Monoclonal antibody against candida albicans csa2 protein and application thereof

Abstract

The application discloses a kind of anti-candida albicans Csa2 protein monoclonal antibody and its application, belong to biotechnology field, the monoclonal antibody includes heavy chain constant region, heavy chain variable region, light chain constant region and light chain variable region, the heavy chain variable region and light chain variable region of the monoclonal antibody include CDR1, CDR2 and CDR3.The monoclonal antibody of the application has higher affinity and specificity, can specifically recognize Csa2 protein, increases the diversity of anti-Csa2 antibody, and lays a solid foundation for developing high-sensitivity detection Csa2 kit.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a monoclonal antibody against Candida albicans Csa2 protein and application thereof. BACKGROUND

[0002] Candida albicans is the most common opportunistic pathogenic fungus of the genus Candida, and has two existence forms of mycelium and yeast. The yeast form exists in the human body and is not pathogenic, which is called colonization. When the human body immunity decreases or the flora is disordered, the yeast form is transformed into the mycelium form, causing superficial and deep tissue and organ infections.

[0003] Distinguishing the colonization and infection of Candida albicans can prevent the abuse of antibacterial drugs and help clinicians take timely antifungal treatment. Csa2 is a member of the common in fungal extracellular membranes (CFEM) protein superfamily and a member of the Rbt5 protein family of Candida albicans, is encoded by the Csa2 gene, consists of 147 amino acids, and has a molecular weight of 15.1 Kd. It mainly participates in the uptake of iron ions in human hemoglobin and hematin protein. It only exists in the supernatant of Candida albicans mycelium culture, and is a new index for diagnosing Candida albicans infection. Therefore, it is of great significance to develop a Csa2 antibody with high sensitivity and specificity. SUMMARY

[0004] The application provides a monoclonal antibody against Candida albicans Csa2 protein and application thereof. Compared with the antibody screened by the traditional hybridoma technology and the rabbit polyclonal antibody, the mouse monoclonal antibody screened by the application has higher affinity and specificity, and the diversity of the anti-Csa2 antibody is increased, which lays a solid foundation for developing a high-sensitivity Csa2 diagnostic kit. The application is realized through the following technologies.

[0005] In a first aspect, the application provides a monoclonal antibody against Candida albicans Csa2 protein, 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 comprises CDR1 with the amino acid sequence shown in SEQ. No. 1, CDR2 with the amino acid sequence shown in SEQ. No. 2, and CDR3 with the amino acid sequence shown in SEQ. No. 3, and the light chain variable region of the first monoclonal antibody comprises CDR1 with the amino acid sequence shown in SEQ. No. 4, CDR2 with the amino acid sequence WAS, and CDR3 with the amino acid sequence shown in SEQ. No. 5.

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

[0008] Further, 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] Further, 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] Further, the nucleotide sequence encoding the heavy chain of the first monoclonal antibody is shown in SEQ. No. 19, 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] In a second aspect of the present application, a biological material is provided, comprising:

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

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

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

[0015] In a third aspect of the present application, the use of the monoclonal antibody against Csa2 protein of Candida albicans described above or the biological material described above in the preparation of a detection product of Csa2 protein of Candida albicans or Candida albicans is provided.

[0016] In a fourth aspect, the present application provides a detection product of C. albicans Csa2 protein or C. albicans, comprising the monoclonal antibody against C. albicans Csa2 protein or the biological material.

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

[0018] Further, the detection kit is a double-antibody sandwich ELISA kit or a colloidal gold kit, and the detection kit comprises the monoclonal antibody against C. albicans Csa2 protein.

[0019] Further, 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] Further, the first monoclonal antibody and the second monoclonal antibody can be used together or separately.

[0021] The present application has the following beneficial effects:

[0022] The present application is directed to C. albicans Csa2 protein, and a monoclonal antibody against C. albicans Csa2 protein is obtained, which has higher specificity and affinity, increases the diversity of anti-Csa2 antibodies, and lays a solid foundation for developing a high-sensitivity detection kit for Csa2. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure 4 is a graph showing the results of identification of the affinity of monoclonal antibodies 2H8 and 6C7 using the ELISA method.

[0024] Figure 2 Figure 5 is a graph showing the results of identification of the affinity of monoclonal antibody 2H8 using the Biacore method.

[0025] Figure 3 Figure 6 is a graph showing the results of identification of the affinity of monoclonal antibody 6C7 using the Biacore method.

[0026] Figure 4 Figure 7 is a graph showing the results of double-antibody sandwich ELISA.

[0027] Figure 5 Figure 8 is the results of the thermal stability experiment of the first antibody.

[0028] Figure 6 Figure 9 is the results of the thermal stability experiment of the second antibody. DETAILED DESCRIPTION

[0029] For further illustrating the technical means adopted by the present application and its effects, the present application is further explained below in conjunction with examples and drawings. It can be understood that the specific implementation described herein is only for the purpose of explaining the present application, but not limiting the present application.

[0030] The specific techniques or conditions not specified in the examples are carried out according to the techniques or conditions described in the literature in the art, or according to the product instructions. The reagents or instruments not specified by the manufacturer are all conventional products that can be commercially available through regular channels.

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

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

[0033] 1. Animal immunization

[0034] In order to obtain mouse monoclonal antibody recognizing Candida albicans Csa2 protein, the company used self-produced recombinant Candida albicans Csa2 protein (Q5A0X8, 34 Asn-147Asn, Pichia pastoris expression) as immunogen to immunize 5 mice; the first dose of each mouse was 50 ug, mixed with equal amount of water adjuvant, 2 points on the back, 1 point in the abdominal cavity, each injection 1 / 3 volume, avoiding injection to the left spleen of the mouse. Then, 2-4 immunizations were carried out 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 mouse with high serum titer was selected for spleen removal.

[0035] 2. Preparation of spleen cells

[0036] (1) Prepare 20 mL sterile PBS buffer in advance into a 50 mL sterile centrifuge tube for standby;

[0037] (2) Sterilely take the spleens of 5 mice, place them on a sterilized metal mesh screen, and place the metal mesh screen on a sterilized beaker, and remove the connective tissue around the spleen;

[0038] (3) According to the size of the spleen, cut several incisions around the spleen with sterile surgical scissors (do not cut the spleen);

[0039] (4) Hold a 1 ml pipette (take the prepared sterile PBS from the 50 mL centrifuge tube) in one hand, and hold a sterile surgical scissors in the other hand, grind the spleen on the sterilized metal mesh screen while slowly adding PBS buffer;

[0040] (5) After the spleen is ground, remove the cell screen, 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, use a pipette to suck out the tissue blocks in the resuspension, centrifuge at 1000 rpm for 5 min;

[0042] (7) Repeat step (6) 1-2 times until the cell suspension is clear of tissue blocks, centrifuge at 1000 rpm for 5 min;

[0043] (8) After centrifugation, resuspend with 20 mL of complete medium for standby, and dilute with an equal volume of PBS, and divide into 2 50 mL centrifuge tubes.

[0044] 3. PBMC cell separation and screening

[0045] (1) Pre-equilibrate the lymphocyte separation medium at room temperature;

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

[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 4 layers: diluent layer, PBMC layer (ring-shaped milky white lymphocytes), transparent separation medium layer, and red blood cell layer. Gently suck the middle white membrane layer and place it in 2 new 50 mL centrifuge tubes, add 2-3 times the volume of PBS in the new centrifuge tubes, mix well by inverting, 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 sorting.

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

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

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

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

[0054] 5. ELISA screening of positive clones

[0055] (1) Plate 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, 37°C, 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, 37°C, 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] The B cells corresponding to the ELISA positive antibodies were collected, and RNA was extracted and reversely transcribed into cDNA by using a conventional method.

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

[0068] Table 1 Heavy chain variable region gene primer sequences

[0069]

[0070] The light chain variable region gene amplification primer sequences are shown in Table 2, and specifically are as follows:

[0071] Table 2 Light chain variable region gene primer sequences

[0072]

[0073] The cDNA was used as a template to amplify the heavy and light chain variable region genes, and the PCR reaction system is shown in Table 3.

[0074] Table 3 PCR amplification system of heavy / light chain variable region

[0075]

[0076] The amplification reaction program was as follows: 95℃ for 3 min, 25 cycles of 95℃ for 1 min, 58℃ for 30 s, 72℃ for 1 min, and finally 72℃ for 5 min. The PCR product was recovered and purified.

[0077] The heavy and light chain variable region genes of the mouse monoclonal antibody were linked to a T carrier for transformation, plating, and finally colony PCR was performed to verify positive clones. The positive clones were subjected to gene sequencing, and the heavy and light chain variable region gene sequences of the mouse monoclonal antibody were obtained.

[0078] The heavy chain variable region gene of the mouse monoclonal antibody with correct sequencing was subcloned into PATX1-Mouse H (developed by Pu Jian Biotechnology (Wuhan) Co., Ltd.), and the light chain variable region gene was subcloned into PATX1-Mouse L (developed by Pu Jian Biotechnology (Wuhan) Co., Ltd.). The two plasmids were co-transfected into Xten CHO cells. After 72 h of transfection, the cells were removed by centrifugation, and the culture supernatant was purified by Protein G resin.

[0079] The finally screened monoclonal antibodies against Candida albicans Csa2 protein were named 2H8 and 6C7.

[0080] The amino acid sequence of CDR1 of the heavy chain variable region of the 2H8 monoclonal antibody against Candida albicans Csa2 protein obtained in the example is GYTFTMYP, as shown in SEQ. No. 1.

[0081] The amino acid sequence of CDR2 of the heavy chain variable region of 2H8 is FHPYNDDT, as shown in SEQ. No. 2.

[0082] The amino acid sequence of CDR3 of the heavy chain variable region of 2H8 is ARWGLYVMDY, as shown in SEQ. No. 3.

[0083] The amino acid sequence of CDR1 of the light chain variable region of 2H8 is QSLLDSSNQQNF, as shown in SEQ. No. 4.

[0084] The amino acid sequence of CDR2 of the light chain variable region of 2H8 is WAS.

[0085] The amino acid sequence of CDR3 of the light chain variable region of 2H8 is HQYYRYLT, as shown in SEQ. No. 5.

[0086] The amino acid sequence of the heavy chain variable region of 2H8 is:

[0087] MKHLWFFLLLVAAPRWVLSQVQLQQSGAELVKPGASVKMSCKAFGYTFTMYPIEWMKQNHGKSLEWIGNFHPYNDDTRYNEKFKGKAKLTVEKSSSTVYLELSRLTSDDSAVYYCARWGLYVMDYWGQGTSVTVSS, as shown in SEQ. No. 11

[0088] The amino acid sequence of the light chain variable region of 2H8 is:

[0089] MVLQTQVFISLLLWISGAYGDIVMSQSPSSLSVSVGEKVTMTCKSSQSLLDSSNQQNFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVEAEDLAVYYCHQYYRYLTFGAGTKLELK, as shown in SEQ. No. 12.

[0090] The amino acid sequence of the heavy chain of 2H8 is:

[0091] MKHLWFFLLLVAAPRWVLSQVQLQQSGAELVKPGASVKMSCKAFGYTFTMYPIEWMKQNHGKSLEWIGNFHPYNDDTRYNEKFKGKAKLTVEKSSSTVYLELSRLTSDDSAVYYCARWGLYVMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK, as set forth in SEQ. No. 15.

[0092] The amino acid sequence of the light chain of 2H8 is:

[0093] MVLQTQVFISLLLWISGAYGDIVMSQSPSSLSVSVGEKVTMTCKSSQSLLDSSNQQNFLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVEAEDLAVYYCHQYYRYLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC, as set forth in SEQ. No. 16.

[0094] The amino acid sequence of CDRl of the heavy chain variable region of 6C7 is: GYTFTTFG, as set forth in SEQ. No. 6.

[0095] The amino acid sequence of CDR2 of the heavy chain variable region of 6C7 is: INMFSGQP, as set forth in SEQ. No. 7.

[0096] The amino acid sequence of CDR3 of the heavy chain variable region of 6C7 is ARSMGGTMDY, as shown in SEQ. No. 8.

[0097] The amino acid sequence of CDR1 of the light chain variable region of 6C7 is QSLLNSRTRKNY, as shown in SEQ. No. 9.

[0098] The amino acid sequence of CDR2 of the light chain variable region of 6C7 is WAS.

[0099] The amino acid sequence of CDR3 of the light chain variable region of 6C7 is KQSYTLFT, as shown in SEQ. No. 10.

[0100] The amino acid sequence of the heavy chain variable region of 6C7 is:

[0101] MKHLWFFLLLVAAPRWVLSQIQLVQSGPELKKPGETVNISCKASGYTFTTFGMNWMKQAPGKTLKWMGWINMFSGQPKYADDFKGRFDFSLETSASTAYLQISNLKNEDMATYFCARSMGGTMDYWGQGTSVTVSS, as shown in SEQ. No. 13.

[0102] The amino acid sequence of the light chain variable region of 6C7 is:

[0103] MVLQTQVFISLLLWISGAYGDIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYTLFTFGSGTKLEIR, as shown in SEQ. No. 14.

[0104] The amino acid sequence of the heavy chain of 6C7 is:

[0105] MKHLWFFLLLVAAPRWVLSQIQLVQSGPELKKPGETVNISCKASGYTFTTFGMNWMKQAPGKTLKWMGWINMFSGQPKYADDFKGRFDFSLETSASTAYLQISNLKNEDMATYFCARSMGGTMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK, as set forth in SEQ. No. 17.

[0106] 6 The amino acid sequence of the light chain of 6C7 is:

[0107] MVLQTQVFISLLLWISGAYGDIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYTLFTFGSGTKLEIRRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC, as set forth in SEQ. No. 18.

[0108] Example 2: Titration of recombinantly expressed antibodies

[0109] Titration of recombinantly expressed antibodies by ELISA

[0110] ELISA method same as step 4 of example 1, antibody detection range: 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, PBS as Blank control.

[0111] The titer detection results of the ELISA method are shown in Table 4 and Table 5. Figure 1 and Table 4,

[0112] From Figure 1 and the results of Table 4, 2H8 has an EC50 of 29.61 ng / mL, and 6C7 has an EC50 of 11.03 ng / mL.

[0113] Table 4 ELISA detection results

[0114]

[0115] Example 3: Affinity identification of recombinantly expressed antibodies

[0116] The method for identifying the affinity of recombinantly expressed antibodies by Biacore is as follows:

[0117] (1) Start the Biacore T200 instrument according to the standard operation.

[0118] (2) Open the Biacore T200 control software and install the CM5 chip according to the standard process.

[0119] (3) Prepare to start the formal experiment, Start running, and the buffer will flush the entire internal flow path system at a high flow rate, and Standby is ready.

[0120] (4) Select the appropriate test tube module according to the sample amount.

[0121] (5) Start capturing the chip, and the coupling buffer is HEPES-ET. According to the sample rack position table, prepare enough volume of sample, EDC / NHS, blocking buffer. Csa2 is placed on the test tube rack according to the software requirements. Cover the test tube rack cover, and send the sample rack back to the sample cabin. The system automatically runs the coupling program.

[0122] (6) After the coupling is completed, sample detection is started, and the ligand Csa2 is set according to the standard program, the contact time is 30 s, the flow rate is 10 μL / min, the analyte Ab is 120 s, the flow rate is 30 μL / min, the dissociation time is 300 s, the regeneration condition is 3 M magnesium chloride, and the regeneration time is 30 s.

[0123] (7) The corresponding sample to be detected is prepared according to the requirements, and is placed in the sample cabin according to the position requirements, and automatic operation program detection is started.

[0124] (8) Result analysis: according to the running results, data fitting analysis is performed to obtain the final affinity fitting KD value.

[0125] The test results are shown in Figure 2 , 3 and Table 5. Finally, through curve fitting and calculation, the affinity of antibody 2H8 is 4.71E-11M, and the affinity of antibody 6C7 is 8.96E-11M.

[0126] Table 5 Biacore method for identifying the affinity of recombinant expressed antibodies

[0127]

[0128] Example 4: Establishment of double antibody sandwich ELISA method

[0129] 1. Detection limit and sensitivity experiment

[0130] After many experiments, the optimal dosage of antibody 2H8 as the capture antibody and antibody 6C7 as the detection antibody was determined, and the antibody detection range was determined to be 31 pg / mL-250 pg / mL. The double antibody sandwich Elisa method was established, and the method is as follows:

[0131] (1) Coating antibody 2H8, 0.2 μg / mL in CBS, 100 μL / well, 4°C overnight.

[0132] (2) Blocking, 3% BSA-PBST, 300 μL / well, 37°C, 90 min.

[0133] (3) Washing the plate, PBST, 300 μL / well, 3 times.

[0134] (4) Antigen Csa2, 400 pg / mL-6.25 pg / ml in PBS, 100 μL / well, 37°C, 1 h.

[0135] (5) Wash plate, PBST, 300 μL / well, 3 times.

[0136] (6) Biotin-labeled antibody 6C7-biotin, 0.1 μg / mL in PBS, 100 μL / well, 37°C, 1 h.

[0137] (7) Wash plate, PBST, 300 μL / well, 3 times.

[0138] (8) Secondary antibody, Streptavidin-HRP, 1:10000 in PBS, 100 μL / well, 37°C, 30 min.

[0139] (9) Wash plate, PBST, 300 μL / well, 3 times.

[0140] (10) TMB color development, 100 μL / well, 37°C, 10 min.

[0141] (11) Stop: 2M HCl, 50 μL / well.

[0142] (12) Read: OD 450 -OD 630 .

[0143] The lowest Csa2 protein concentration with an average absorbance value greater than three times the average absorbance value of the blank control is the sensitivity of the double antibody sandwich ELISA method. The experimental results show that the double antibody sandwich ELISA method based on the first antibody and the second antibody of the anti-Csa2 protein mouse monoclonal antibody has a Csa2 protein detection range of 31 pg / mL-250 pg / mL, and the detection sensitivity reaches 31 pg / mL.

[0144] 2. Heat stability test

[0145] 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 the double antibody sandwich ELISA method, and the untreated first antibody and second antibody (i.e., the first antibody 2H8 and the second antibody 6C7-biotin were stored at -20°C environment all the time) were used as blank controls. The standard curves were established for the antibody samples treated at 37°C for 14 days and the blank controls.

[0146] As Figure 5、 6 As shown in Table 1, the first antibody 2H8 and the second antibody 6C7-biotin provided by the present application are observed without obvious precipitation or deterioration, without obvious decrease in concentration, and without obvious decrease in signal of enzyme-linked immunoassay by double-antibody sandwich method after treatment at 37℃ for 14 days, and the result shows that the signal reduction is less than 5%. This indicates that the first antibody 2H8 and the second antibody 6C7-biotin provided by the present application have strong thermal stability.

[0147] Although the embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and alterations can be made thereto without departing from the principles and spirit of the present application, the scope of which 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 includes a first monoclonal antibody or a second monoclonal antibody; The heavy chain variable region of the first monoclonal antibody includes CDR1 with an amino acid sequence as shown in SEQ.NO.1, CDR2 with an amino acid sequence as shown in SEQ.NO.2, and CDR3 with an amino acid sequence as shown in SEQ.NO.3; the light chain variable region of the first monoclonal antibody includes CDR1 with an amino acid sequence as shown in SEQ.NO.4, CDR2 with an amino acid sequence of WAS, and CDR3 with an amino acid sequence as shown in SEQ.NO.

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

10.

2. The 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 1-4; (2) A carrier comprising the polynucleotide described in (1); (3) A host cell containing the polynucleotide described in (1) or the vector described in (2).

6. The monoclonal antibody against Candida albicans Csa2 protein as described in any one of claims 1-4, or the biomaterial as described in claim 5, in the preparation of a detection product for Candida albicans Csa2 protein.

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

8. The detection product for Candida albicans Csa2 protein according to claim 7, characterized in that, The testing product is a test kit.

9. The testing 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 includes a monoclonal antibody against Candida albicans Csa2 protein as described in any one of claims 1-4.

10. The testing product according to claim 9, characterized in that, The double-antibody sandwich ELISA kit includes 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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