Monoclonal antibody for resisting Candida albicans Csa2 protein and application thereof

Abstract

The invention discloses a monoclonal antibody for resisting Candida albicans Csa2 protein and application of the monoclonal antibody, and belongs to the technical field of biology, the monoclonal antibody comprises a heavy chain constant region, a heavy chain variable region, a light chain constant region and a light chain variable region, and the heavy chain variable region and the light chain variable region of the monoclonal antibody comprise CDR1, CDR2 and CDR3. The monoclonal antibody provided by the invention has higher affinity and specificity, can specifically recognize Csa2 protein, increases the diversity of anti-Csa2 antibodies, and lays a solid foundation for developing a high-sensitivity kit for detecting Csa2.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a monoclonal antibody against Candida albicans Csa2 protein and application thereof. Background Art

[0002] Candida albicans is the most common conditionally pathogenic fungus of the genus Candida, and exists in two forms: hyphae and yeast. The presence of yeast in the human body without causing disease is called colonization. When the human immune system is weakened or the flora is imbalanced, it changes from yeast to hyphae, causing superficial and deep tissue and organ infections.

[0003] Differentiating between colonization and infection of Candida albicans can not only prevent the abuse of antimicrobial drugs, but also help clinicians take timely antifungal treatment. Csa2 is a member of the common in fungal extracellu-larmembranes (CFEM) protein superfamily and a member of the Candida albicans Rbt5 protein family. It is encoded by the Csa2 gene, consists of 147 amino acids, and has a molecular weight of 15.1 Kd. It is mainly involved in the uptake of iron ions from human hemoglobin and heme proteins by Candida albicans. It only exists in the supernatant of Candida albicans hyphae culture and is a new indicator for diagnosing Candida albicans infection. Therefore, it is of great significance to develop highly sensitive and specific Csa2 antibodies. Summary of the invention

[0004] The present invention provides a monoclonal antibody against Candida albicans Csa2 protein and its application. The mouse monoclonal antibody screened by the present invention has higher affinity and specificity than the antibody screened by traditional hybridoma technology and rabbit polyclonal antibody, and increases the diversity of anti-Csa2 antibodies, laying a solid foundation for the development of a high-sensitivity Csa2 diagnostic kit, which is specifically achieved through the following technologies.

[0005] In a first aspect of the present invention, a monoclonal antibody against Candida albicans Csa2 protein is provided, wherein the monoclonal antibody comprises a first monoclonal antibody or a second monoclonal antibody;

[0006] The heavy chain variable region of the first monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ.NO.1, a CDR2 with an amino acid sequence as shown in SEQ.NO.2, and a CDR3 with an amino acid sequence as shown in SEQ.NO.3, and the light chain variable region of the first monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ.NO.4, a CDR2 with an amino acid sequence of WAS, and a CDR3 with an amino acid sequence as shown in SEQ.NO.5;

[0007] The heavy chain variable region of the second monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ.NO.6, a CDR2 with an amino acid sequence as shown in SEQ.NO.7, and a CDR3 with an amino acid sequence as shown in SEQ.NO.8; the light chain variable region of the second monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ.NO.9, a CDR2 with an amino acid sequence of WAS, and a CDR3 with an amino acid sequence as shown in SEQ.NO.10.

[0008] Furthermore, the amino acid sequence of the heavy chain variable region of the first monoclonal antibody is shown in SEQ.NO.11, and the amino acid sequence of the light chain variable region of the first monoclonal antibody is shown in SEQ.NO.12; the amino acid sequence of the heavy chain variable region of the second monoclonal antibody is shown in SEQ.NO.13, and the amino acid sequence of the light chain variable region of the second monoclonal antibody is shown in SEQ.NO.14.

[0009] Furthermore, the amino acid sequence of the heavy chain of the first monoclonal antibody is shown in SEQ.NO.15, the amino acid sequence of the light chain of the first monoclonal antibody is shown in SEQ.NO.16, the amino acid sequence of the heavy chain of the second monoclonal antibody is shown in SEQ.NO.17, and the amino acid sequence of the light chain of the second monoclonal antibody is shown in SEQ.NO.18.

[0010] Furthermore, the nucleotide sequence encoding the heavy chain of the first monoclonal antibody is shown in SEQ.NO.19, and the nucleotide sequence encoding the light chain of the first monoclonal antibody is shown in SEQ.NO.20; the nucleotide sequence encoding the heavy chain of the second monoclonal antibody is shown in SEQ.NO.21, and the nucleotide sequence encoding the light chain of the second monoclonal antibody is shown in SEQ.NO.22.

[0011] A second aspect of the present invention provides a biomaterial comprising:

[0012] (1) a polynucleotide encoding the above-mentioned monoclonal antibody;

[0013] (2) a vector comprising the polynucleotide;

[0014] (3) A host cell comprising the polynucleotide or the vector.

[0015] The third aspect of the present invention provides a use of the above-mentioned monoclonal antibody against Candida albicans Csa2 protein or the above-mentioned biological material in the preparation of a detection product for Candida albicans Csa2 protein or Candida albicans.

[0016] In a fourth aspect, the present invention provides a detection product of Candida albicans Csa2 protein or Candida albicans, comprising the above-mentioned monoclonal antibody against Candida albicans Csa2 protein or the above-mentioned biological material.

[0017] Furthermore, the detection product is a detection reagent, a detection kit, a detection test strip, a detection probe or a detection chip.

[0018] Furthermore, the detection kit is a double antibody sandwich ELISA kit or a colloidal gold kit; the detection kit includes the above-mentioned monoclonal antibody against Candida albicans Csa2 protein.

[0019] Furthermore, the double antibody sandwich ELISA kit comprises a coating antibody and a detection antibody, the coating antibody is the first monoclonal antibody, and the detection antibody is the second monoclonal antibody.

[0020] Furthermore, the first monoclonal antibody and the second monoclonal antibody can be used together or separately.

[0021] The beneficial effects of the present invention are:

[0022] The present invention targets the Candida albicans Csa2 protein, studies and obtains a monoclonal antibody against the Candida albicans Csa2 protein, which has higher specificity and affinity, increases the diversity of anti-Csa2 antibodies, and lays a solid foundation for the development of a highly sensitive kit for detecting Csa2. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the result of affinity identification of monoclonal antibodies 2H8 and 6C7 using ELISA method.

[0024] Figure 2 This is the result of affinity identification of monoclonal antibody 2H8 using Biacore method.

[0025] Figure 3 This is the result of affinity identification of monoclonal antibody 6C7 using Biacore method.

[0026] Figure 4 This is the result diagram of double antibody sandwich Elisa.

[0027] Figure 5 The thermal stability test results of the first antibody.

[0028] Figure 6 The thermal stability test results of the second antibody. DETAILED DESCRIPTION

[0029] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0030] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0031] Example 1: Screening, separation and purification of monoclonal antibodies against Candida albicans Csa2 protein

[0032] This example is about the screening, separation and purification of monoclonal antibodies against Candida albicans Csa2 protein, and the steps include:

[0033] 1. Animal immunization

[0034] In order to obtain mouse monoclonal antibodies that recognize the Csa2 protein of Candida albicans, our company used the self-produced recombinant Csa2 protein of Candida albicans (Q5A0X8, 34 Asn-147Asn, expressed in Pichia pastoris) as the immunogen to immunize 5 mice; the first dose for each mouse was 50ug, mixed with an equal amount of water adjuvant, injected at 2 points on the back and 1 point in the abdominal cavity, and 1 / 3 volume was injected each time to avoid injecting into the left spleen of the mouse. Then, 2-4 immunizations were performed at intervals of 1 week, and the immunization antigen dose and treatment method were the same as the first immunization. After the fourth immunization, the serum titer was determined by ELISA method, and the spleen of the mouse with high serum titer was taken.

[0035] 2. Preparation of splenocytes

[0036] (1) Prepare 20 mL of sterile PBS buffer in a 50 mL sterile centrifuge tube for later use;

[0037] (2) Aseptically remove spleens from 5 mice and place them on a sterilized metal mesh sieve placed on a sterilized beaker to remove the connective tissue around the spleens.

[0038] (3) Depending on the size of the spleen, use sterile surgical scissors to make several incisions around the spleen (do not cut the spleen off);

[0039] (4) Hold a 1 ml pipette in one hand (take the prepared sterile PBS from a 50 mL centrifuge tube) and a pair of sterile surgical scissors in the other hand, and slowly add PBS buffer while grinding on a sterile metal mesh;

[0040] (5) After the spleen is ground, remove the cell screen and pour the cell suspension in the beaker into the centrifuge tube containing the remaining PBS. Centrifuge at 1000 rpm for 5 min.

[0041] (6) After centrifugation, discard the supernatant, resuspend the cell pellet with sterile PBS, remove the tissue pieces in the resuspension with a pipette, and centrifuge at 1000 rpm for 5 min.

[0042] (7) Repeat step (6) 1-2 times until the cell suspension is clear and free of tissue clumps. Centrifuge at 1000 rpm for 5 min.

[0043] (8) After centrifugation, add 20 mL of complete culture medium to resuspend the sample for later use, dilute with an equal volume of PBS, and divide equally into two 50 mL centrifuge tubes.

[0044] 3. PBMC cell isolation and screening

[0045] (1) Pre-equilibrate the lymph separation fluid at room temperature;

[0046] (2) Take 20 mL of separation solution into a new 50 mL centrifuge tube, and slowly add 2 times the volume of diluted spleen cell suspension along the tube wall on the upper layer of the separation solution, so that it is spread evenly on the upper layer of the separation solution;

[0047] (3) Centrifuge at 700-800 g for 20-30 min at room temperature;

[0048] (4) After centrifugation, the liquid in the centrifuge tube is divided into four layers: diluent layer, PBMC layer (ring-shaped milky white lymphocytes), transparent separation liquid layer, and red blood cell layer. Gently aspirate the middle white film layer and place it in two new 50 mL centrifuge tubes. Add 2-3 times the volume of PBS to the new centrifuge tubes, mix them upside down, centrifuge at 250 g for 10 min at room temperature, and discard the supernatant.

[0049] (5) Wash 1-2 times with PBS, count, and use for flow cytometry.

[0050] 4. Single B cell flow cytometry sorting and culture

[0051] (1) Resuspend PBMC in PBS and adjust the cell concentration to 1×10 7 cells / mL, add flow cytometry antibodies to stain B cells according to the volume of resuspended cells, add Goat Anti-mouse CD19-FITC, Goat Anti-mouse IgM APC and Csa2-specific-IF405 at a ratio of 1:100, pay attention to setting up blank control and single staining control, incubate at room temperature in the dark for 30 minutes; wash twice with PBS, centrifuge at 300 g for 5 min, and 4°C.

[0052] (2) Resuspend the cells in PBS and filter them with a 100-mesh filter before loading. 5 min before loading, add 2% PI for staining to remove dead cells. Use the control to adjust the voltage and set the fluorescence compensation. Use FSC-A / SSC-A to select lymphocytes and use FSC-W / FSC-A to remove adherent cells. Select CD19-positive, IgM-negative, antigen-positive, and PI-negative B cells.

[0053] (3) By flow cytometry, add one B cell to each well of a 96-well plate containing 200 μL of complete 1640 medium (containing 10% FBS, 1% penicillin-streptomycin, 1% glutamine, 0.2 ng / mL IL-1β, and 0.2 ng / mL TNFα). Centrifuge the 96-well plate at 300 g for 5 min and culture at 37°C in 5% CO2 for 7 days.

[0054] 5. ELISA screening of positive clones

[0055] (1) Coating: Csa2 antigen, 5 μg / mL, 100 μL / well, 4°C overnight.

[0056] (2) Blocking: 3% BSA-PBS, 300 μL / well, 37°C, 1.5 h.

[0057] (3) Washing: PBST, 300 μL / well, 2 times.

[0058] (4) Sample addition: Take 80 μL of 96-well plate culture supernatant and incubate at 37°C for 1 h

[0059] (5) Washing: PBST, 300 μL / well, 3 times.

[0060] (6) Add Goat-anti Mouse IgG secondary antibody: 100 μL / well, 37°C, 30 min.

[0061] (7) Washing: PBST, 300 μL / well, 3 times.

[0062] (8) Add TMB Substrate Solution, 100 μL / well, incubate at 37°C for 5-10 min.

[0063] (9) Termination: 2M HCl, 50 μL / well.

[0064] (10) Reading: OD 450 -OD 630 .

[0065] 6. Sequencing and expression of specific mouse monoclonal antibodies

[0066] B cells corresponding to ELISA-positive antibodies were collected, and RNA was extracted and reverse transcribed into cDNA using conventional methods.

[0067] The heavy chain variable region gene primer sequences are shown in Table 1, specifically:

[0068] Table 1 Primer sequences for heavy chain variable region genes

[0069] The light chain variable region gene amplification primer sequences are shown in Table 2, specifically:

[0070] Table 2 Primer sequences for light chain variable region genes

[0071] The heavy and light chain variable region genes were amplified using cDNA as a template, and the PCR reaction system is shown in Table 3 below.

[0072] Table 3 PCR amplification system for heavy chain / light chain variable regions

[0073] The amplification reaction program was: 95°C for 3 min, (95°C for 1 min, 58°C for 30 s, 72°C for 1 min) for a total of 25 cycles, and finally 72°C for 5 min, and the PCR product was recovered and purified.

[0074] The heavy and light chain variable region genes of the mouse monoclonal antibody are connected to the T vector for transformation, plated, and finally colony PCR is performed to verify the positive clones. The positive clones are sequenced to obtain the heavy and light chain variable region gene sequences of the mouse monoclonal antibody.

[0075] The heavy chain variable region gene of the correctly sequenced mouse monoclonal antibody was subcloned into PATX1-Mouse H (independently developed by Pujian Biotechnology (Wuhan) Technology Co., Ltd.), and the light chain variable region gene was subcloned into PATX1-Mouse L (independently developed by Pujian Biotechnology (Wuhan) Technology Co., Ltd.). The two plasmids were co-transfected into Xten CHO cells. 72 hours after transfection, the cells were removed by centrifugation and the culture supernatant was purified with Protein G resin.

[0076] The monoclonal antibodies against Candida albicans Csa2 protein were finally screened out and named 2H8 and 6C7.

[0077] The amino acid sequence of CDR1 in the heavy chain variable region of 2H8, the monoclonal antibody against Candida albicans Csa2 protein obtained in this example, is: GYTFTMYP, as shown in SEQ.NO.1.

[0078] The amino acid sequence of CDR2 of the heavy chain variable region of 2H8 is: FHPYNDDT, as shown in SEQ.NO.2.

[0079] The amino acid sequence of CDR3 of the heavy chain variable region of 2H8 is: ARWGLYVMDY, as shown in SEQ.NO.3.

[0080] The amino acid sequence of CDR1 of the light chain variable region of 2H8 is: QSLLDSSNQQNF, as shown in SEQ.NO.4.

[0081] The amino acid sequence of CDR2 of the light chain variable region of 2H8 is: WAS.

[0082] The amino acid sequence of CDR3 of the light chain variable region of 2H8 is: HQYYRYLT, as shown in SEQ.NO.5.

[0083] The amino acid sequence of the heavy chain variable region of 2H8 is:

[0084] MKHLWFFLLLVAAPRWVLSQVQLQQSGAELVKPGASVKMSCKAFGYTFTMYPIEWMKQNHGKSLEWIGNFHPYNDDTRYNEKFKGKAKLTVEKSSSTVYLELSRLTSDDSAVYYCARWGLYVMDYWGQGTSVTVSS, as shown in SEQ.NO.11

[0085] The amino acid sequence of the light chain variable region of 2H8 is:

[0086] MVLQTQVFISLLLWISGAYGDIVMSQSPSSLSVSVGEKVTMTCKSSQSLLDSSNQQNFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVEAEDLAVYYCHQYYRYLTFGAGTKLELK, as shown in SEQ.NO.12.

[0087] The amino acid sequence of the heavy chain of 2H8 is:

[0088] MKHLWFFLLLVAAPRWVLSQVQLQQSGAELVKPGASVKMSCKAFGYTFTMYPIEWMKQNHGKSLEWIGNFHPYNDDTRYNEKFKGKAKLTVEKSSSTVYLELSRLTSDDSAVYYCARWGL YVMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTI KPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKG SVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK, as shown in SEQ.NO.15.

[0089] The amino acid sequence of the light chain of 2H8 is:

[0090] MVLQTQVFISLLLWISGAYGDIVMSQSPSSLSVSVGEKVTMTCKSSQSLLDSSNQQNFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVEAEDLAVYYCHQYYRYLTFGAG TKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC, as shown in SEQ.NO.16.

[0091] The amino acid sequence of CDR1 of the heavy chain variable region of 6C7 is: GYTFTTFG, as shown in SEQ.NO.6.

[0092] The amino acid sequence of CDR2 of the heavy chain variable region of 6C7 is: INMFSGQP, as shown in SEQ.NO.7.

[0093] The amino acid sequence of CDR3 of the heavy chain variable region of 6C7 is: ARSMGGTMDY, as shown in SEQ.NO.8.

[0094] The amino acid sequence of CDR1 of the light chain variable region of 6C7 is: QSLLNSRTRKNY, as shown in SEQ.NO.9.

[0095] The amino acid sequence of CDR2 of the light chain variable region of 6C7 is: WAS.

[0096] The amino acid sequence of CDR3 of the light chain variable region of 6C7 is: KQSYTLFT, as shown in SEQ.NO.10.

[0097] The amino acid sequence of the heavy chain variable region of 6C7 is:

[0098] MKHLWFFLLLVAAPRWVLSQIQLVQSGPELKKPGETVNISCKASGYTFTTFGMNWMKQAPGKTLKWMGWINMFSGQPKYADDFKGRFDFSLETSASTAYLQISNLKNEDMATYFCARSMGGTMDYWGQGTSVTVSS, as shown in SEQ.NO.13.

[0099] The amino acid sequence of the light chain variable region of 6C7 is:

[0100] MVLQTQVFISLLLWISGAYGDIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYTLFTFGSGTKLEIR, as shown in SEQ.NO.14.

[0101] The amino acid sequence of the heavy chain of 6C7 is:

[0102] MKHLWFFLLLVAAPRWVLSQIQLVQSGPELKKPGETVNISCKASGYTFTTFGMNWMKQAPGKTLKWMGWINMFSGQPKYADDFKGRFDFSLETSASTAYLQISNLKNEDMATYFCARSMG GTMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTI KPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKG SVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK, as shown in SEQ.NO.17.

[0103] The amino acid sequence of the light chain of 6C7 is:

[0104] MVLQTQVFISLLLWISGAYGDIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYTLFTFGSG TKLEIRRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC, as shown in SEQ.NO.18.

[0105] Example 2: Titer detection of recombinantly expressed antibodies

[0106] ELISA method to detect the titer of recombinant expressed antibodies

[0107] The ELISA method was the same as step 4 of Example 1. The antibody detection range was: 1.953125 ng / mL, 3.90625 ng / mL, 7.8125 ng / mL, 15.625 ng / mL, 31.25 ng / mL, 62.5 ng / mL, 125 ng / mL, 250 ng / mL, 500 ng / mL, 1000 ng / mL, and PBS was used as a Blank control.

[0108] The titer test results of ELISA method are as follows Figure 1 As shown in Table 4,

[0109] from Figure 1 From the results in Table 4, it can be seen that the EC50 of 2H8 is 29.61 ng / mL, and the EC50 of 6C7 is 11.03 ng / mL.

[0110] Table 4 Elisa test results

[0111] Example 3: Affinity identification of recombinantly expressed antibodies

[0112] The Biacore method for identifying the affinity of recombinantly expressed antibodies is as follows:

[0113] (1) Turn on the Biacore T200 instrument according to standard operating procedures.

[0114] (2) Open the Biacore T200 control software and install the CM5 chip according to the standard process.

[0115] (3) Prepare to start the formal experiment. Start the run. The buffer will flush the flow system inside the entire system at a high flow rate and prepare for standby.

[0116] (4) Select the appropriate test tube module based on the sample size.

[0117] (5) Start capturing the chip, and the coupling buffer is HEPES-ET. According to the sample rack position table, prepare sufficient volume of sample, EDC / NHS, and blocking buffer. Place Csa2 on the test tube rack according to the corresponding position required by the software. Cover the test tube rack lid and return the sample rack to the sample chamber. The system officially automatically runs the coupling program.

[0118] (6) After coupling was completed, sample detection was started. According to the standard procedure, the ligand Csa2 was set to a contact time of 30 s and a flow rate of 10 μL / min. The analyte Ab was set to a contact time of 120 s, a flow rate of 30 μL / min, a dissociation time of 300 s, and a regeneration condition of 3 M magnesium chloride with a regeneration time of 30 s.

[0119] (7) Prepare the corresponding samples to be tested as required, place them in the sample compartment according to the location requirements, and start the automatic running program for testing.

[0120] (8) Result analysis: Based on the running results, perform data fitting analysis to obtain the final affinity fitting KD value.

[0121] Test results such as Figure 2 , 3 As shown in Table 5, through curve fitting and calculation, the affinity of antibody 2H8 is 4.71E-11M, and the affinity of antibody 6C7 is 8.96E-11M.

[0122] Table 5 Affinity identification results of recombinant expressed antibodies by Biacore

[0123] Example 4: Establishment of double antibody sandwich ELISA method

[0124] 1. Detection limit and sensitivity experiment

[0125] Antibody 2H8 was used as the capture antibody and antibody 6C7 was used as the detection antibody. After multiple tests, the optimal dosage was determined. The antibody detection range was determined to be 31 pg / mL-250 pg / mL. A double antibody sandwich Elisa method was established. The method is as follows:

[0126] (1) Coating antibody 2H8, 0.2 μg / mL in CBS, 100 μL / well, 4℃ overnight.

[0127] (2) Blocking, 3% BSA-PBST, 300 μL / well, 37°C, 90 min.

[0128] (3) Wash the plate with PBST, 300 μL / well, 3 times.

[0129] (4) Antigen Csa2, 400 pg / mL-6.25 pg / ml in PBS, 100 μL / well, 37°C, 1 h.

[0130] (5) Wash the plate with PBST, 300 μL / well, 3 times.

[0131] (6) Biotin-labeled antibody 6C7-biotin, 0.1 μg / mL in PBS, 100 μL / well, 37°C, 1 h.

[0132] (7) Wash the plate with PBST, 300 μL / well, 3 times.

[0133] (8) Secondary antibody, Streptavidin-HRP, 1:10,000 in PBS, 100 μL / well, 37°C, 30 min.

[0134] (9) Wash the plate with PBST, 300 μL / well, 3 times.

[0135] (10) TMB color development, 100 μL / well, 37°C, 10 min.

[0136] (11) Termination: 2M HCl, 50 μL / well.

[0137] (12) Reading: OD 450 -OD 630 .

[0138] The sensitivity of the double antibody sandwich ELISA method was determined by taking the lowest Csa2 protein concentration at which the average absorbance value was greater than three times the average absorbance value of the blank control. The experimental results showed that the double antibody sandwich ELISA method established based on the first antibody and the second antibody of the anti-Csa2 protein mouse monoclonal antibody had a detection range of 31 pg / mL-250pg / mL for Csa2 protein, and the detection sensitivity reached 31pg / mL.

[0139] 2. Thermal stability test

[0140] 2H8 and 6C7-biotin were placed in a 37°C incubator and samples were taken on the 14th day. The Csa2 standard protein was detected by double antibody sandwich enzyme-linked immunosorbent assay, with untreated first antibody and second antibody (i.e., first antibody 2H8 and second antibody 6C7-biotin were kept in -20°C environment) as blank controls. Standard curves were established for antibody samples treated at 37°C for 14 days and blank controls.

[0141] like Figure 5 , 6As shown, the first antibody 2H8 and the second antibody 6C7-biotin of the anti-Csa2 protein mouse monoclonal antibody provided by the present invention were treated at 37°C for 14 days. The antibodies showed no obvious precipitation or deterioration, no obvious decrease in concentration, and no obvious decrease in the double antibody sandwich enzyme-linked immunosorbent assay signal. The results showed that the signal decrease was less than 5%. This shows that the first antibody 2H8 and the second antibody 6C7-biotin of the anti-Csa2 protein mouse monoclonal antibody provided by the present invention have strong thermal stability.

[0142] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A monoclonal antibody against Candida albicans Csa2 protein, characterized in that: The monoclonal antibody comprises a first monoclonal antibody or a second monoclonal antibody; The heavy chain variable region of the first monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ.NO.1, a CDR2 with an amino acid sequence as shown in SEQ.NO.2, and a CDR3 with an amino acid sequence as shown in SEQ.NO.3, and the light chain variable region of the first monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ.NO.4, a CDR2 with an amino acid sequence of WAS, and a CDR3 with an amino acid sequence as shown in SEQ.NO.5; The heavy chain variable region of the second monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ.NO.6, a CDR2 with an amino acid sequence as shown in SEQ.NO.7, and a CDR3 with an amino acid sequence as shown in SEQ.NO.8; the light chain variable region of the second monoclonal antibody includes a CDR1 with an amino acid sequence as shown in SEQ.NO.9, a CDR2 with an amino acid sequence of WAS, and a CDR3 with an amino acid sequence as shown in SEQ.NO.

10.

2. A monoclonal antibody against Candida albicans Csa2 protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the first monoclonal antibody is shown in SEQ.NO.11, and the amino acid sequence of the light chain variable region of the first monoclonal antibody is shown in SEQ.NO.12; the amino acid sequence of the heavy chain variable region of the second monoclonal antibody is shown in SEQ.NO.13, and the amino acid sequence of the light chain variable region of the second monoclonal antibody is shown in SEQ.NO.

14.

3. The monoclonal antibody against Candida albicans Csa2 protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain of the first monoclonal antibody is shown in SEQ.NO.15, the amino acid sequence of the light chain of the first monoclonal antibody is shown in SEQ.NO.16, the amino acid sequence of the heavy chain of the second monoclonal antibody is shown in SEQ.NO.17, and the amino acid sequence of the light chain of the second monoclonal antibody is shown in SEQ.NO.

18.

4. The monoclonal antibody against Candida albicans Csa2 protein according to claim 1, characterized in that: The nucleotide sequence encoding the heavy chain of the first monoclonal antibody is shown in SEQ.NO.19, and the nucleotide sequence encoding the light chain of the first monoclonal antibody is shown in SEQ.NO.20; the nucleotide sequence encoding the heavy chain of the second monoclonal antibody is shown in SEQ.NO.21, and the nucleotide sequence encoding the light chain of the second monoclonal antibody is shown in SEQ.NO.

22.

5. A biomaterial, characterized in that: include: (1) A polynucleotide encoding the monoclonal antibody according to any one of claims 1 to 4; (2) a vector comprising the polynucleotide; (3) A host cell comprising the polynucleotide or the vector.

6. Use of the monoclonal antibody against Candida albicans Csa2 protein according to any one of claims 1 to 4, or the biological material according to claim 5 in the preparation of a Candida albicans Csa2 protein or a Candida albicans detection product.

7. A detection product for Candida albicans Csa2 protein or Candida albicans, characterized in that: It comprises the monoclonal antibody against Candida albicans Csa2 protein as described in any one of claims 1 to 4, or the biological material as described in claim 5.

8. The Candida albicans Csa2 protein or the Candida albicans detection product according to claim 7, characterized in that: The detection product is a detection reagent, a detection kit, a detection test strip, a detection probe or a detection chip.

9. The detection product according to claim 8, characterized in that: The detection kit is a double antibody sandwich ELISA kit or a colloidal gold kit; the detection kit comprises the monoclonal antibody against Candida albicans Csa2 protein according to any one of claims 1 to 4.

10. The detection product according to claim 9, characterized in that: The double antibody sandwich ELISA kit comprises a coating antibody and a detection antibody, wherein the coating antibody is the first monoclonal antibody and the detection antibody is the second monoclonal antibody.

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