Anti-cryptococcus antigen-antibody pair and application thereof in chemiluminescence method kit

Abstract

The invention belongs to the technical field of biological medicine, and provides an anti-cryptococcus antigen-antibody pair and application thereof in a chemiluminescence method kit. The anti-cryptococcus antigen and antibody pair comprises an anti-cryptococcus capsular polysaccharide monoclonal antibody GMX-2H12 and an anti-cryptococcus capsular polysaccharide monoclonal antibody GMX-4B09; the anti-cryptococcus capsular polysaccharide monoclonal antibody GMX-2H12 has a light chain variable region of which the amino acid sequence is as shown in SEQ ID NO.1 and a heavy chain variable region of which the amino acid sequence is as shown in SEQ ID NO.2; the anti-cryptococcus capsular polysaccharide monoclonal antibody GMX-4B09 has a light chain variable region of which the amino acid sequence is as shown in SEQ ID NO.3 and a heavy chain variable region of which the amino acid sequence is as shown in SEQ ID NO.4. According to the present invention, the anti-cryptococcus capsular polysaccharide monoclonal antibody GMX-2H12 is combined with the conformation-dependent epitope of the beta-1, 2-xylose side chain, the anti-cryptococcus capsular polysaccharide monoclonal antibody GMX-4B09 recognizes the O-acetylation modified linear epitope, and the detection blind area caused by antigen oligomerization is effectively avoided through the double-epitope sandwich method design;

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to an anti-Cryptococcus antigen-antibody pair and its application in a chemiluminescence kit. Background Art

[0002] Cryptococcus neoformans and Cryptococcus gattii can cause Cryptococcus infections. Their pathogenic bacteria are widely distributed in nature, and are highly enriched especially in pigeon droppings, rotten plants and soil. The pathogen mainly invades the human body through the respiratory tract, and is prone to cause disseminated infections in immunocompromised populations (such as HIV / AIDS patients, organ transplant recipients or those who have long-term used immunosuppressants), manifested as cryptococcal meningitis, pneumonia and skin and mucosal lesions, etc. The pathogenicity of Cryptococcus neoformans is closely related to capsular polysaccharides. Its capsule is composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM) and mannoprotein (MP), among which glucuronoxylomannan accounts for up to 90% and is the core component of diagnostic markers. This polysaccharide structure inhibits the phagocytosis of host immune cells by forming a physical barrier, and can be released into body fluids as a soluble antigen, making it an ideal detection target.

[0003] The detection techniques for Cryptococcus capsular polysaccharide antigens mainly include latex agglutination test, enzyme-linked immunosorbent assay and colloidal gold immunochromatography. Among them, the colloidal gold method is widely used because of its simple operation and rapidity (results can be obtained within 10 minutes). However, the existing colloidal gold detection methods still have significant limitations. First, the sensitivity is limited by the analytical performance of the detection system, and the lowest detection limit is 1.0 - 1.5 ng / mL, which is prone to false negatives in the early stage of infection or when the antigen concentration is low. Secondly, the problems of repeatability and batch-to-batch difference are prominent. Research shows that the positive coincidence rate of different batches of reagents for detecting weakly positive samples can fluctuate by up to 5%, and the coefficient of variation (CV) of quantitative detection is as high as 15% - 20%, which is difficult to meet the requirements of clinical precise monitoring. Moreover, the high-dose hook effect causes strongly positive samples to be possibly misjudged as negative, and accurate quantification can only be achieved by dilution and retesting, significantly prolonging the detection process. In addition, sample matrix interference is significant. Hemolysis, lipemia or hyperbilirubinemia will cause abnormal background color development of colloidal gold, resulting in a false negative rate of about 3% - 5%. Finally, the existing technologies lack the ability of multi-index joint detection and cannot distinguish between Cryptococcus neoformans and Cryptococcus gattii infections, while there are differences in the treatment plan selection for the two. Summary of the Invention

[0004] Aiming at the deficiencies of the existing technologies, the present invention provides an anti-Cryptococcus antigen-antibody pair and its application in a chemiluminescence kit, aiming to solve the problems mentioned in the background art.

[0005] In a first aspect, the present invention provides an anti-Cryptococcus antigen antibody pair, comprising a monoclonal antibody GMX-2H12 against Cryptococcus capsular polysaccharide and a monoclonal antibody GMX-4B09 against Cryptococcus capsular polysaccharide; The monoclonal antibody GMX-2H12 against Cryptococcus capsular polysaccharide has a light chain variable region with an amino acid sequence as shown in SEQ ID NO.1, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.2; The monoclonal antibody GMX-4B09 against Cryptococcus capsular polysaccharide has a light chain variable region with an amino acid sequence as shown in SEQ ID NO.3, and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.4.

[0006] Furthermore, the nucleotide sequence encoding the light chain variable region of the monoclonal antibody GMX-2H12 against Cryptococcus capsular polysaccharide is as shown in SEQ ID NO.5; the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody GMX-2H12 against Cryptococcus capsular polysaccharide is as shown in SEQ ID NO.6.

[0007] Furthermore, the nucleotide sequence encoding the light chain variable region of the monoclonal antibody GMX-4B09 against Cryptococcus capsular polysaccharide is as shown in SEQ ID NO.7; the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody GMX-4B09 against Cryptococcus capsular polysaccharide is as shown in SEQ ID NO.8.

[0008] In a second aspect, the present invention provides the application of the anti-Cryptococcus antigen antibody pair in a chemiluminescence kit, wherein in the chemiluminescence kit, the monoclonal antibody GMX-2H12 against Cryptococcus capsular polysaccharide binds to a conformation-dependent epitope of the β-1,2-xylose side chain, and the monoclonal antibody GMX-4B09 against Cryptococcus capsular polysaccharide recognizes an O-acetylated modified linear epitope.

[0009] Furthermore, the chemiluminescence kit further comprises a calibrator and a calibrator diluent; The preparation method of the calibrator is as follows: Using human serum matrix, after removing interference substances from human serum, standard Cryptococcus capsular polysaccharide antigens are added respectively to prepare calibrators with concentrations of 0 pg / mL, 50 pg / mL, 150 pg / mL, 700 pg / mL, 2500 pg / mL and 10000 pg / mL; The preparation method of the calibrator diluent is as follows: 1000 mL of newborn bovine serum, 2.0 g of NaN3, 43 mg of tetracycline, 0.1% of ProClin 300, mix and stir, after testing the pH value to be 5.5 ± 0.1, filter with a 0.22 μm filter paper.

[0010] Further, the preparation method of the chemiluminescence kit comprises the following steps: Step 1: Preparation of calibrators: Using human serum matrix, after removing interfering substances from human serum, cryptococcal capsular polysaccharide antigen standards are added respectively to prepare calibrators with concentrations of 0 pg / mL, 50 pg / mL, 150 pg / mL, 700 pg / mL, 2500 pg / mL, and 10000 pg / mL; Step 2: Preparation of calibrator diluent: 1000 mL of newborn bovine serum, 2.0 g of , 43 mg of tetracycline, 0.1% of , mix and stir evenly, after testing the pH value to be 5.5 ± 0.1, filter with a 0.22 μm filter paper; Step 3: Preparation of antibody-coated magnetic bead reagent: Select carboxyl magnetic beads with a particle size of 2.8 μm, activate the surface of the carboxyl magnetic beads with EDC and NHS esters in a MES 5.0 buffer solution, then wash with phosphate buffer, and then mix the 2 mg / mL anti-cryptococcal capsular polysaccharide monoclonal antibody GMX-2H12 solution with the carboxyl magnetic beads, incubate at 37 °C for 2 h, then use 10 mg / mL bovine serum albumin to block the surface of the magnetic beads, and finally suspend the magnetic beads in a mixed solution containing 0.1% bovine serum albumin and 0.05 M, pH = 7.4 buffer solution; Step 4: Preparation of antibody detection reagent: Label acridinium ester onto the anti-cryptococcal capsular polysaccharide monoclonal antibody GMX-4B09 by chemical synthesis method, and dilute the combined label with calibrator diluent.

[0011] Further, the chemiluminescence kit is used for diagnosing whether cryptococcus infection occurs.

[0012] Further, the chemiluminescence kit is used for diagnosing whether cryptococcus infection occurs, including the following detection steps: Gently invert and mix the antibody-coated magnetic bead reagent to make the magnetic beads evenly suspended; Pipette 100 μL of the antibody-coated magnetic bead reagent into a reaction tube, then add 50 μL of the test sample and 50 μL of the antibody detection reagent, incubate at 37 °C for 7 min, after the incubation ends, use a magnetic separator to separate the magnetic beads, then wash the magnetic beads with phosphate buffer containing Tween, wash 3 times, 300 μL each time; Add 100 μL of pre-excitation solution to resuspend the magnetic beads, then immediately place the reaction tube into a chemiluminescence detector, start the chemiluminescence reaction, and obtain the chemiluminescence signal intensity of the test sample.

[0013] Further, the test sample includes cerebrospinal fluid or bronchoalveolar lavage fluid.

[0014] The present invention has the following beneficial effects: (1) The anti-Cryptococcus neoformans capsular polysaccharide monoclonal antibody GMX-2H12 binds to the conformation-dependent epitope of the β-1,2-xylose side chain, and the anti-Cryptococcus neoformans capsular polysaccharide monoclonal antibody GMX-4B09 recognizes the O-acetylated modified linear epitope. Through the design of the double epitope sandwich method, the detection blind area caused by antigen oligomerization is effectively avoided.

[0015] (2) The antibody GMX-2H12 has a high affinity for the β-1,2-xylose side chain of Cryptococcus neoformans capsular polysaccharide, and its KD is M, the antibody GMX-4B09 has a high affinity for the O-acetylation of Cryptococcus neoformans capsular polysaccharide, and its KD is M. Using magnetic beads as the solid-phase carrier and combining the direct labeling technology of acridinium ester, the detection sensitivity of the reagent is finally increased to 0.01 ng / mL, which is 100 times higher than that of the colloidal gold method.

[0016] (3) Using chemiluminescence for full-automatic detection, the first sample can be detected within 9 minutes (including 1 minute of lysis pretreatment). It supports on-demand detection of multi-matrix samples such as cerebrospinal fluid and bronchoalveolar lavage fluid, and the hook effect threshold breaks through 5000 ng / mL without manual dilution intervention.

[0017] (4) Through a full-automatic platform, high-throughput detection of 40 - 120 test samples per hour is achieved, providing an accurate tool for the early screening and treatment monitoring of Cryptococcus infection. Description of the Drawings

[0018] The exemplary embodiments of the present invention can be more fully understood by referring to the following drawings: Figure 1 It is the dose-response curve of the Cryptococcus neoformans capsular polysaccharide antigen kit of the present invention. Detailed Embodiments

[0019] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0021] The embodiments of the present invention provide an anti-Cryptococcus antigen antibody pair, including the anti-Cryptococcus neoformans capsular polysaccharide monoclonal antibody GMX-2H12 and the anti-Cryptococcus neoformans capsular polysaccharide monoclonal antibody GMX-4B09; The monoclonal antibody GMX-2H12 against Cryptococcus capsular polysaccharide has a light chain variable region with an amino acid sequence as shown in SEQ ID NO.1 and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.2; The monoclonal antibody GMX-4B09 against Cryptococcus capsular polysaccharide has a light chain variable region with an amino acid sequence as shown in SEQ ID NO.3 and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.4.

[0022] In some embodiments, the nucleotide sequence encoding the light chain variable region of the monoclonal antibody GMX-2H12 against Cryptococcus capsular polysaccharide is as shown in SEQ ID NO.5; the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody GMX-2H12 against Cryptococcus capsular polysaccharide is as shown in SEQ ID NO.6.

[0023] In some embodiments, the nucleotide sequence encoding the light chain variable region of the monoclonal antibody GMX-4B09 against Cryptococcus capsular polysaccharide is as shown in SEQ ID NO.7; the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody GMX-4B09 against Cryptococcus capsular polysaccharide is as shown in SEQ ID NO.8.

[0024] The embodiments of the present invention also provide the application of the anti-Cryptococcus antigen-antibody pair in a chemiluminescence kit. In the chemiluminescence kit, the monoclonal antibody GMX-2H12 against Cryptococcus capsular polysaccharide binds to the conformation-dependent epitope of the β-1,2-xylose side chain, and the monoclonal antibody GMX-4B09 against Cryptococcus capsular polysaccharide recognizes the O-acetylated modified linear epitope.

[0025] In some embodiments, the chemiluminescence kit further includes a calibrator and a calibrator diluent;

[0026] The preparation method of the calibrator is as follows: Using human serum matrix, after removing interfering substances from human serum, Cryptococcus capsular polysaccharide antigen standards are added respectively to prepare calibrators with concentrations of 0 pg / mL, 50 pg / mL, 150 pg / mL, 700 pg / mL, 2500 pg / mL, and 10000 pg / mL;

[0027] The preparation method of the calibrator diluent is as follows: 1000 mL of newborn bovine serum, 2.0 g of , 43 mg of tetracycline, 0.1% of , mix and stir, after testing the pH value to be 5.5 ± 0.1, filter with a 0.22 μm filter paper.

[0028] In some embodiments, the preparation method of the chemiluminescence kit includes the following steps:

[0029] Step 1: Preparation of calibrator: Using human serum matrix, after removing interfering substances from human serum, cryptococcal capsular polysaccharide antigen standards were added respectively to prepare calibrators with concentrations of 0 pg / mL, 50 pg / mL, 150 pg / mL, 700 pg / mL, 2500 pg / mL, and 10000 pg / mL; Step 2: Preparation of calibrator diluent: 1000 mL of newborn bovine serum, 2.0 g of NaN3, 43 mg of tetracycline, 0.1% of , mixed and stirred evenly. After testing the pH value to be 5.5 ± 0.1, it was filtered through a 0.22 μm filter paper; Step 3: Preparation of antibody-coated magnetic bead reagent: Carboxyl magnetic beads with a particle size of 2.8 μm were selected. The surface of the carboxyl magnetic beads was activated with EDC and NHS esters in a MES 5.0 buffer solution, then washed with phosphate buffer solution. Then, a 2 mg / mL solution of anti-cryptococcal capsular polysaccharide monoclonal antibody GMX-2H12 was mixed with the carboxyl magnetic beads and incubated at 37 °C for 2 h. Then, the surface of the magnetic beads was blocked with 10 mg / mL bovine serum albumin. Finally, the magnetic beads were suspended in a mixed solution containing 0.1% bovine serum albumin and 0.05 M, pH = 7.4 Tris-HCl buffer solution; Step 4: Preparation of antibody detection reagent: Acridinium ester was labeled onto the anti-cryptococcal capsular polysaccharide monoclonal antibody GMX-4B09 by chemical synthesis method, and the combined label was diluted with calibrator diluent.

[0030] In some embodiments, the chemiluminescence assay kit is used to diagnose whether a person is infected with Cryptococcus.

[0031] In some embodiments, the chemiluminescence assay kit is used to diagnose whether a person is infected with Cryptococcus, including the following detection steps: Gently invert and mix the antibody-coated magnetic bead reagent to make the magnetic beads evenly suspended; Pipette 100 μL of the antibody-coated magnetic bead reagent into a reaction tube, then add 50 μL of the test sample and 50 μL of the antibody detection reagent, and incubate at 37 °C for 7 min. After the incubation, use a magnetic separator to separate the magnetic beads, then wash the magnetic beads with phosphate buffer solution containing Tween 3 times, 300 μL each time; Add 100 μL of pre-excitation solution to resuspend the magnetic beads, then immediately place the reaction tube into a chemiluminescence detector to initiate the chemiluminescence reaction and obtain the chemiluminescence signal intensity of the test sample.

[0032] In some embodiments, the test sample includes cerebrospinal fluid or bronchoalveolar lavage fluid.

[0033] Example 1: Preparation of rabbit monoclonal antibody against cryptococcal capsular polysaccharide (1)Immunogen Preparation First, inoculate Cryptococcus neoformans into YM broth medium containing yeast extract, peptone, and glucose, and culture it with shaking at 30 °C until the late logarithmic growth phase; then lyse the bacteria by autoclaving (121 °C, 25 minutes) and centrifuge to remove the bacteria, retaining the supernatant rich in capsular polysaccharides. In the crude extraction stage, after adjusting the pH to 5.0 with 5% calcium acetate, precipitate the polysaccharides with 3 volumes of 95% ethanol, and then purify by selective precipitation with cetyltrimethylammonium bromide (CTAB) - CTAB binds to negatively charged polysaccharides to form a complex, which is washed with 10% ethanol to remove residual CTAB and then dissolved in 1 M NaCl solution, and the polysaccharides are precipitated with ethanol for the second time to obtain high-purity glucuronoxylomannan (GXM). The final product is dialyzed to remove small molecule impurities and freeze-dried, and its monosaccharide composition (mannose, xylose, glucuronic acid) and molecular weight (1000 - 2000 kDa) are verified by HPLC and gel permeation chromatography. Covalently conjugate the Cryptococcus capsular polysaccharide antigen to a carrier protein (BSA or KLH) to enhance immunogenicity, and then add an equal volume of complete Freund's adjuvant, mix and emulsify to form a stable emulsion to extend the antigen release time.

[0034] (2)Immunization Protocol Select healthy adult rabbits, and perform primary immunization by subcutaneous multi-point injection, and boost immunize every 2 weeks for a total of 5 times. Monitor the serum titer (ELISA method) during immunization to ensure that the antibody titer reaches above.

[0035] (3)Antigen-Specific B Cell Sorting After immunization, prepare single-cell suspensions from rabbit spleens or peripheral blood, and remove red blood cells and dead cells by density gradient centrifugation. Sort B cells carrying antigen-specific BCR on the surface using fluorescently labeled capsular polysaccharide antigen by flow cytometry (FACS). Distribute the sorted B cells into 96-well plates (1 cell per well), add a medium containing cytokines (IL-6, IL-21) for short-term culture to activate antibody secretion, amplify the antibody variable region genes, namely the heavy chain variable region / light chain variable region (VH / VL) genes, by single-cell RT-PCR, clone the amplified heavy chain variable region / light chain variable region genes into the expression vector pcDNA3.4 and send them to Genewiz for sequencing simultaneously, and construct a full-length IgG expression framework by overlap extension PCR. The nucleotide sequences of the light and heavy chain variable regions of the anti-Cryptococcus capsular polysaccharide monoclonal antibody GMX-2H12 are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively, and the nucleotide sequences of the light and heavy chain variable regions of GMX-4B09 are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively.

[0036] (4)Antibody Expression and Screening Transient transfection was performed using HEK293 cells, and the supernatant was collected to detect the antibody expression level. By coating Cryptococcus capsular polysaccharide antigen, antibody clones with high binding activity were screened, and cross-reactivity detection was carried out to exclude cross-reactivity with host polysaccharides or other pathogen antigens. The selected antibody pairs were GMX-2H12 and GMX-4B09. The amino acid sequences of the light and heavy chain variable regions of the anti-Cryptococcus capsular polysaccharide monoclonal antibody GMX-2H12 are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively, and the amino acid sequences of the light and heavy chain variable regions of GMX-4B09 are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0037] By using surface plasmon resonance to analyze their affinities, the KD of GMX-2H12 is M, and the KD of the antibody GMX-4B09 is M, as shown in Table 1.

[0038] Table 1 Results of antibody affinity determination

[0039] Example 2: Preparation of a chemiluminescence assay kit for detecting Cryptococcus capsular polysaccharide antigen (1) Preparation of calibrators: Human serum was used as the matrix. First, the human serum was treated to remove interfering substances through an antibody affinity separation column (to eliminate endogenous substances in the human serum matrix that affect the test results), and then Cryptococcus capsular polysaccharide antigen standards (purity above 99%) were added respectively to prepare calibrators with concentrations of 0 pg / mL, 50 pg / mL, 150 pg / mL, 700 pg / mL, 2500 pg / mL, and 10000 pg / mL. During the preparation process, high-precision pipettes and balances were used to ensure the accuracy and repeatability of the calibrator concentrations.

[0040] (2) Calibrator diluent: 1000 mL of newborn bovine serum, 2.0 g, 43 mg of tetracycline (tetracycline was dissolved in 0.1 M, pH = 7.5 Tris and then added), 0.1% , mixed and stirred evenly. After testing the pH value to be 5.5 ± 0.1, it was filtered through a 0.22 μm filter paper and stored at 4 °C.

[0041] (3)Preparation of antibody-coated magnetic bead reagent: Select carboxyl magnetic beads with a particle size of 2.8 μm. In MES 5.0 buffer solution, activate the surface of the magnetic beads with EDC and NHS ester, and then wash them with phosphate buffer. Next, mix the GMX-2H12 antibody solution (concentration 2 mg / mL) with the carboxyl magnetic beads, incubate at 37 °C for 2 h, and then block the surface of the magnetic beads with 10 mg / mL bovine serum albumin. Finally, suspend the magnetic beads in a solution containing 0.1% bovine serum albumin and 0.05 M buffer solution (pH = 7.4).

[0042] (4)Preparation of antibody detection reagent: Label acridinium ester onto the GMX-4B09 antibody by chemical synthesis method, and dilute the combined label with calibrator diluent. During the labeling process, optimize the reaction conditions (temperature, time, and reagent concentration) to ensure that the labeling efficiency reaches over 90%.

[0043] Example 3: Detection steps of the chemiluminescence assay kit for cryptococcal capsular polysaccharide antigen: Gently invert and mix the antibody-coated magnetic bead reagent to evenly suspend the magnetic beads. Pipette 100 μL of the antibody-coated magnetic bead reagent into a reaction tube, then add 50 μL of the test sample and 50 μL of the antibody detection reagent, and incubate at 37 °C for 7 min. After incubation, use a magnetic separator to separate the magnetic beads from the reaction solution, and then wash the magnetic beads with phosphate buffer containing Tween 3 times, 300 μL each time. After washing, add 100 μL of pre-excitation solution to the reaction tube, immediately place the reaction tube into a chemiluminescence detector, and inject 100 μL of excitation solution by the instrument to initiate the chemiluminescence reaction and obtain the chemiluminescence signal intensity of the test sample.

[0044] According to the concentration of the calibrator and the corresponding chemiluminescence signal intensity, plot a standard curve, and use the standard curve to calculate the content of cryptococcal capsular polysaccharide antigen in the test sample based on the chemiluminescence signal intensity of the test sample.

[0045] Example 4: Performance evaluation of the cryptococcal capsular polysaccharide antigen kit Dilute the cryptococcal capsular polysaccharide antigen in Example 1 with standard product diluent to prepare calibrator solutions with different concentrations, which are 0 pg / mL, 50 pg / mL, 150 pg / mL, 700 pg / mL, 2500 pg / mL, and 10000 pg / mL respectively, and store them at -20 °C for later use. Then use the chemiluminescence assay kit for cryptococcal capsular polysaccharide antigen provided in Example 2 of the present invention to detect the calibrators, and read the luminescence intensity values corresponding to each calibrator respectively.

[0046] The test range determination results of the cryptococcal capsular polysaccharide antigen kit are shown in Table 2; with the concentration as the abscissa and the luminescence intensity as the ordinate, a standard curve was fitted as shown in Figure 1 . It can be seen that the linear relationship of the standard curve is good, and the linear regression equation after fitting is , . According to the zero value plus 2 times the SD, its lowest detection limit was calculated to be 14.9 pg / mL.

[0047] Table 2 Determination of the test range of the cryptococcal capsular polysaccharide antigen kit

[0048] Specific substances were configured into corresponding concentrations, and the kit in Example 2 of the present invention was used for determination to evaluate its cross-reactivity rate. The results are shown in Table 3. It can be seen that the cross-reactivity rates are all less than 0.1%.

[0049] Table 3 Detection results of the specificity experiment of the cryptococcal capsular polysaccharide antigen kit

[0050] Amino acid sequence: SEQ ID NO:1: WDTVHSSAEGPRWWSKLLIYDAGAPAALAPRCHICASADPDSILRVCSCGRHSHHQLPGQLERLQSSLILVSAETRAASQAPRCHICASADPDSILRVCSCWGSCCSGSSVACSVTMLPLTIVLVVTALAVML

[0051] SEQ ID NO:2: GHSSLFGGGTEVVVKMDTRAPTQLLGLLLLWLPGATFAQVLTQTPSSVSAAVGDTVTINCQASESVYNNRLSWYQQKPGQPPKLPGATFAQVLTQTPSSVSAAAGAPAALAHQWRAVSRCCHLLLSKWLLLW

[0052] SEQ ID NO:3: DAATYYCLSGYCFGGYASTFGGGTEVVVKMDTRAPTQLLGLLLLWLLVSAETRAASQAPRCHICASADPDSILRVCSCWGSCCSGSSVACSVTFTLTISGVQCDEGPGSLPSSLSTMQHPNCLGSHPGSKAV

[0053] SEQ ID NO:4: GRLVTPGTPLTLTCTASGFSLSYYMSTWVRQAPGKGLEWIGYASWTKIHAGGSAYYGRFTISRTSTTVDLKVTSLTTEDTAMYFCAREYLNNFGLWGPGTLVTVSSMETGLRWLLLVAVLKGVQCQSLEES

[0054] Nucleotide sequence: SEQ ID NO.5: tgggacacagttcactcttcggcggagggaccgaggtggtggtcaaagctcttgatctacgatgcaggggctcctgctgctctggctcccaggtgccacatttgcgcaagtgctgacccagactccatcctccgtgtctgcagctgtgggagacacagtcaccatcaattgccaggccagttagagcgtttacaatcatcgcttatcctggtatcagcagaaaccagggcagcctcccaagctcccaggtgccacatttgcgcaagtgctgacccagactccatcctccgtgtctgcagctgctggggctcctgctgctctggctcatcagtggcgtgcagtgtgacgatgctgccacttactattgtcttagtggttactgctttggcggttatgcta

[0055] SEQ ID NO.6: ggacacagttcactcttcggcggagggaccgaggtggtggtcaaaatggacacgagggcccccactcagctgctggggctcctgctgctctggctcccaggtgccacatttgcgcaagtgctgacccagactccatcctccgtgtctgcagctgtgggagacacagtcaccatcaattgccaggccagtgagagcgtttataataatcgcttatcctggtatcagcagaaaccagggcagcctcccaagctcccaggtgccacatttgcgcaagtgctgacccagactccatcctccgtgtctgcagctgctggggctcctgctgctctggctcatcagtggcgtgcagtgtcacgatgctgccacttactattgtctaagtggttactgctttgg

[0056] SEQ ID NO.7: gatgctgccacttactattgtctaagtggttactgctttggcggttatgctagtactttcggcggagggaccgaggtggtggtcaaaatggacacgagggcccccactcagctgctggggctcctgctgctctggctcctggtatcagcagaaaccagggcagcctcccaagctcccaggtgccacatttgcgcaagtgctgacccagactccatcctccgtgtctgcagctgctggggctcctgctgctctggctcatcagtggcgtgcagtgtgacgttcactctcaccatcagtggcgtgcagtgtgacgagggcccaggcagcctcccaagctctttatctacgatgcagcatccaaattgtctggggtcccatcccggttcaaaggcagtg

[0057] SEQ ID NO.8: ggtcgcctggtcacgcctgggacacccctgacactcacctgcacagcctctggattctccctcagttactacatgagcacctgggtccgccaggctccagggaaggggctggagtggatcggatacgcgagctggacaaaaattcatgctggtggtagcgcatactacggccgattcaccatctccagaacctcgaccacggtggatctgaaagtgaccagtctgacaacggaggacacggccatgtatttctgtgccagagaatatttgaataattttggtttgtggggcccaggcaccctggtcaccgtctcctcaatggagactgggctgcgctggcttctcctggtcgctgtgctcaaaggtgtccagtgtcagtcgctggaggagtcc

[0058] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. An anti-Cryptococcus antigen-antibody pair, characterized in that: It includes the anti-Cryptococcus neoformans capsular polysaccharide monoclonal antibody GMX-2H12 and the anti-Cryptococcus neoformans capsular polysaccharide monoclonal antibody GMX-4B09; The anti-Cryptococcus neoformans capsular polysaccharide monoclonal antibody GMX-2H12 has a light chain variable region with the amino acid sequence shown in SEQ ID NO.1 and a heavy chain variable region with the amino acid sequence shown in SEQ ID NO.2; The anti-Cryptococcus neoformans capsular polysaccharide monoclonal antibody GMX-4B09 has a light chain variable region with the amino acid sequence shown in SEQ ID NO.3 and a heavy chain variable region with the amino acid sequence shown in SEQ ID NO.

4.

2. The cryptococcus antigen-antibody pair according to claim 1, wherein: The nucleotide sequence encoding the light chain variable region of the anti-Cryptococcus neoformans capsular polysaccharide monoclonal antibody GMX-2H12 is shown in SEQ ID NO.5; the nucleotide sequence encoding the heavy chain variable region of the anti-Cryptococcus neoformans capsular polysaccharide monoclonal antibody GMX-2H12 is shown in SEQ ID NO.

6.

3. The cryptococcus antigen-antibody pair according to claim 2, wherein: The nucleotide sequence encoding the light chain variable region of the anti-Cryptococcus neoformans capsular polysaccharide monoclonal antibody GMX-4B09 is shown in SEQ ID NO.7; the nucleotide sequence encoding the heavy chain variable region of the anti-Cryptococcus neoformans capsular polysaccharide monoclonal antibody GMX-4B09 is shown in SEQ ID NO.

8.

4. Application of cryptococcus antigen-antibody pair in chemiluminescence kit, characterized in that: In the chemiluminescence kit, the anti-Cryptococcus neoformans capsular polysaccharide monoclonal antibody GMX-2H12 binds to the conformation-dependent epitope of the β-1,2-xylose side chain, and the anti-Cryptococcus neoformans capsular polysaccharide monoclonal antibody GMX-4B09 recognizes the O-acetylated modified linear epitope.

5. Use of the cryptococcus antigen-antibody pair according to claim 4 in a chemiluminescence assay kit, characterized in that: The chemiluminescence kit also includes a calibrator and a calibrator diluent; The preparation method of the calibrator is as follows: Using human serum matrix, after removing interfering substances from human serum, Cryptococcus neoformans capsular polysaccharide antigen standards are added respectively to prepare calibrators with concentrations of 0 pg / mL, 50 pg / mL, 150 pg / mL, 700 pg / mL, 2500 pg / mL, and 10000 pg / mL; The preparation method of the calibration diluent is as follows: 1000 mL of newborn bovine serum, 2.0 g of , 43 mg of tetracycline, 0.1% of ProClin 300, are mixed and stirred. After testing the pH value to be 5.5 ± 0.1, it is filtered through a 0.22 μm filter paper.

6. Use of the cryptococcus antigen-antibody pair according to claim 4 in a chemiluminescence kit, characterized in that: The preparation method of the chemiluminescence kit includes the following steps: Step 1: Preparation of the calibrator: Using human serum matrix, after removing interfering substances from human serum, Cryptococcus neoformans capsular polysaccharide antigen standards are added respectively to prepare calibrators with concentrations of 0 pg / mL, 50 pg / mL, 150 pg / mL, 700 pg / mL, 2500 pg / mL, and 10000 pg / mL; Step 2: Preparation of the calibrator diluent: 1000 mL of newborn bovine serum, 2.0 g of , 43 mg of tetracycline, 0.1% ProClin 300, mixed and stirred evenly. After testing the pH value to be 5.5 ± 0.1, filter through a 0.22 μm filter paper; Step 3: Preparation of the antibody-coated magnetic bead reagent: Select carboxyl magnetic beads with a particle size of 2.8 μm. In a MES 5.0 buffer solution, activate the surface of the carboxyl magnetic beads with EDC and NHS ester, then wash with phosphate buffer solution. Then mix the 2 mg / mL anti-Cryptococcus neoformans capsular polysaccharide monoclonal antibody GMX-2H12 solution with the carboxyl magnetic beads, incubate at 37 °C for 2 h, then use 10 mg / mL bovine serum albumin to block the surface of the magnetic beads, and finally suspend the magnetic beads in a mixed solution containing 0.1% bovine serum albumin and 0.05 M, pH = 7.4 Tris-HCl buffer solution; Step 4: Preparation of the antibody detection reagent: The acridinium ester was labeled onto the monoclonal antibody GMX-4B09 against Cryptococcus capsular polysaccharide by chemical synthesis method, and the conjugated labeling agent was diluted with the calibrator diluent.

7. Use of the cryptococcus antigen-antibody pair according to claim 4 in a chemiluminescence kit, characterized in that: The chemiluminescence kit is used for diagnosing whether Cryptococcus is infected.

8. Use of the cryptococcus antigen-antibody pair according to claim 7 in a chemiluminescence assay kit, characterized in that: The chemiluminescence kit is used for diagnosing whether Cryptococcus is infected, and includes the following detection steps: gently invert and mix the antibody-coated magnetic bead reagent to make the magnetic beads evenly suspended; Aspirate 100 μL of the antibody-coated magnetic bead reagent into a reaction tube, then add 50 μL of the test sample and 50 μL of the antibody detection reagent, incubate at 37 °C for 7 min. After the incubation, use a magnetic separator to separate the magnetic beads, and then wash the magnetic beads with phosphate buffer containing Tween, wash 3 times, 300 μL each time; add 100 μL of the pre-excitation solution to resuspend the magnetic beads, then immediately place the reaction tube into a chemiluminescence detector, start the chemiluminescence reaction, and obtain the chemiluminescence signal intensity of the test sample.

9. Use of the cryptococcus antigen-antibody pair according to claim 8 in a chemiluminescence assay kit, characterized in that: The test sample includes cerebrospinal fluid or bronchoalveolar lavage fluid.

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