are only found in some basement membranes. IV-C not only plays a crucial role in the formation of the BM skeleton but also participates in important physiological processes such as cell adhesion, migration, growth, proliferation, and differentiation. For example, in gastric cancer tissue, the α5 and α6 peptide chains of BMs are lost to varying degrees as the disease progresses. The type of IV-C in BMs also changes during the development of tumors such as colorectal cancer, pancreatic cancer, and extrahepatic bile duct cancer. For example, during the embryonic development of the kidneys of patients with Alport syndrome (AS), due to mutations in the gene encoding the α-peptide chain, the α3α4α5 heterotrimer cannot effectively replace the α1α1α2 heterotrimer. The glomerular filtration network is relatively weak and susceptible to oxidative damage and pressure damage, resulting in progressive renal failure. Therefore, the biological characteristics of IV-C are closely related to many major diseases such as tumors and kidney disease.

[0003] Type IV collagen is a primary indicator for observing liver cirrhosis, and its concentration correlates with the degree of liver fibrosis. In fibrotic liver tissue, IV-C expression increases approximately 14-fold, and the degree of cirrhosis can be estimated from serum type IV collagen concentration. In acute hepatitis, despite extensive liver cell destruction, there is no significant connective tissue proliferation, so serum type IV collagen concentrations are not significantly different from those in healthy individuals. Serum type IV collagen levels are significantly elevated in patients with chronic hepatitis, cirrhosis, and liver cancer, with the degree of increase decreasing in primary liver cancer, cirrhosis, chronic active hepatitis, chronic persistent hepatitis, and acute viral hepatitis.

[0004] Common methods currently available for IV collagen testing include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), time-resolved fluorescence immunoassay (TRFIA), latex-enhanced immunoturbidimetry (LTTI), and chemiluminescence. ELISA is a manual procedure, unsuitable for batch testing, and requires a long testing time. RIA requires labeling with rare metals, while chemiluminescence requires specialized equipment and is susceptible to interference. LETTI, however, is difficult to detect with small antigen-antibody complexes unless they are left for a long time. If larger complexes form, the amount of antigen and antibody required is also large, clearly not meeting the requirements for microquantification. Consequently, the LETTI (latex-enhanced immunoturbidimetry) assay, now widely used in biochemical analyzers, was developed. This modified turbidimetric assay, based on polyclonal antibodies, utilizes genetic engineering to bind antibodies to latex particles. Upon antigen-antibody binding, an antigen-antibody-latex particle complex is formed, enhancing the absorbance of the reaction. The reaction solution is turbidimetrically measured at a specific wavelength and compared with a similarly treated standard solution to calculate the antigen content in the specimen. The turbidimetric determination is performed using a biochemical analyzer, and the entire analysis process only takes a few minutes. Compared with the traditional immunoturbidimetric method, this method has higher sensitivity.

[0005] However, the IV collagen latex-enhanced immunoturbidimetric detection kit currently on the market has the disadvantages of a minimum detection limit of 20-30 ng / ml for IV collagen and a high false positive rate due to the lack of highly specific and sensitive monoclonal antibodies. Summary of the Invention

[0006] In response to the shortcomings of the prior art, the present invention aims to provide a method for preparing a monoclonal antibody against IV collagen and its application in a detection kit. The present invention expresses a highly specific conserved fragment of IV collagen and prepares a polyclonal antibody against this fragment. Simultaneously, a pair of highly specific, high-affinity, and highly sensitive IV monoclonal antibodies are screened and obtained. Furthermore, a highly sensitive and specific IV collagen detection kit and method are developed, with a minimum detection limit of approximately 3 ng / ml, a clinical false-positive rate of less than 5‰, and a detection range exceeding 600.00 ng / ml, contributing to improved clinical diagnostic accuracy.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a IV collagen monoclonal antibody, wherein the IV collagen monoclonal antibody comprises the monoclonal antibody shown in any one of the following (a)-(b):

[0009] (a) Monoclonal antibody IV Col Ab1 comprises heavy and light chain variable regions; wherein the three complementarity determining region sequences of the heavy chain variable region are:

[0010] CDR1-H1: GKIRGATEYGM (as shown in SEQ ID NO. 1),

[0011] CDR1-H2: PGLRGTTGLA (as shown in SEQ ID NO. 2),

[0012] CDR1-H3: AYRRGDPGYRFGDAY (as shown in SEQ ID NO. 3);

[0013] Three complementarity-determining regions of the light chain variable region:

[0014] CDR1-L1: SGVARTTILFP (as shown in SEQ ID NO. 4),

[0015] CDR1-L2: GMGRQTPG (as shown in SEQ ID NO. 5),

[0016] CDR1-L3: ALEAYSKPP (as shown in SEQ ID NO. 6);

[0017] (b) Monoclonal antibody IV Col Ab2 comprises heavy and light chain variable regions; wherein the three complementarity determining region sequences of the heavy chain variable region are:

[0018] CDR2-H1: GSSIMFGGAYTTRGGYATM (as shown in SEQ ID NO. 7),

[0019] CDR2-H2: PAYTLYIPGGNVTSLS (as shown in SEQ ID NO. 8),

[0020] CDR2-H3: ASYARDTGPYGFRDPY (as shown in SEQ ID NO. 9);

[0021] Three complementarity-determining regions of the light chain variable region:

[0022] CDR2-L1: STAGGAVTDTIGNPGFRA (as shown in SEQ ID NO. 10),

[0023] CDR2-L2: GATRGRNQPYGIRN (as shown in SEQ ID NO. 11),

[0024] CDR2-L3: ADLEANPYSGWTWGV (as shown in SEQ ID NO. 12).

[0025] As one embodiment of the present invention, the variable region sequences of the heavy and light chains of the monoclonal antibody IV Col Ab1 are shown as SEQ ID NOs. 13-14, respectively; the variable region sequences of the heavy and light chains of the monoclonal antibody IV Col Ab2 are shown as SEQ ID NOs. 15-16, respectively.

[0026] As one embodiment of the present invention, the monoclonal antibody is selected from a Fab fragment, a Fab' fragment, a F(ab')2 fragment or a single-chain antibody.

[0027] As one embodiment of the present invention, the monoclonal antibody is prepared by hybridoma technology and obtained by screening; the antigen used in the hybridoma technology is prepared by eukaryotic expression, and the antigen sequence (i.e., the amino acid sequence expressed in eukaryotes) is as follows:

[0028] sgpygakgprgehglkgekgascygpmkpgapgikgekgepassfpvkpthtvmgprgdmgqkgepglvgrkgep gpegdtgldgqkgekglpggpgdrgrqgnfgppgstgqkgdrgepglnglpgnpgqkgepgragatgkpgllgppgppgggr gtpgppgpkgprgyvgapgpqgl (SEQ ID NO. 17).

[0029] In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleic acid encoding the monoclonal antibody.

[0030] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule.

[0031] In a fourth aspect, the present invention provides a recombinant comprising the nucleic acid molecule or the expression vector.

[0032] In a fifth aspect, the present invention provides a use of the monoclonal antibody, the nucleic acid molecule, the expression vector or the recombinant in preparing a test kit for IV collagen. The test kit of the present invention is a latex-enhanced immunoturbidimetric test kit.

[0033] In a sixth aspect, the present invention provides an IV collagen detection kit, which comprises R1 reagent, R2 reagent, and a calibrator; wherein the R2 reagent comprises the monoclonal antibody.

[0034] As one embodiment of the present invention, the R1 reagent is a Tris buffer, which comprises: 20-50 mM Tris pH 7.2-7.6, 30-300 mM NaCl, 0.1-2% (v / v) Tween20, 0.5-5% (w / v) BSA, 10-500 mM polyethylene glycol 6000, 20-25 mg / L blocking agent, and 0.5-1.5% (w / v) preservative.

[0035] Furthermore, the preservative includes sodium azide; and the blocking agent includes HBR5.

[0036] As one embodiment of the present invention, the R2 reagent is an antibody-sensitized particle suspension, which comprises: antibody-sensitized particles 0.1-0.8 g / l, 20-50 mM MOPSO pH 7.2-7.6, NaCl 30-300 mM, BSA 0.1-4.0% (w / v); the antibody-sensitized particles are liposome particles coated with monoclonal antibodies.

[0037] Furthermore, the preparation method of the antibody-sensitized particles comprises the following steps:

[0038] S1. Centrifuge the liposome particles, take the supernatant, resuspend in glycine buffer, and disperse by ultrasonication to obtain a resuspension;

[0039] S2. Add NHS solution to the resuspension and mix thoroughly, then add EDC solution and mix thoroughly, stir (at room temperature), and centrifuge;

[0040] S3, dissolving and suspending the precipitate obtained by centrifugation in step S2 with glycine buffer, sonicating, adding monoclonal antibody, stirring (at room temperature), and centrifuging;

[0041] S4. Dissolve and suspend the precipitate obtained after centrifugation in step S3 with a glycine buffer, perform sonication, and centrifuge to obtain the antibody-sensitized particles.

[0042] Furthermore, in step S1, the diameter of the liposome particles is 100-250 nm; wherein the diameter of the liposome particles coated with monoclonal antibody IV Col Ab1 is 200-250 nm, and the diameter of the liposome particles coated with monoclonal antibody IV Col Ab2 is 100-120 nm.

[0043] Furthermore, in step S3, the monoclonal antibody includes at least one of monoclonal antibodies IV Col Ab1 and IV Col Ab2; wherein the mass ratio of the added amount of the monoclonal antibody IV Col Ab1 to the liposome particles is 0.2-0.4 mg:100 mg, and the mass ratio of the added amount of the monoclonal antibody IV Col Ab2 to the liposome particles is 0.5-0.8 mg:100 mg.

[0044] As an embodiment of the present invention, the calibrator is a bovine serum matrix, including Proclin-300 Tween 200

[0045] 2-2.2% (v / v), BSA1-3% (w / v).

[0046] The above percentages in the present invention are percentages of human serum matrix volume.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The human body contains multiple collagens, including collagen I, II, III, IV, and V. Currently available marketed antibodies against collagen IV suffer from poor sensitivity, poor specificity, and high false positive rates, making them unsuitable for use in detection kit development. This invention selects a highly specific peptide chain exposed on the surface of natural collagen IV, expresses it in prokaryotes, and prepares monoclonal antibodies against the peptides. The resulting monoclonal antibodies, IV Col Ab1 and 2, are screened for high specificity, high affinity, and high sensitivity, making them suitable for use in detection kits.

[0049] 2. This invention further develops the latex-enhanced immunoturbidimetry method, using transparent liposome particles instead of white latex microspheres. The light transmittance of the liposome particles significantly increases the detection signal range of the reagent, resolving the high background issue of latex-enhanced immunoturbidimetry. Simultaneously, the dual-size liposome particles are coated with monoclonal antibodies IV Col Ab1 and 2, thereby increasing both detection sensitivity and the detection range of the reagent, achieving a sensitivity of approximately 3 ng / ml, a clinical false-positive rate of less than 5‰, and a detection range exceeding 600.00 ng / ml.

[0050] 3) The IV collagen detection kit of the present invention has good repeatability, stable reagents, simple operation, high sensitivity, good specificity, rapid determination, accurate and reliable determination results, and helps to improve the accuracy of clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0052] Figure 1 This is the linear range test result of Example 1;

[0053] Figure 2 The linear range test results of Example 2 are shown below:

[0054] Figure 3 The linear range test results of Example 3 are shown below:

[0055] Figure 4 is the calibration curve of the type IV collagen detection kit of Example 3;

[0056] Figure 5 This is the construction map of the recombinant plasmid. DETAILED DESCRIPTION

[0057] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, provide detailed implementation methods and specific operating procedures, and will help those skilled in the art to further understand the present invention. It should be pointed out that the scope of protection of the present invention is not limited to the following embodiments, and a number of adjustments and improvements made under the premise of the concept of the present invention all fall within the scope of protection of the present invention.

[0058] The present invention provides a method for preparing a monoclonal antibody, comprising the following steps:

[0059] First, the synthesis of recombinant antigens includes the following steps:

[0060] A. Plasmid construction: Design and synthesize recombinant plasmids, determine the designed antigen amino acid sequence (as shown in SEQ ID NO.17), insert the antigen target gene (as shown in SEQ ID NO.18) into the PET32a expression vector (purchased from EMD Biosciences (Novagen)), and introduce the plasmid. The plasmid construction map is shown in Figure 5 shown.

[0061] B. Antigen expression: Transform the recombinant plasmid into competent E. coli (one Shot TM BL21 Star TM(DE3)), high efficiency expression. The construction of recombinant plasmid generally selects strains with genetic stability, high transformation efficiency and high plasmid yield as recipient bacteria. The present invention selects competent Escherichia coli (one Shot TM BL21 Star TM (DE3)) for expression.

[0062] C. Antigen Purification: The expression product is purified using a Ni affinity chromatography column to obtain the target protein, i.e., the recombinant antigen. The expression vector pET32a contains a polyhistidine sequence, known as the His-Tag. This yields a recombinant target protein with a His-Tag. The His-Tag binds to metallic Ni ions, facilitating purification of the target protein. The His-Tag-attached protein can be purified using a Ni affinity chromatography column under non-denaturing conditions.

[0063] Secondly, the preparation method of IV collagen monoclonal antibody comprises the following steps:

[0064] A. Immunization of Animals: Primary immunization: 6-8 week old female BALB / c mice were injected subcutaneously with antigen (60 μg / mouse) emulsified in Freund's complete adjuvant at multiple sites three weeks apart. Second immunization: the same dosage and route as above, with Freund's incomplete adjuvant, three weeks apart. Third immunization: the same dosage and route as above, without adjuvant, via intraperitoneal injection. Booster immunization: 60 μg intraperitoneally, with spleen fusion three days later.

[0065] B. Cell fusion: Immunized BALB / c mice were taken. The mice were killed by cervical dislocation and soaked in 75% alcohol for 5 minutes. The spleen was removed and ground into a cell suspension. A single cell suspension was prepared with 1*10 cells per mouse. 8 -3*10 8 Splenocytes. 8 Splenocytes and 1*10 7 After myeloma cells are fused, they are subcultured in complete culture medium.

[0066] C. Screening and cloning of hybridoma positive clones: Hybridomas can be screened approximately two weeks after cell fusion using the ELISA method for initial screening. Positive clones that pass the initial screening can be cloned. Repeat 2-3 rounds of cloning to obtain hybridomas targeting the desired antigen.

[0067] D. Large scale preparation of monoclonal antibodies: Monoclonal antibodies are prepared in large quantities using in vitro culture method.

[0068] E. Antibody Purification and Identification: Monoclonal antibodies were purified using a two-step caprylic acid-ammonium sulfate precipitation method. Specificity was detected using native antigens.

[0069] Two type IV collagen monoclonal antibodies, IV Col Ab1 and IV Col Ab2, were obtained and sequenced and immunoglobulin domain sequence analyzed. The sequences are as follows:

[0070] Among them, the three heavy chain complementary determining regions of monoclonal antibody IV Col Ab1 are:

[0071] CDR1-H1: GKIRGATEYGM (as shown in SEQ ID NO. 1),

[0072] CDR1-H2: PGLRGTTGLA (as shown in SEQ ID NO. 2),

[0073] CDR1-H3: AYRRGDPGYRFGDAY (as shown in SEQ ID NO. 3);

[0074] Three light chain complementarity determining regions of monoclonal antibody IV Col Ab1:

[0075] CDR1-L1: SGVARTTILFP (as shown in SEQ ID NO. 4),

[0076] CDR1-L2: GMGRQTPG (as shown in SEQ ID NO. 5),

[0077] CDR1-L3: ALEAYSKPP (as shown in SEQ ID NO. 6);

[0078] Three heavy chain complementarity determining regions of monoclonal antibody IV Col Ab2:

[0079] CDR2-H1: GSSIMFGGAYTTRGGYATM (as shown in SEQ ID NO. 7),

[0080] CDR2-H2: PAYTLYIPGGNVTSLS (as shown in SEQ ID NO. 8),

[0081] CDR2-H3: ASYARDTGPYGFRDPY (as shown in SEQ ID NO. 9);

[0082] Three light chain complementarity determining regions of monoclonal antibody IV Col Ab2:

[0083] CDR2-L1: STAGGAVTDTIGNPGFRA (as shown in SEQ ID NO. 10),

[0084] CDR2-L2: GATRGRNQPYGIRN (as shown in SEQ ID NO. 11),

[0085] CDR2-L3: ADLEANPYSGWTWGV (as shown in SEQ ID NO. 12).

[0086] The variable region sequences of the heavy chain and light chain of the monoclonal antibody IV Col Ab1 are shown in SEQ ID NOs. 13-14, respectively; the variable region sequences of the heavy chain and light chain of the monoclonal antibody IV Col Ab2 are shown in SEQ ID NOs. 15-16, respectively.

[0087] Example 1

[0088] This embodiment provides a IV collagen detection kit, the preparation method of which is as follows:

[0089] First, antibody-sensitized particles are prepared, including the following steps:

[0090] Take 10 ml (1000 mg) of 100 nm liposome particles and coat them with the treated IV Col Ab2 antibody according to the following steps (1)-(5):

[0091] (1) Take 10 ml of liposome particles and centrifuge at 20,000 rpm for 30 min at 4°C; remove the supernatant and resuspend in 50 mM glycine buffer (5 times the amount of liposome particles), disperse by ultrasonication for 2 min, and set aside;

[0092] (2) Take 10 ml of the resuspended liposome particles, add 0.690 ml of 100 mg / ml NHS solution and mix well, then add 1.435 ml of 10 mg / ml EDC solution and mix well. Stir at room temperature for 30 min, and then centrifuge at 4°C and 20,000 rpm for 30 min.

[0093] (3) The precipitate obtained after centrifugation in step (2) was resuspended in 10 ml of 25 mM glycine buffer (purchased from Xibao Biotechnology), sonicated for 1 minute, and 6 mg of IV Col Ab1 was added. The mixture was stirred at room temperature for 90 minutes and centrifuged at 20,000 rpm at 4°C for 15 minutes. The precipitate was resuspended in 10 ml of 25 mM glycine buffer, sonicated for 1 minute, and centrifuged again at 20,000 rpm at 4°C for 20 minutes.

[0094] (4) The precipitate obtained after centrifugation in step (3) was suspended in 10 ml of 0.5% glycine buffer, sonicated for 1 minute, and then centrifuged at 4°C and 20,000 rpm for 25 minutes to obtain the antibody-sensitized particles IV Col Ab2.

[0095] Next, the IV collagen test kit, the components of which are as follows:

[0096] R1 reagent:

[0097] The R1 reagent is a Tris buffer composed of 50 mM Tris pH 7.6, 300 mM NaCl, 1% (v / v) Tween20, 2% (w / v) BSA, 500 mM polyethylene glycol 6000, 0.9% (w / v) sodium azide, and 20 mg / L HBR5 blocker (purchased from Nanjing Jingda Biotechnology).

[0098] R2 reagent:

[0099] R2 reagent is an antibody-sensitized particle suspension, which consists of: antibody-sensitized particles (IV Col Ab2-sensitized particles) 0.6 g / l, MOPSO 50 mM / L, NaCl 300 mM / L, BSA 1.0% (w / v), pH 7.4;

[0100] The calibrator was a bovine serum matrix, including: ProClin-300 Tween-20 2% (v / v), BSA 1% (w / v);

[0101] The above ingredients can be added sequentially at room temperature, or added simultaneously, or packaged separately and prepared immediately before testing.

[0102] Example 2

[0103] This embodiment provides a IV collagen detection kit, the preparation method of which is as follows:

[0104] First, antibody-sensitized particles are prepared, including the following steps:

[0105] Take 10 ml (1000 mg) of 220 nm liposome particles and coat them with the treated IV Col Ab1 antibody according to the following steps (1)-(5):

[0106] (1) Take 10 ml of liposome particles and centrifuge at 20,000 rpm for 30 min at 4°C; remove the supernatant and resuspend in 50 mM glycine buffer (5 times the amount of liposome particles), disperse by ultrasonication for 2 min, and set aside;

[0107] (2) Take 10 ml of the resuspended liposome particles, add 0.30 ml of 100 mg / ml NHS solution and mix well, then add 1.435 ml of 10 mg / ml EDC solution and mix well. Stir at room temperature for 30 minutes, and then centrifuge at 4°C and 20,000 rpm for 30 minutes.

[0108] (3) The precipitate obtained after centrifugation in step (2) was resuspended in 10 ml of 25 mM glycine buffer, sonicated for 1 minute, and 2.5 mg of IV Col Ab1 was added. The mixture was stirred at room temperature for 90 minutes and centrifuged at 20,000 rpm at 4°C for 15 minutes. The precipitate was resuspended in 10 ml of 25 mM glycine buffer, sonicated for 1 minute, and centrifuged again at 20,000 rpm at 4°C for 20 minutes.

[0109] (4) The precipitate obtained after centrifugation in step (3) was suspended in 10 ml of 0.5% glycine buffer, sonicated for 1 minute, and then centrifuged at 4°C and 20,000 rpm for 25 minutes to obtain the antibody-sensitized particles IV Col Ab1.

[0110] Next, the IV collagen test kit, the components of which are as follows:

[0111] R1 reagent:

[0112] The R1 reagent is a Tris buffer composed of 50 mM Tris pH 7.6, 300 mM NaCl, 1% (v / v) Tween20, 2% (w / v) BSA, 500 mM polyethylene glycol 6000, 0.9% (w / v) sodium azide, and 20 mg / L HBR5 blocker (purchased from Nanjing Jingda Biotechnology).

[0113] R2 reagent:

[0114] R2 reagent is an antibody-sensitized particle suspension, which consists of: antibody-sensitized particles (IV Col Ab1-sensitized particles) 0.5 g / l, MOPSO 50 mM / L, NaCl 300 mM / L, BSA 1.0% (w / v), pH 7.4;

[0115] The calibrator was a bovine serum matrix, including: ProClin 300 Tween-20 2% (v / v), BSA 1% (w / v);

[0116] The above ingredients can be added sequentially at room temperature, or added simultaneously, or packaged separately and prepared immediately before testing.

[0117] Example 3

[0118] This embodiment provides an IV collagen detection kit, and its R2 preparation method is basically the same as that of Example 1 and Example 2, except that: the R2 reagent is a mixture of the R2 reagent prepared in Example 1 and the R2 reagent prepared in Example 2 in a 1:1 (volume ratio).

[0119] R1 reagent:

[0120] The R1 reagent is a Tris buffer composed of 50 mM Tris pH 7.6, 300 mM NaCl, 1% (v / v) Tween20, 2% (w / v) BSA, 500 mM polyethylene glycol 6000, 0.9% (w / v) sodium azide, and 20 mg / L HBR5 blocker (purchased from Nanjing Jingda Biotechnology).

[0121] R2 reagent;

[0122] Antibody-sensitized particles (IV Col Ab1-sensitized particles) 0.25 g / l, antibody-sensitized particles (IV Col Ab2-sensitized particles) 0.3 g / l, MOPSO 50 mM / L, NaCl 300 mM / L, BSA 1.0% (w / v), pH 7.4;

[0123] The calibrator was a bovine serum matrix, including: ProClin-300 Tween-20 2% (v / v), BSA 1% (w / v);

[0124] The above ingredients can be added sequentially at room temperature, or added simultaneously, or packaged separately and prepared immediately before testing.

[0125] Example 4

[0126] This embodiment provides a IV collagen detection kit, and its R2 preparation method is basically the same as that of Example 1, except that the diameter of the liposome particles used is 150 nm, and the antibody used for coating is a sheep anti-human IV collagen polyclonal antibody purchased from Biospacific.

[0127] This embodiment also provides a IV collagen detection kit, the components of which are as follows:

[0128] R1 reagent:

[0129] The R1 reagent is a Tris buffer composed of 50 mM Tris pH 7.6, 300 mM NaCl, 1% (v / v) Tween20, 2% (w / v) BSA, 500 mM polyethylene glycol 6000, 0.9% sodium azide, and 20 mg / L HBR5 blocker (purchased from Nanjing Jingda Biotechnology).

[0130] R2 reagent;

[0131] R2 reagent is an antibody-sensitized particle suspension, which consists of: antibody-sensitized particles (goat anti-human IV collagen polyclonal antibody-sensitized particles) 0.5 g / l, MOPSO 50 mM / L, NaCl 300 mM / L, BSA 1.0% (w / v) pH 7.4;

[0132] The calibrator was a bovine serum matrix, including: ProClin 300 Tween-20 2% (v / v), BSA 1% (w / v);

[0133] The above ingredients can be added sequentially at room temperature, or added simultaneously, or packaged separately and prepared immediately before testing.

[0134] Add them sequentially, or add them simultaneously, or package them separately and prepare them immediately before testing.

[0135] Example 5

[0136] This example provides a IV collagen detection kit, and the preparation method of R2 is basically the same as that of Example 3, except that IV Col Ab1 and IV Col Ab2 are replaced by a pair of mouse anti-human IV collagen monoclonal antibodies purchased from Biospacific.

[0137] This embodiment also provides a IV collagen detection kit, the components of which are as follows:

[0138] R1 reagent:

[0139] The R1 reagent is a Tris buffer composed of 50 mM Tris pH 7.6, 300 mM NaCl, 1% (v / v) Tween20, 2% (w / v) BSA, 500 mM polyethylene glycol 6000, 0.9% (w / v) sodium azide, and 20 mg / L HBR5 blocker (purchased from Nanjing Jingda Biotechnology).

[0140] R2 reagent;

[0141] R2 reagent is an antibody-sensitized particle suspension, which consists of: antibody-sensitized particles (mouse anti-human IV collagen monoclonal antibody-sensitized particles) 0.5 g / l, MOPSO 50 mM / L, NaCl 300 mM / L, BSA 1.0% (w / v) pH 7.4;

[0142] The calibrator was a bovine serum matrix, including: Prolin 300 Tween-20 2% (v / v), BSA 1% (w / v);

[0143] The above ingredients can be added sequentially at room temperature, or added simultaneously, or packaged separately and prepared immediately before testing.

[0144] Add them sequentially, or add them simultaneously, or package them separately and prepare them immediately before testing.

[0145] Effect test

[0146] A Hitachi 7180 fully automatic biochemical analyzer was used for analysis. The analysis method was a two-point endpoint method, i.e., the amounts of reagents R1 and R2 were 180ul and 60ul, respectively, and the sample volume was 6ul. 180ul of reagent R1 was added to 3ul of sample and incubated at 37°C for 5 minutes, followed by 60ul of R2. A1 was read after a delay of 0.5min, and A2 was read after incubating at 37°C for 4.5min. The dominant wavelength for detection was 570nm. The detection conditions and determination method are shown in Table 1 below (the calibration curve of the type IV collagen detection kit in Example 3 is referenced). Figure 4 ):

[0147] Table 1: Detection parameters

[0148]

[0149]

[0150] Comparison and verification data

[0151] 1. Minimum detection limit comparison test results

[0152] The purpose of this experiment is to compare the minimum detection sensitivity of the reagents of Example 1, Example 2, and Example 3 in clinical samples.

[0153] The reagents of Example 1, Example 2, and Example 3, standard products, blank solutions (generally physiological saline and purified water), and normal human serum samples were used.

[0154] Machine: Hitachi 7170 automatic biochemical analyzer.

[0155] Operation steps: Dissolve the sample in physiological saline or deionized water, then dilute it by 50% into 5 points. Test each sample 5 times together with the zero point, calculate the average value, and obtain the SD value.

[0156] Result interpretation: As shown in Table 2, based on the test data, calculate the SD value and CV value, calculate 1SD and 2SD respectively, starting from the smallest, the value of its average value - 2SD is above the zero point average value + 2SD, which is the minimum detection sensitivity of the reagent.

[0157] Table 2: Minimum detection limit comparison results

[0158]

[0159]

[0160] The experimental results show that the minimum detection limit of Example 1 of the present invention is 50 ng / ml, and the minimum detection limit of Examples 2 and 3 is 3.12 ng / ml. The minimum detection line of Implementation Cases 2 and 3 is better than that of Implementation Case 1. The use of large-particle liposome microspheres can improve the detection sensitivity of the reagent. The detection limit of Implementation Case 5 is greater than 50 ng / ml. A pair of mouse anti-human type IV collagen monoclonal antibodies were purchased. Although dual-particle liposome particle coating technology was also used, the reaction sensitivity was low, which may be due to the weak antibody-antigen binding affinity.

[0161] 2. Comparison of linear range

[0162] Take a high-value sample with a concentration of about 600.00 ng / mL and use 0.9% NaCl solution as a diluent. Dilute it into 5 points at a ratio of 0.025, 0.2, 0.4, 0.6, and 0.8. Add the high-value sample, and a total of 6 samples are measured 3 times according to the standard experimental operation steps. The average value is calculated respectively. The linear range curves of Examples 1-3 are shown as follows: Figure 1-3 The test results are shown in Table 3 below.

[0163] Table 3: Linearity test results

[0164]

[0165] Experimental results show that the linear peak values ​​of the reagents provided in Examples 1 and 3 of the present invention can reach a concentration of 600 ng / mL, which is superior to that of Example 2. The use of small-particle liposome microspheres can extend the linear range of the reagents. The use of a mixture of large and small-particle liposome microspheres can improve both detection sensitivity and the detection range of the reagents.

[0166] 3. Cross-reactivity

[0167] Cross-reactivity studies consider the impact of structural analogs of the analyte, nucleic acid fragments with homologous sequences, other pathogens that may cause the same or similar clinical symptoms, other microorganisms that normally inhabit or are prone to complications at the sampling site, previously reported cross-reactivity substances, and cross-reactivity substances introduced during raw material production on product testing results. This study analyzed the cross-interference of type I collagen, type II collagen, type III collagen, and type V collagen on the reagents.

[0168] Table 4: Interference sample preparation

[0169]

[0170] Table 5: Cross-reactivity assay results

[0171]

[0172] The experimental results showed that Example 4, which used a commercially available sheep anti-human type IV collagen polyclonal antibody for coating, had varying degrees of interference with homologous proteins such as type I collagen, type II collagen, type III collagen, and type V collagen. Example 5, which used a commercially available pair of mouse anti-human type IV collagen monoclonal antibodies for coating, had no cross-reaction with type I collagen and type III collagen, but had a certain degree of cross-reaction with type II collagen and type V collagen. Example 3 is a reagent of this scheme, which had no cross-reaction with homologous proteins such as type I collagen, type II collagen, type III collagen, and type V collagen.

Claims

1. A monoclonal antibody against IV collagen, characterized in that: The IV collagen monoclonal antibody includes the monoclonal antibody shown in any one of the following (a)-(b): (a) Monoclonal antibody IV Col Ab1 comprises heavy and light chain variable regions; the three complementarity determining region sequences of the heavy chain variable region are: CDR1-H1: GKIRGATEYGM, CDR1-H2:PGLRGTTGLA, CDR1-H3:AYRRGDPGYRFGDAY; The three complementarity determining region sequences of the light chain variable region are: CDR1-L1: SGVARTTILFP, CDR1-L2:GMGRQTPG, CDR1-L3: ALEAYSKPP; (b) Monoclonal antibody IV Col Ab2 comprises heavy and light chain variable regions; the three complementarity determining region sequences of the heavy chain variable region are: CDR2-H1:GSSIMFGGAYTTRGGYATM, CDR2-H2: PAYTLYIPGGNVTSLS, CDR2-H3:ASYARDTGPYGFRDPY; The three complementarity determining region sequences of the light chain variable region are: CDR2-L1: STAGGAVDTIGNPGFRA, CDR2-L2:GATRGRNQPYGIRN, CDR2-L3:ADLEANPYSGWTWGV.

2. The IV collagen monoclonal antibody according to claim 1, characterized in that The variable region sequences of the heavy chain and light chain of the monoclonal antibody IV ColAb1 are shown in SEQ ID NOs. 13-14, respectively; the variable region sequences of the heavy chain and light chain of the monoclonal antibody IV Col Ab2 are shown in SEQ ID NOs. 15-16, respectively.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleic acid encoding the monoclonal antibody according to any one of claims 1-2.

4. An expression vector, characterized in that The expression vector comprises the nucleic acid molecule of claim 3.

5. A recombinant, characterized in that The recombinant comprises the nucleic acid molecule according to claim 3 or the expression vector according to claim 4.

6. Use of the monoclonal antibody according to any one of claims 1 to 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4 or the recombinant according to claim 5 in preparing a kit for detecting IV collagen.

7. A IV collagen detection kit, characterized in that: The kit comprises R1 reagent, R2 reagent, and a calibrator; wherein the R2 reagent comprises the monoclonal antibody according to any one of claims 1-2.

8. The IV collagen detection kit according to claim 7, characterized in that The R1 reagent is a Tris buffer, which comprises: 20-50 mM Tris pH 7.2-7.6, 30-300 mM NaCl, 0.1-2% v / v Tween20, 0.5-5% w / v BSA, 10-500 mM polyethylene glycol 6000, 20-25 mg / L blocking agent, 0.5-1.5% w / v preservative; the preservative comprises sodium azide; the blocking agent comprises HBR5; The R2 reagent is an antibody-sensitized particle suspension, which comprises: 0.1-0.8 g / l of antibody-sensitized particles, 20-50 mM MOPSO pH 7.2-7.6, 30-300 mM NaCl, and 0.1-4.0% w / v of BSA; the antibody-sensitized particles are liposome particles coated with monoclonal antibodies. The calibrator is a bovine serum matrix, including Proclin-300 Tween 200 2-2.2% v / v, and BSA 1-3% w / v.

9. The IV collagen detection kit according to claim 8, characterized in that The preparation method of the antibody-sensitized particles comprises the following steps: S1. Centrifuge the liposome particles, take the supernatant, resuspend in glycine buffer, and disperse by ultrasonication to obtain a resuspension; The diameter of the liposome particles is 100-250 nm; wherein the diameter of the liposome particles coated with monoclonal antibody IV Col Ab1 is 200-250 nm, and the diameter of the liposome particles coated with monoclonal antibody IV Col Ab2 is 100-120 nm; S2. Add NHS solution to the resuspension and mix thoroughly, then add EDC solution and mix thoroughly, stir, and centrifuge; S3, dissolving and suspending the precipitate obtained by centrifugation in step S2 with glycine buffer, sonicating, adding monoclonal antibody, stirring, and centrifuging; The monoclonal antibody comprises at least one of monoclonal antibodies IV Col Ab1 and IV Col Ab2; wherein the mass ratio of the monoclonal antibody IV Col Ab1 to the liposome particles is 0.2-0.4 mg:100 mg, and the mass ratio of the monoclonal antibody IV Col Ab2 to the liposome particles is 0.5-0.8 mg:100 mg; S4. Dissolve and suspend the precipitate obtained after centrifugation in step S3 with a glycine buffer, perform sonication, and centrifuge to obtain the antibody-sensitized particles.

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