WO2017148330A1 - 一种新型隐球菌荚膜多糖gxm的制备方法及gxm抗原免疫检测试剂盒及其应用 - Google Patents

一种新型隐球菌荚膜多糖gxm的制备方法及gxm抗原免疫检测试剂盒及其应用 Download PDF

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WO2017148330A1
WO2017148330A1 PCT/CN2017/074700 CN2017074700W WO2017148330A1 WO 2017148330 A1 WO2017148330 A1 WO 2017148330A1 CN 2017074700 W CN2017074700 W CN 2017074700W WO 2017148330 A1 WO2017148330 A1 WO 2017148330A1
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gxm
solution
buffer
capsular polysaccharide
preparation
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PCT/CN2017/074700
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French (fr)
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彭洁
史东东
李宁
粟艳
周泽奇
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丹娜（天津）生物科技有限公司
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Priority claimed from CN201610112389.8A external-priority patent/CN105646703B/zh
Priority claimed from CN201610112390.0A external-priority patent/CN105784987A/zh
Priority claimed from CN201610112315.4A external-priority patent/CN105651996A/zh
Application filed by 丹娜（天津）生物科技有限公司 filed Critical 丹娜（天津）生物科技有限公司
Publication of WO2017148330A1 publication Critical patent/WO2017148330A1/zh
Priority to US15/924,170 priority Critical patent/US10670598B2/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts

Definitions

  • the invention relates to the field of immunoassay technology, in particular to a preparation method of a novel cryptococcal capsular polysaccharide glucuronoxylomannan (GXM), a polyclonal antibody against the GXM antigen, and a preparation method thereof.
  • Crytococcus Neoformans is an important conditional pathogen, often infected with patients with low immunity or impaired immunity, leading to deep fungal infections with central nervous system infections and high mortality.
  • CDC Centers for Disease Control and Prevention
  • the new type of cryptococcal meningitis (hereinafter referred to as "hidden brain") is the most common central nervous system disease caused by Cryptococcus neoformans, accounting for about 80% of cryptococcosis.
  • the clinical manifestations of occult brain are complex, the symptoms are atypical, so it is difficult to diagnose. About 80% of patients with occult brain are misdiagnosed as tuberculous meningitis.
  • the main pathogenic factors of Cryptococcus neoformans are capsule, melanin production and its ability to grow in the host temperature environment.
  • the capsule is one of the main determinants of toxicity. Its main component is capsular polysaccharide, which has the effect of inhibiting phagocytosis. And the role of activation of complement, the most abundant is glucuronoxymannan (GXM).
  • GXM glucuronoxymannan
  • the sensitivity of direct microscopic examination was 91.1%, 69.6%, and 73.2%, respectively, and the specificities were 96.0%, 100%, and 100%, respectively. It is considered that the latex agglutination test can detect cryptococcal capsular polysaccharide antigen as a novel cryptococcal Early diagnosis of meningitis.
  • the cryptococcal latex agglutination test for cryptococcal meningitis is highly specific for the diagnosis of cryptococcal meningitis, which improves the early diagnosis rate (Wang H, Yuan X, Zhang L. Latex agglutination: Diagnose the early cryptococcus neoformans test of capsular) Polysaccharide antigen. Pakistan journal of pharmaceutical sciences, 2015, 28 (1 Suppl): 307-311.). Therefore, the novel cryptococcal capsular polysaccharide is very important for the early diagnosis of cryptococcal meningitis.
  • the precipitate is dissolved in deionized water, dialyzed, and lyophilized to obtain a purified capsular polysaccharide (Kozel T R, Cazin J. Nonencapsulated variant of Cryptococcus neoformans I. Virulence studies and characterization of soluble polysaccharide. Infection and immunity, 1971, 3 (2) ): 287-294.).
  • a large amount of chloroform is required in the above method, and chloroform is a toxic and harmful chemical dangerous product, which has a carcinogenic risk, and has high requirements on working conditions, protection of operators, and sewage treatment.
  • Maxson et al. centrifuged the cryptococcal cell culture solution, separated the cell pellet, and concentrated the obtained supernatant with a polyethersulfone ultrafiltration disk for about 20 times. The mixture was continuously stirred during the concentration process, and a viscous film was formed on the filter disk. Fluid phase, recovery of gelling material with a cell scraper, and finally lyophilization to obtain capsular polysaccharide (Maxson M E, Dadachova E, Casadevall A, et al. Radial mass density, charge, and epitope distribution in the Cryptococcus neoformans capsule. Eukaryotic cell, 2007, 6(1): 95-109.). The above method is simple in operation, and the preparation cycle is short, but the yield of the product is not high.
  • Fraes et al. found that the extracellular polysaccharides and capsular polysaccharides of Cryptococcus were structurally different, and found that the polysaccharide obtained by precipitation of cetyltrimethylammonium bromide was precipitated and concentrated by ultrafiltration.
  • the obtained polysaccharide (refer to Maxson M E, Dadachova E, Casadevall A, et al. Radial mass density, charge, and epitope distribution in the Cryptococcus neoformans capsule. Eukaryotic cell, 2007, 6(1): 95-109.
  • Neoformans capsular polysaccharide and exopolysaccharide fractions manifest physical, chemical, and antigenic differences. Eukaryotic cell, 2008, 7(2): 319-327.).
  • the invention overcomes the deficiencies of the prior art, and provides a preparation method of the novel cryptococcal capsular polysaccharide GXM and the prepared GXM, and the GXM preparation test kit is used, and the prepared GXM has high purity and specificity, and utilizes The detection kit prepared by the antigen has high detection sensitivity.
  • the invention provides a preparation method of a novel cryptococcal capsular polysaccharide GXM immunoaffinity chromatography column, which is prepared by using a novel monoclonal antibody of cryptococcal capsular polysaccharide GXM.
  • the preparation method comprises the following steps:
  • step (3) resuspend the mixture of the antibody obtained in step (2) and the affinity chromatography matrix with a crosslinking buffer, add a bifunctional binding agent to the obtained suspension, incubate, mix, and liquid-solid separation to obtain a pro And a chromatography matrix-antibody cross-linking complex;
  • (7) preserving: the affinity chromatography matrix-antibody cross-linking complex obtained in step (6) or the filled cryptococcal capsular polysaccharide GXM immunoaffinity chromatography column obtained in step (6) is 4 Stored under °C conditions, stable performance within 1 year;
  • the chromatography column stored in the step (7) at 4 ° C or the used chromatography column is buffered with 10-25 times the volume of the sample to be purified before use.
  • the liquid column is regenerated.
  • the affinity chromatography matrix-antibody cross-linking complex obtained in the step (5) is preservatively treated and then filled into the chromatography column, and the antiseptic treatment is the affinity chromatography obtained in the step (5).
  • the matrix-antibody cross-linking complex was washed with PBS buffer and resuspended in PBS buffer and added with thimerosal; the concentration of thimerosal was 0.005-0.015%.
  • the affinity chromatography matrix described in the step (1) is selected from the group consisting of protein A microbeads, protein G microbeads, active microbeads, preferably protein A microbeads.
  • the solution described in step (1) is selected from the group consisting of pH 8.0-9.0 carbonate buffer.
  • the ratio of the affinity chromatography matrix and the solution in the step (1) is: 0.5-2.0 mL is added per 10 mL of the solution.
  • the ratio of the affinity chromatography matrix to the affinity chromatography matrix to the antibody is: 1-4 mg of the monoclonal antibody is bound per 1 mL of the affinity chromatography matrix; further preferably, the ratio of the affinity chromatography matrix and the solution in the step (1) To: 1 mL of affinity chromatography matrix was added to each 10 mL of solution, and the ratio of affinity chromatography matrix to antibody was: 2 mg of monoclonal antibody per 1 mL of affinity chromatography matrix.
  • the crosslinking buffer described in the step (2) is 0.1-0.3 mol/L of a sodium borate solution having a pH of 8.0-9.5, and the amount is 5-15 times the affinity chromatography matrix volume, further preferably, The crosslinking buffer is a sodium borate solution having a concentration of 0.2 mol/L of pH 9.0, and the amount is 10 times the affinity chromatography matrix volume; the number of washings is 1-3 times, and the centrifugation condition is 3000 g centrifugation 2 Centrifuge for 5 seconds at -5 minutes or 10000g.
  • the crosslinking buffer in the step (3) is 0.1-0.3 mol/L of a sodium borate solution having a pH of 8.3-9.5, and the amount is 5-15 times the affinity chromatography matrix volume; further preferably, The cross-linking buffer is a sodium borate solution having a concentration of 0.2 mol/L and a pH of 9.0, and the amount is 10 times the affinity chromatography matrix volume.
  • the bifunctional binder of step (3) is selected from the group consisting of dimethyl pimelate, carbonyl diimidazole, cyanogen bromide, hydroxysuccinimide, acetyl iodide, preferably dimethylpimelic acid.
  • the ester is used in a final concentration of 15-25 mmol/L, preferably 20 mmol/L, in the affinity chromatography matrix suspension.
  • the blocking solution according to the steps (4) and (5) is selected from the group consisting of ethanolamine, aminoethane and the like, and a solution containing a small molecule capable of binding an amino group, preferably an ethanolamine solution, further preferably having a concentration of 0.1. - 0.25 mol/L, an ethanolamine solution having a pH of 7.5 to 8.5, more preferably 0.2 mol/L of an ethanolamine solution having a pH of 8.0.
  • the detection process of the crosslinking efficiency of the affinity chromatography matrix-antibody cross-linking complex described in the step (6) is: taking the affinity chromatography matrix sample and the affinity chromatography matrix and the antibody after cross-linking
  • the samples were separately boiled in LaemmLi buffer, and two samples corresponding to 1 mL and 9 mL were taken out respectively, and electrophoresed in a 10% SDS-polyacrylamide gel, stained with Coomassie blue, such as a heavy chain before cross-linking.
  • the zone 55 kDa
  • the cross-linked sample is absent, indicating that the cross-linking was successful.
  • the container is rinsed with PBS buffer to collect the residual affinity chromatography matrix, and if possible, only the affinity chromatography required for all GXM in the sample to be purified is used. Matrix-antibody cross-linking complex.
  • the invention also provides a novel Cryptococcus capsular polysaccharide GXM immunoaffinity chromatography column prepared by the above method.
  • the invention also provides a preparation method of a novel cryptococcal capsular polysaccharide GXM, which comprises the steps of purifying GXM by using the above GXM immunoaffinity chromatography column;
  • the preparation method comprises the following steps:
  • the crude extraction of the novel cryptococcal capsular polysaccharide GXM comprises the following steps:
  • the culture condition of the Cryptococcus neoformans in the crude extraction step (1) of the novel cryptococcal capsular polysaccharide is: the medium is a YM broth medium, the culture temperature is 30 ° C, and the shaking speed is 200 rpm.
  • the culture time was 32-40 hours, and the YM broth medium formulation contained: 3 g of yeast extract, 5 g of peptone, 3 g of maltose extract, and 10 g of D-glucose per liter of medium.
  • the calcium acetate powder described in the step (3) is added in an amount such that its final concentration in the supernatant reaches 2-8%, and glacial acetic acid adjusts the pH to 4.6-5.2.
  • the concentration of the ethanol in the step (4) is 90-98%, and the amount added is 2-4 times the volume of the solution obtained in the step (3).
  • novel cryptococcal capsular polysaccharide GXM is carried out by using the novel cryptococcal capsular polysaccharide GXM immunoaffinity chromatography column. Purification of the crude extract, including the following steps:
  • (7) using 10-25 times the bed volume of the starting buffer to flow through the column to regenerate, and adding thimerosal to a final concentration of 0.005-0.015%, and storing the column at 4 ° C .
  • the flow rate of the sample to be purified described in the step (2) is 0.5-1.5 mL/h, and the flow rate is controlled by a pump, preferably using a peristaltic pump.
  • the binding buffer described in the step (3) is selected from the group consisting of PBS buffer of pH 6.0-8.0, Tris-HCl buffer of pH 6.0-8.0, and acetic acid-sodium acetate buffer of pH 6.0-8.0.
  • PBS buffer of pH 6.0-8.0 Tris-HCl buffer of pH 6.0-8.0
  • acetic acid-sodium acetate buffer of pH 6.0-8.0 One is preferably a PBS buffer having a pH of 6.0 to 8.0; the binding buffer is used in the step (3) in an amount of 10 to 25 times the bed volume, preferably 20 times the bed volume.
  • the pre-elution buffer described in the step (4) is a pH 8.5-9.5 carbonate buffer, and the amount is 10-25 times the bed volume, and further preferably 20 times the bed volume.
  • the elution buffer described in the step (5) is a buffer of pH 3.0, preferably 0.1 M glycine buffer at pH 3.0, citrate-phosphate buffer at pH 3.0, pH 3.0. Citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer at pH 3.0, in an amount of 0.4-0.8 bed volume.
  • the GXM eluate obtained in the step (6) is subjected to dialysis desalting treatment or the pH thereof is adjusted with a buffer.
  • the invention provides a novel cryptococcal capsular polysaccharide GXM prepared by the above method.
  • the invention also provides an application of a novel cryptococcal capsular polysaccharide GXM immunoaffinity chromatography column in the early diagnosis of the purified Cryptococcus capsular polysaccharide GXM and the novel cryptococcal meningitis.
  • the invention also provides an application of a novel cryptococcal capsular polysaccharide GXM in an early diagnostic reagent for novel cryptococcal meningitis.
  • the present invention also provides a polyclonal antibody against Cryptococcus neoformans capsular polysaccharide GXM, which is prepared by the following method: separating and purifying serum obtained by using GXM as an immunogen to obtain an antibody; the purification method is Saturated ammonium sulfate salting-precipitation method and/or affinity chromatography method, the immunization method may be subcutaneous injection, intrasplenic injection, intravenous injection or intraperitoneal injection, the immunization dose is 0.01-1 mg/head, and the immunized animal is a rat. , mouse, guinea pig, rabbit, chicken, sheep, horse, pig or donkey, preferably rabbit.
  • the anti-GXM polyclonal antibody prepared by the above steps can specifically bind to GXM, and is shown as a uniform antibody product by SDS-PAGE.
  • the indirect ELISA assay shows that the titer is greater than 1:1 ⁇ 10 6 .
  • the invention also provides a novel Cryptococcus capsular polysaccharide GXM antigen immunodetection kit, the kit comprising: a GXM coated enzyme label carrier, an anti-GXM polyclonal enzyme label antibody, and a GXM standard.
  • the GXM standard consists of a series of different concentrations of GXM solution which is a solution of GXM dissolved in a suitable solvent to form a specific concentration of GXM.
  • step 2 adding a blocking solution to the well of the microplate obtained in step 1 for blocking;
  • step 3 Discard the enzyme-labeled plate obtained in step 2, and incubate to obtain a GXM-coated enzyme-labeled vector.
  • the enzyme label carrier is a microplate, a plastic tube; more preferably, the enzyme label carrier is a microplate;
  • the enzyme for labeling is horseradish peroxidase (HRP), alkaline phosphatase (AP); more preferably, The enzyme for labeling is horseradish peroxidase;
  • the GXM standard has at least three different concentrations in the range of 0-100 ng/mL;
  • the GXM standard has five different concentrations, which are respectively recorded as a, b, c, d and e, and their concentrations are respectively recorded. They are 100, 32, 10, 6.4, and 3.2 ng/mL, respectively.
  • the above novel cryptococcal capsular polysaccharide antigen immunoassay kit further comprises a sample treatment solution, a concentrated washing solution, a sample diluent, a substrate solution and a stop solution;
  • the components of the concentrated washing liquid, the sample diluent, the sample processing liquid, the substrate solution and the stopping liquid are as follows:
  • Concentrated lotion phosphate buffer containing 0.4-1.0% Tween;
  • Sample dilution artificial cerebrospinal fluid or artificial serum
  • the sample treatment solution is selected from the group consisting of a protein denaturation solution of pH 2.0-10.0; 0.05-0.2 mol/L of EDTA (disodium edetate) solution of pH 4.0-4.8; 0.07 of pH 2.2-2.8. -0.2 mol/L glycine-HCL (glycine-hydrochloric acid) solution; 0.05-15 mg/mL chain enzyme protease at pH 8.0-9.0; 0.01-0.1 mol/L SDS (dodecane at pH 8.5-1.0) Sodium sulfonate) solution or 1-8 mol/L urea at pH 7.2-8.0;
  • Substrate solution TMB (tetramethylbenzidine), OPD (o-phenylenediamine), OT (o-toluidine), ABTS (2,2'-azino-bis(3-ethylbenzothiazole-6) -sulfonic acid)) or p-NPP (p-nitrophenyl phosphate); preferably TMB;
  • Stop solution 1-20 mol/L sulfuric acid solution, preferably 2 mol/L sulfuric acid solution.
  • the composition and ratio of the concentrated washing liquid are: 96.0 parts of sodium chloride, 2.40 parts of potassium chloride, and disodium hydrogen phosphate dodecahydrate 42.96 by weight. Parts, 2.88 parts of potassium dihydrogen phosphate, Tween-20 0.05 parts, and 1000 parts of ultrapure water.
  • the invention also provides a preparation method of a novel cryptococcal capsular polysaccharide antigen immunoassay kit, the specific steps of which are as follows:
  • step 2 adding a blocking solution to the well of the microplate obtained in step 1 for blocking;
  • step 3 Discard the enzyme-labeled plate obtained in step 2, and incubate to obtain a GXM-coated enzyme-labeled carrier;
  • the enzyme used for labeling is horseradish peroxidase, and the anti-GXM polyclonal enzyme-labeled antibody is prepared by periodate oxidation method; or the enzyme used for labeling is alkaline phosphatase, and glutaraldehyde cross-linking method is used. Preparation of an anti-GXM polyclonal enzyme-labeled antibody;
  • the anti-GXM polyclonal enzyme-labeled antibody is diluted with an enzyme conjugate stabilizer to prepare an anti-GXM polyclonal enzyme-labeled antibody solution.
  • the preparation method of the novel cryptococcal capsular polysaccharide antigen immunoassay kit comprises the following specific steps:
  • step 2 Add the blocking solution to the wells of the microplate obtained in step 1, add 60-200 ⁇ L blocking solution to each well, and block at 30 ° C for 30-90 min;
  • step 3 Discard the enzyme label obtained in step 2 and remove the blocking solution, and place it at a constant temperature of 37 ° C for 30-90 min to obtain a GXM-coated enzyme-labeled carrier;
  • the enzyme used for labeling is horseradish peroxidase, and the anti-GXM polyclonal enzyme-labeled antibody is prepared by periodate oxidation method; or the enzyme used for labeling is alkaline phosphatase, which is cross-linked with glutaraldehyde. Preparation of anti-GXM polyclonal enzyme-labeled antibody;
  • the anti-GXM polyclonal enzyme-labeled antibody is diluted with an enzyme conjugate stabilizer at a ratio of 1:2000-1:20000 to prepare an anti-GXM polyclonal enzyme-labeled antibody solution;
  • the preparation method of the novel cryptococcal capsular polysaccharide antigen immunoassay kit further comprises:
  • the coating buffer is selected from the group consisting of: 0.01-0.20 mol/L PBS (phosphate) buffer solution of pH 7.0-8.0, 0.05-0.20 mol/L of CBS (carbon of pH 9.0-9.6). Acidate) buffer solution or 0.05 mol/L Tris (trishydroxymethylaminomethane) buffer at pH 10.0-10.6.
  • the composition and ratio of the blocking solution are: 3-5% by weight of skim milk powder or 1-4% BSA (bovine serum albumin) of pH 7.0-8.0 of 0.01-0.20 mol/L. PBS buffer solution.
  • BSA bovine serum albumin
  • the GXM standard is obtained by diluting GXM with a sample diluent, and has five different concentrations, which are respectively recorded as a, b, c, d, and e, and the concentrations thereof are 100, 32, respectively. 10, 6.4, 3.2 ng / mL.
  • Another object of the present invention is to provide a novel cryptococcal capsular polysaccharide antigen immunoassay kit for detecting GXM concentration.
  • the application of the above-mentioned novel cryptococcal capsular polysaccharide antigen immunoassay kit for detecting GXM concentration is a competitive ELISA method (one-step method), and the specific steps are as follows:
  • step (2) mixing the substance to be detected in step (1) with an anti-GXM polyclonal enzyme-labeled antibody in an equal volume and adding it to a GXM-coated ELISA plate, incubating for 20-60 min, and washing the plate after the incubation;
  • the application of the above-mentioned novel cryptococcal capsular polysaccharide antigen immunoassay kit for detecting GXM concentration is a competitive ELISA method (two-step method), and the specific steps are as follows:
  • step (2) mixing the analyte of step (1) with an anti-GXM polyclonal enzyme-labeled antibody in an equal volume, and incubating for 20-60 min;
  • step (3) adding the mixture of step (2) to the GXM-coated ELISA plate and incubating for 20-60 minutes, and washing the plate after the incubation;
  • GXM is coated on a solid phase carrier to form a solid phase antigen
  • reaction substrate TMB of the enzyme is added, and the color depth is negatively correlated with the concentration of GXM in the sample to be tested;
  • the absorbance (A value) is measured at a certain wavelength by a microplate reader, and the antigen is detected by a standard curve.
  • the novel cryptococcus used in the present invention is purchased from the China Medical Microbial Culture Collection Management Center.
  • the capsular polysaccharide monoclonal antibody used in the present invention is provided by Dana (Tianjin) Biotechnology Co., Ltd.
  • the preparation method of the novel cryptococcal capsular polysaccharide GXM provided by the invention uses the immunoaffinity chromatography to purify the crude extract of the cryptococcal capsular polysaccharide GXM, thereby effectively avoiding the use of toxic chemical reagents and ensuring the operation of the operator. It is safe, avoids environmental pollution, and has high specificity. It can prepare high-purity cryptococcal capsular polysaccharide while simplifying the preparation process.
  • the prepared polyclonal antibody is produced by using the prepared GXM as an immunogen, and the obtained polyclonal antibody has the characteristics of high potency and good specificity, and the polyclonal antibody has stable properties and has a strong application prospect.
  • the GXM antigen immunoassay kit is prepared by using the above GXM and anti-GXM polyclonal antibodies, and the kit is competitive. The method firstly coats the GXM on the ELISA plate, and then competes with the coated antigen for binding to the coated antigen to bind a limited antibody binding site, and then reacts with the substrate to develop a color reaction, and then performs detection and calculation. Antigen concentration.
  • the detection kit has good sensitivity, specificity, repeatability and stability, and has high recovery rate for the target compound, and can provide more accurate and reliable test results.
  • the kit is easy to use and fast to detect. Sensitive and inexpensive, it provides an effective tool for clinical testing of GXM.
  • Figure 1 shows the results of quantitative detection of the novel cryptococcal capsular polysaccharide GXM provided by the present invention.
  • Fig. 3 shows the results of SDS-PAGE heteroprotein content analysis of the novel cryptococcal capsular polysaccharide GXM provided by the present invention.
  • Figure 4 shows the results of SDS-PAGE detection of rabbit IgG type polyclonal antibodies provided by the present invention.
  • Fig. 5 shows the results of titer determination of a rabbit IgG type polyclonal antibody provided by the present invention.
  • Figure 6 is a diagram showing the standard curve of the GXM immunodetection kit provided by the present invention.
  • a YL broth medium of 0.6 L was prepared, and its composition was 1.8 g of yeast extract, 3 g of peptone, 1.8 g of maltose extract, and 6 g of D-glucose. Cultivating Cryptococcus neoformans, the culture temperature is 30 ° C, the shaking speed is 200 rpm, and the culture time is 36 h;
  • the novel Cryptococcus capsular polysaccharide GXM monoclonal antibody is bound to the protein A microbeads.
  • the antibody and protein A can be mixed into a thin homogenate with about 2 mg of monoclonal antibody per milliliter of wet microbeads.
  • About 1 mL of microbeads are added to a total amount of 10 mL of the solution, incubated at room temperature for 1 h, and gently shaken to mix;
  • microbead-antibody cross-linking complex obtained in the step (4) is resuspended in a 0.2 mol/L ethanolamine solution, incubated at room temperature for 2 h, and gently mixed;
  • microbead-antibody cross-linking complex obtained in the step (5) is washed with PBS, resuspended in PBS, and added with thimerosal until the final concentration is 0.01%;
  • microbead-antibody cross-linking complex obtained in the step (6) is tested and cross-linked successfully, it is filled into a chromatography column to prepare a novel cryptococcal capsular polysaccharide immunoaffinity chromatography column, and PBS is used. Rinse the container and collect the remaining beads. If possible, use only the antibody microbead matrix required for all GXM in the preparation to be purified.
  • the starting buffer, the binding buffer, the pre-elution buffer, and the elution buffer were respectively high-purity water, pH 7 PBS buffer, pH 9 carbonate buffer, and pH 3.0 0.1 M glycine buffer.
  • the sugar content is determined by the A. Dubois-sulfate phenol method, and the specific steps are as follows:
  • test tube water bath for about 20 minutes, cooling to room temperature
  • the standard curve is shown in Figure 1.
  • the linearity is very good and can be used to calculate the concentration of the sample to be tested.
  • Sensitivity setting range 1.0 ⁇ 10 -6 -5.0 ⁇ 10 -4 RIU
  • the HPLC injection was carried out by a refractive index detector. The results are shown in Fig. 2. A single absorption peak appeared, indicating that the sample was of good purity.
  • the gel chart is shown in Fig. 3. As can be seen from the comparison, the cryptococcal capsular polysaccharide sample prepared in Example 3 has high purity and no impurity protein.
  • the phenolic acid method was used for quantification, and the results are shown in Table 1.
  • the total amount of sugar obtained after purification was CV ⁇ 10%;
  • the SDS-PAGE heteroprotein content analysis showed no impurity protein bands.
  • the specific cryptococcal GXM immunoaffinity chromatography column prepared according to the procedure of Example 2 has high specificity and recovery rate for the target compound, and has good repeatability and stability, and is used for novel hiddenness.
  • the purified preparation of the capsular polysaccharide GXM can effectively reduce the purification preparation cost, simplify the preparation steps and improve the preparation efficiency.
  • Control substance a novel cryptococcal capsular polysaccharide prepared according to the method disclosed in the patent document CN201110240065.X.
  • the capsular polysaccharide purified by the two methods was coated onto the ELISA plate at 10 ng/well, 25 ng/well, 50 ng/well, 100 ng/well;
  • Termination 50 ⁇ L of the stop solution was added to each well, and after mixing, the absorbance at 450 nm was read.
  • the GXM antigen is mixed with the Freund's complete adjuvant in an equal volume to the appropriate volume. After full emulsification, New Zealand big ears rabbits were injected subcutaneously, and the immune dose of each rabbit was controlled at 0.01-1 mg. Ear blood was taken 3 days before immunization, and serum was separated as a negative control. The immunization was performed once every 2 weeks after the initial immunization, and the method was the same as the first time.
  • a polyclonal antibody against the Cryptococcus neoformans capsular polysaccharide GXM antigen was obtained by eluting with 2-5 bed volumes of an elution buffer (0.1 mol/L glycine-hydrochloric acid buffer at pH 2.8).
  • the anti-GXM polyclonal antibody prepared in Example 7 was subjected to SDS-PAGE electrophoresis, and the obtained gel was subjected to Coomassie blue staining.
  • the experimental results are shown in Figure 4 (pAb lane is anti-GXM polyclonal antibody prepared in Example 7, and M lane is protein Marker).
  • pAb lane is anti-GXM polyclonal antibody prepared in Example 7, and M lane is protein Marker.
  • Antibody titers were determined by indirect ELISA.
  • the enzyme-labeled secondary antibody used was horseradish peroxidase-labeled goat anti-rabbit IgG, and the negative control was PBS solution.
  • the test results are shown in Figure 5. As can be seen from the results, the antibody titer is very high, greater than 1:1 ⁇ 10 6 .
  • the coating buffer is a 0.01 mol/L PBS buffer solution having a pH of 7.0-7.4;
  • the preparation of the blocking solution 0.01 mol/L PBS buffer solution containing 3% skim milk powder at pH 7.0-7.4;
  • the GXM antigen was diluted to a concentration of 100, 32, 10, 6.4, and 3.2 ng/mL with a sample diluent;
  • the enzyme used for labeling is AP, and the preparation of anti-GXM polyclonal enzyme-labeled antibody is carried out by glutaraldehyde cross-linking method;
  • the AP-labeled anti-GXM polyclonal enzyme-labeled antibody is diluted with an AP conjugate stabilizer at a ratio of 1:2000;
  • Sample treatment solution dissolving disodium edetate in ultrapure water to prepare 0.05 mol/L EDTA solution, the pH value is 4.0-4.8;
  • Concentrated lotion 96.0 parts of sodium chloride, 2.40 parts of potassium chloride, 42.96 parts of disodium hydrogen phosphate dodecahydrate, 2.88 parts of potassium dihydrogen phosphate, Tween-20 0.05 parts, ultrapure water 1000 Share
  • Substrate solution p-NPP solution
  • Stop solution 54.7mL concentrated sulfuric acid was dissolved in high purity water and diluted to 100mL to obtain 10mol/L sulfuric acid solution;
  • the above anti-GXM polyclonal enzyme antibody, GXM standard, sample treatment solution, concentrated washing solution, sample diluent, substrate solution and stop solution are respectively filled into corresponding reagent bottles, and the above reagent bottle is made of sponge
  • the rack is fixed and placed in the kit case together with the GXM coated enzyme carrier and the sealing membrane.
  • the GXM coating solution was diluted to 25 ng/100 ⁇ L with a 0.1 mol/L PBS buffer solution of pH 7.6-8.0, and the remaining steps were the same as in Example 9.
  • the GXM coating solution was diluted to 25 ng/100 ⁇ L with a 0.2 mol/L PBS buffer solution of pH 7.6-8.0, and the remaining steps were the same as in Example 9.
  • the GXM coating solution was diluted to 25 ng/100 ⁇ L with a 0.05 mol/L CBS buffer solution of pH 9.0-9.6, and the remaining steps were the same as in Example 9.
  • the GXM coating solution was diluted to 25 ng/100 ⁇ L with a 0.1 mol/L CBS buffer solution of pH 9.0-9.6, and the remaining steps were the same as in Example 9.
  • the GXM coating solution was diluted to 25 ng/100 ⁇ L with a 0.2 mol/L CBS buffer solution of pH 9.0-9.6, and the remaining steps were the same as in Example 9.
  • the GXM coating solution was diluted to 25 ng/100 ⁇ L with a 0.05 mol/L Tris buffer solution having a pH of 10.0-10.6, and the remaining steps were the same as in Example 9.
  • the coating buffer is a 0.01 mol/L PBS buffer solution having a pH of 7.2-7.4;
  • the preparation of the blocking solution a 0.1 mol/L PBS buffer solution containing 4% skim milk powder at pH 7.2-7.4;
  • the GXM antigen was diluted to a concentration of 100, 32, 10, 6.4, and 3.2 ng/mL with a sample diluent;
  • the enzyme used for labeling is HRP, and the preparation of anti-GXM polyclonal enzyme-labeled antibody is carried out by periodate oxidation method;
  • HRP-labeled anti-GXM polyclonal enzyme-labeled antibody is diluted with HRP conjugate stabilizer at a ratio of 1:10000;
  • Sample treatment solution dissolving disodium edetate in ultrapure water to prepare 0.1 mol/L EDTA solution, the pH value is 4.0-4.8;
  • Concentrated lotion 96.0 parts of sodium chloride, 2.40 parts of potassium chloride, 42.96 parts of disodium hydrogen phosphate dodecahydrate, 2.88 parts of potassium dihydrogen phosphate, Tween-20 0.05 parts, ultrapure water 1000 Share
  • Sample diluent artificial cerebrospinal fluid
  • Substrate solution TMB solution
  • Stopping liquid diluting concentrated sulfuric acid and ultrapure water in a volume ratio of 1:8 to obtain a 2 mol/L sulfuric acid solution;
  • the above anti-GXM polyclonal enzyme antibody, GXM standard, sample treatment solution, concentrated washing solution, sample diluent, substrate solution and stop solution are respectively filled into corresponding reagent bottles, and the above reagent bottle is made of sponge
  • the rack is fixed and placed in the kit case together with the GXM coated enzyme carrier and the sealing membrane.
  • the blocking solution was a 0.2 mol/L PBS buffer solution containing 5% non-fat dry milk at pH 7.6-8.0, and the remaining steps were the same as in Example 16.
  • the blocking solution was a 0.2 mol/L PBS buffer solution containing 1% BSA at pH 7.6-8.0, and the remaining steps were the same as in Example 16.
  • the blocking solution was a 0.2 mol/L PBS buffer solution containing 2% BSA at pH 7.6-8.0, and the remaining steps were the same as in Example 16.
  • the blocking solution was a 0.2 mol/L PBS buffer solution containing 4% BSA at pH 7.6-8.0, and the remaining steps were the same as in Example 16.
  • the coating buffer is a 0.1 mol/L PBS buffer solution of pH 7.4 (4.25 parts of sodium chloride, 15.40 parts of disodium hydrogen phosphate dodecahydrate, 0.95 parts of potassium dihydrogen phosphate, by weight, 500 parts of ultrapure water);
  • the preparation of the blocking solution a 0.1 mol/L PBS buffer solution containing 4% of BSA at pH 7.2-7.4;
  • the GXM antigen was diluted to a concentration of 100, 32, 10, 6.4, and 3.2 ng/mL with a sample diluent;
  • the enzyme used for labeling is HRP, and the preparation of anti-GXM polyclonal enzyme-labeled antibody is carried out by periodate oxidation method;
  • HRP-labeled anti-GXM polyclonal ELISA antibody was diluted with HRP conjugate stabilizer at a ratio of 1:20000;
  • Sample treatment solution dissolving disodium edetate in ultrapure water to prepare a 0.2 mol/L EDTA solution, the pH of which is 4.0-4.8;
  • Concentrated lotion 96.0 parts of sodium chloride, 2.40 parts of potassium chloride, 42.96 parts of disodium hydrogen phosphate dodecahydrate, 2.88 parts of potassium dihydrogen phosphate, Tween-20 0.05 parts, ultrapure water 1000 Share
  • Substrate solution TMB solution
  • the above anti-GXM polyclonal enzyme antibody, GXM standard, sample treatment solution, concentrated washing solution, sample diluent, substrate solution and stop solution are respectively filled into corresponding reagent bottles, and the above reagent bottle is made of sponge
  • the rack is fixed and placed in the kit case together with the GXM coated enzyme carrier and the sealing membrane.
  • the sample treatment liquid was prepared by dissolving sodium dodecyl sulfate in ultrapure water to prepare a 0.01 mol/L SDS solution having a pH of 8.5 to 10.0, and the remaining steps were the same as those in Example 21.
  • the sample treatment liquid was prepared by dissolving sodium dodecyl sulfate in ultrapure water to prepare a 0.05 mol/L SDS solution having a pH of 8.5 to 10.0, and the remaining steps were the same as those in Example 21.
  • the sample treatment liquid was prepared by dissolving sodium dodecyl sulfate in ultrapure water to prepare a 0.1 mol/L SDS solution having a pH of 8.5 to 10.0, and the remaining steps were the same as those in Example 21.
  • the sample treatment solution was prepared by dissolving glycine in ultrapure water, preparing a 0.07 mol/L glycine solution, and adjusting the pH to pH 2.2-2.8 with a concentrated HCL solution. The remaining steps were the same as in Example 21.
  • the sample treatment solution was prepared by dissolving glycine in ultrapure water, preparing 0.13 mol/L of glycine, and adjusting the pH to pH 2.2-2.8 with concentrated HCL solution. The other steps were the same as in Example 21.
  • the sample treatment solution was prepared by dissolving glycine in ultrapure water, preparing 0.2 mol/L glycine, and adjusting the pH to pH 2.2-2.8 with concentrated HCL solution. The other steps were the same as in Example 21.
  • the sample treatment liquid is prepared by dissolving the chain enzyme protease in ultrapure water to prepare a 0.05 mg/mL chain enzyme protease solution having a pH of 8.0-9.0, and the remaining steps are the same as in Example 21.
  • the sample treatment solution is prepared by dissolving the chain enzyme protease in ultrapure water to prepare a 5 mg/mL chain enzyme protease solution having a pH of 8.0-9.0, and the remaining steps are the same as in Example 21.
  • the sample treatment liquid was prepared by dissolving the chain enzyme protease in ultrapure water to prepare a 15 mg/mL chain enzyme protease solution having a pH of 8.0-9.0, and the remaining steps were the same as those in Example 21.
  • the sample treatment liquid is prepared by dissolving urea in ultrapure water to prepare 1 mol/L urea, and the pH is pH 7.2-8.0, and the remaining steps are the same as in Example 21.
  • the sample treatment liquid is prepared by dissolving urea in ultrapure water to prepare 4 mol/L urea, and the pH is pH 7.2-8.0, and the remaining steps are the same as in Example 21.
  • the preparation method of the sample treatment liquid is: dissolving urea in ultrapure water, and preparing urea of 8 mol/L, and the pH value thereof is The pH was 7.2-8.0, and the remaining steps were the same as in Example 21.
  • Standard curve group each standard curve point (3.2, 6.4, 10, 32 and 100 ng/mL);
  • Sample group to be tested the sample to be tested after the step 1 is repeatedly tested 10 times;
  • the two groups were separately mixed with an anti-GXM enzyme-labeled antibody in an equal volume and incubated at 37 ° C for 20 min.
  • Washing remove the liquid in the well of the microplate, add not less than 300 ⁇ L of working lotion per well, lap dry after standing for 40 s, repeat the above washing operation, and wash a total of 5 times;
  • Termination 50 ⁇ L of stop solution was added to each well, and after mixing, the absorbance at 405 nm was read;
  • the test results of the kits prepared in Examples 9-15 are shown in Table 4. As can be seen from the data in Table 6, the CV values of the test results of the kits are less than 7%, indicating that the kits are sample pairs. The detection results are less discrete and reproducible, and can be used for immunodetection of the cryptococcal capsular polysaccharide antigen; and the kit prepared in Example 10 has the smallest CV value of 2.6%, indicating that the present embodiment is present in the present embodiment. In the test of the example, the kit was the most reproducible.
  • Standard curve group each standard curve point (3.2, 6.4, 10, 32 and 100 ng/mL);
  • Sample group to be tested the tested sample to be tested is repeatedly tested 10 times;
  • the two groups were separately mixed with an anti-GXM enzyme-labeled antibody in an equal volume and incubated at 37 ° C for 40 min.
  • Washing remove the liquid in the well of the microplate, add not less than 300 ⁇ L of working lotion per well, lap dry after standing for 40 s, repeat the above washing operation, and wash a total of 5 times;
  • Termination 50 ⁇ L of stop solution was added to each well, and after mixing, the absorbance at 450 nm was read;
  • the test results of the kits prepared in Examples 16-20 are shown in Table 7. As can be seen from the data in Table 7, the CV values of the test results of the kits are less than 6%, indicating that each kit is a sample. The detection result is less discrete and reproducible, and can be used for immunodetection of the novel cryptococcal capsular polysaccharide antigen; and the kit prepared in Example 16 has the smallest CV value of 2.3%, indicating that the present embodiment is present in the present embodiment. In the test of the example, the kit was the most reproducible.
  • Standard curve group each standard curve point (3.2, 6.4, 10, 32 and 100 ng/mL);
  • Sample group to be tested the tested sample to be tested is repeatedly tested 10 times;
  • the two groups were separately mixed with an anti-GXM enzyme-labeled antibody in an equal volume and incubated at 37 ° C for 60 min.
  • Washing remove the liquid in the well of the microplate, add not less than 300 ⁇ L of working lotion per well, lap dry after standing for 40 s, repeat the above washing operation, and wash a total of 5 times;
  • Termination 50 ⁇ L of stop solution was added to each well, and after mixing, the absorbance at 450 nm was read;
  • the test results of the kits prepared in Examples 21-33 for the same sample are shown in Table 8.
  • the CV values of the test results of the kits for each sample were less than 7%, indicating that each kit was against the sample.
  • the detection result is less discrete and reproducible, and can be used for immunodetection of the novel cryptococcal capsular polysaccharide antigen; and the kit prepared in Example 26 has the smallest CV value of 2.8%, indicating that the present embodiment is present in the present embodiment.
  • the kit was the most reproducible.
  • Example 16 The kit prepared in Example 16 was subjected to the procedure of Example 35 to obtain measurement values of each standard curve point (3.2, 6.4, 10, 32, and 100 ng/mL) as shown in Table 9, using the data of Table 9 to sample
  • the logarithm of the concentration of the GXM antigen is the horizontal axis (x-axis), and the absorbance measured at 450 nm is the vertical axis (y-axis).
  • the standard curve is shown in Fig. 6.
  • the standard curve equation is:
  • the concentration of the detected antigen was calculated based on the results of the standard curve.
  • the concentration of the antigen in the 95% confidence interval was the Cut-off upper limit:
  • the concentration of the antigen in the 95% confidence interval was the Cut-off lower limit:
  • the concentration of the antigen is between 6 ng/ml and 10 ng/ml as a suspected patient. That is, the judgment standard reference value of the GXM immunoassay kit is shown in Table 11.
  • test result of the sample falls within the suspected interval, a second test is required to determine the result.
  • Example 19 Using the kit prepared in Example 19, 20 clinically confirmed samples were tested according to the procedure of Example 35.
  • Diagnostic sensitivity number of positive samples detected / total number of positive samples ⁇ 100%, the experimental results are shown in Table 12, from the data in Table 12, the sensitivity of the GXM immunoassay kit used in this experiment is above 85%. .
  • Example 19 The kit prepared in Example 19 was tested and tested for 20 healthy human samples as in Example 35.
  • the antigen concentration was measured according to the procedure of Example 35 using the kit prepared in Example 19, and the ratio of the actual value to the expected value was calculated to obtain a recovery. Rate, recovery rate between 80-120% is considered qualified.
  • the experimental results are shown in Table 14. The data in Table 14 shows that the recovery rate of the GXM immunoassay kit used in this experiment is between 80% and 120%, and the recovery rate is good.
  • Eligibility Criteria The same sample was tested in parallel for 10 sets of data in the same batch of experiments, each time measured twice, averaged, and the corresponding antigen concentration was calculated. The mean value M, the standard deviation SD and the coefficient of variation CV were calculated, and the coefficient of variation CV ⁇ 15% was qualified.
  • the intra-assay precision (ie, coefficient of variation CV) of the GXM immunoassay kit provided by the present invention is 7%, less than 15%, meets the standard, and is qualified.
  • the GXM immunoassay kit prepared in Example 18 was placed in an environment of 37 ° C, and a standard control solution (55 ⁇ g / L) of a known concentration was detected by a standard curve every day for 5 days, and the rate of change of the detection value (ie, variation) The coefficient CV) is less than 20%, demonstrating that the kit is stable.
  • the experimental results are shown in Table 17. From the data in Table 17, the 5D coefficient of variation CV ⁇ 20% of the GXM immunoassay kit prepared in Example 9 was used to demonstrate the stability of the GXM immunoassay kit provided by the present invention. Good sex.

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Abstract

一种隐球菌荚膜多糖GXM的制备方法及GXM抗原免疫检测试剂盒。制备方法包括新型隐球菌荚膜多糖GXM的粗提和利用GXM免疫亲和层析柱进行隐球菌荚膜多糖GXM粗提物的纯化步骤。利用该方法制备得到的纯化后的GXM制备GXM抗原免疫检测试剂盒，为GXM的临床检测提供了一种有效工具。

Description

一种新型隐球菌荚膜多糖GXM的制备方法及GXM抗原免疫检测试剂盒及其应用 技术领域
本发明涉及免疫检测技术领域，具体涉及一种新型隐球菌荚膜多糖葡萄糖醛酸木糖甘露聚糖(Glucuronoxylomannan，GXM)的制备方法、针对该GXM抗原的多克隆抗体及其制备方法、针对该GXM抗原的免疫检测试剂盒及其制备方法与应用。
发明背景
新型隐球菌(Crytococcus Neoformans)是一种重要的条件致病菌，常感染免疫能力低下或免疫能力缺陷的患者，导致以中枢神经系统感染为主的深部真菌感染，病死率很高。美国疾病预防与控制中心(CDC)于1992-1993年进行的大系列流行病学研究显示，深部真菌感染的年发病率为178.3/百万。其中，隐球菌病为65.5/百万，约占36.7％。
近年来由于广谱抗菌药物、肾上腺皮质激素、肿瘤化疗、放疗和器官移植后免疫抑制剂的长期广泛应用，以及艾滋病的流行，隐球菌病明显增多。新型隐球菌性脑膜炎(以下简称“隐脑”)就是由新型隐球菌所致的最常见的中枢神经系统疾病，占隐球菌病的80％左右。隐脑的临床表现复杂，症状不典型，因而很难诊断，约80％的隐脑患者会被误诊为结核性脑膜炎。
新型隐球菌的主要致病因素为荚膜、产黑素及其在宿主体温环境生长的能力等，其中荚膜为其毒性的主要决定因子之一，其主要成分是荚膜多糖，具有抑制吞噬和激活补体的作用，其中含量最多的是葡萄糖醛酸木糖甘露聚糖(GXM)。在隐球菌性脑膜炎的早期诊断方法上，Wang等采用乳胶凝集试验检测隐球菌荚膜多糖抗原，用真菌培养和直接镜检法评估诊断的灵敏度和特异性，结果发现乳胶凝集试验、真菌培养和直接镜检的灵敏度分别为91.1％、69.6％和73.2％，特异性分别为96.0％、100％和100％，由此认为乳胶凝集试验检测隐球菌荚膜多糖抗原可作为对新型隐球菌性脑膜炎早期诊断方法。对隐球菌性脑膜炎进行隐球菌乳胶凝集试验对诊断隐球菌性脑膜炎特异性高，使早期诊断率得以提高(Wang H,Yuan X,Zhang L.Latex agglutination:Diagnose the early cryptococcus neoformans test of capsular polysaccharide antigen.Pakistan journal of pharmaceutical sciences,2015,28(1Suppl):307-311.)。因此，新型隐球菌荚膜多糖对隐球菌性脑膜炎的早期诊断非常重要。
Kozel等用培养罐培养新型隐球菌，培养液经过高压灭菌后，离心除去菌体，上清液先用0.45um滤膜过滤，然后超滤浓缩，并向浓缩液中加入醋酸钠和冰醋酸，接下来用乙醇沉淀出粗制荚膜多糖，然后用氯仿/正丁醇(v:v＝5:1)进行反复抽提，除去蛋白，再用乙醇将荚膜多糖沉淀出来，离心收集沉淀，将沉淀溶于去离子水，透析，冻干得到精制荚膜多糖(Kozel T R,Cazin J.Nonencapsulated variant of Cryptococcus neoformans I.Virulence studies and characterization of soluble polysaccharide.Infection and immunity,1971,3(2):287-294.)。上述方法中需使用大量的氯仿，氯仿是有毒有害的化学危险品，有致癌的危险性，对工作条件、操作人员的保护、污水处理等有较高要求。
Bryan等将新型隐球菌细胞用蒸馏水洗涤3次，离心收集细胞，用15mL二甲基亚砜(DMSO)重悬收集的湿细胞并孵育30分钟，然后通过离心将细胞沉淀分离，将DMSO上清液透析12小时，每2小时用水置换，得到的样品用蒸馏水或1mM EDTA充分透析3天，将透析得到的多糖溶液冷冻干燥得到荚膜多糖(Bryan R A,Zaragoza O,Zhang T,等。Radiological studies reveal radial differences in the architecture of the polysaccharide capsule of Cryptococcus neoformans.Eukaryotic Cell,2005,4(2):465-475.)。上述方法的制备周期较长，产品的产率不高。
Maxson等将隐球菌细胞培养液离心，分离细胞沉淀，将所得上清液用聚醚砜超滤盘进行浓缩约20倍，浓缩的过程中不断搅拌，在滤盘上形成黏性薄膜后，排出流体相，用细胞刮刀回收胶凝材料，最后进行冷冻干燥得到荚膜多糖(Maxson M E,Dadachova E, Casadevall A,等。Radial mass density,charge,and epitope distribution in the Cryptococcus neoformans capsule.Eukaryotic cell,2007,6(1):95-109.)。上述方法操作简单，制备周期较短，但产品的产率也不高。
另外，Frases等通过实验发现隐球菌的胞外多糖和荚膜多糖在结构上是不同的，并在试验中发现利用十六烷基三甲基溴化铵沉淀得到的多糖与通过浓缩和超滤得到的多糖(参照Maxson M E,Dadachova E,Casadevall A,等。Radial mass density,charge,and epitope distribution in the Cryptococcus neoformans capsule.Eukaryotic cell,2007,6(1):95-109.中的提取方法)相比，质量更大，二者构象也不同，由此判断常用的隐球菌荚膜多糖的提取方法会显著影响产物的结构和抗原性(Frases S,Nimrichter L,Viana N B,等.Cryptococcus neoformans capsular polysaccharide and exopolysaccharide fractions manifest physical,chemical,and antigenic differences.Eukaryotic cell,2008,7(2):319-327.)。
目前，本领域迫切需要开发一种简便、高效、低成本、特异性高的新型隐球菌荚膜多糖的制备方法，并能够有效地避免使用有毒有害的化学试剂。本发明克服现有技术的不足，提供了一种新型隐球菌荚膜多糖GXM的制备方法及制备得到的GXM，并利用该GXM制备检测试剂盒，制备得到的GXM纯度和特异性较高，利用该抗原制备的检测试剂盒检测灵敏度较高。
发明内容
本发明提供一种新型隐球菌荚膜多糖GXM免疫亲和层析柱的制备方法，所述亲和层析柱利用新型隐球菌荚膜多糖GXM单克隆抗体制备得到。
优选的，所述制备方法包括如下步骤：
(1)将新型隐球菌荚膜多糖GXM单克隆抗体溶于溶液中，并与亲和层析基质混合成稀薄的匀浆；
(2)用交联缓冲液洗涤步骤(1)得到的匀浆，并离心，得到抗体与亲和层析基质的混合物；
(3)用交联缓冲液重悬步骤(2)得到的抗体与亲和层析基质的混合物，向得到的混悬液中加入双功能结合剂，孵育，混匀，液固分离，得到亲和层析基质-抗体交联复合物；
(4)用封闭溶液洗涤步骤(3)得到的亲和层析基质-抗体交联复合物来封闭层析基质中多余的活性基团以终止交联反应；
(5)将步骤(4)得到的亲和层析基质-抗体交联复合物重悬于封闭溶液中，孵育，混匀；
(6)装柱：将步骤(5)得到的亲和层析基质-抗体交联复合物经检测交联成功后，填充入层析柱中，制得新型隐球菌荚膜多糖GXM免疫亲和层析柱；
优选的，(7)保存：步骤(6)得到的亲和层析基质-抗体交联复合物或步骤(6)得到的填充好的新型隐球菌荚膜多糖GXM免疫亲和层析柱在4℃条件下保存，1年内性能稳定；
优选的，(8)再生：步骤(7)中在4℃条件下保存的层析柱或使用后的层析柱在使用前用10-25倍柱床体积的待纯化样品的溶剂相同的缓冲液洗柱再生。
优选的，将步骤(5)得到的亲和层析基质-抗体交联复合物进行防腐处理后再填充入层析柱中，所述的防腐处理为将步骤(5)得到的亲和层析基质-抗体交联复合物用PBS缓冲液洗涤，并重悬于PBS缓冲液中，加入硫柳汞保存；所述的硫柳汞的浓度为0.005-0.015％。
优选的，步骤(1)中所述的亲和层析基质选自蛋白A微珠、蛋白G微珠、活性微珠，优选蛋白A微珠。
优选的，步骤(1)中所述的溶液选自pH8.0-9.0碳酸盐缓冲液。
优选的，步骤(1)中亲和层析基质和溶液的比例为：每10mL溶液中加入0.5-2.0mL 亲和层析基质，亲和层析基质与抗体的比例为：每1mL亲和层析基质结合1-4mg单克隆抗体；进一步优选的，步骤(1)中亲和层析基质和溶液的比例为：每10mL溶液中加入1mL亲和层析基质，亲和层析基质与抗体的比例为：每1mL亲和层析基质结合2mg单克隆抗体。
优选的，步骤(2)中所述的交联缓冲液为0.1-0.3mol/L的pH8.0-9.5的硼酸钠溶液，用量为5-15倍亲和层析基质体积，进一步优选的，所述交联缓冲液为浓度为0.2mol/L的pH9.0的硼酸钠溶液，用量为10倍亲和层析基质体积；所述的洗涤次数为1-3次，离心条件为3000g离心2-5分钟或10000g离心30秒。
优选的，步骤(3)中所述交联缓冲液为0.1-0.3mol/L的pH8.3-9.5的硼酸钠溶液，用量为5-15倍亲和层析基质体积；进一步优选的，所述交联缓冲液为浓度为0.2mol/L的pH9.0的硼酸钠溶液，用量为10倍亲和层析基质体积。
优选的，步骤(3)所述的双功能结合剂选自二甲基庚二酸酯、羰基二咪唑、溴化氰、羟基丁二酰亚胺、乙酰基碘，优选二甲基庚二酸酯，用量为使其在亲和层析基质混悬液中的终浓度为15-25mmol/L，优选为20mmol/L。
优选的，步骤(4)和(5)所述的封闭溶液选自乙醇胺、氨基乙烷等含可结合氨基的活性基团的小分子物质的溶液，优选为乙醇胺溶液，进一步优选为浓度为0.1-0.25mol/L，pH值为7.5-8.5的乙醇胺溶液，更优选为0.2mol/L的pH8.0的乙醇胺溶液。
优选的，步骤(6)中所述的亲和层析基质-抗体交联复合物的交联效率的检测过程为：取亲和层析基质样品及亲和层析基质与抗体交联后的样品，分别加入LaemmLi缓冲液中煮沸，分别取出相当于1mL和9mL的两种样品，在10％SDS-聚丙烯酰胺凝胶中进行电泳，用考马斯亮蓝染色，如交联前样品呈现重链区带(55kDa)，而交联后样品则无，表明交联成功。
优选的，步骤(6)中所述的装柱完成后，用PBS缓冲液冲洗容器，收集残留的亲和层析基质，如果可能，仅使用待纯化样品中全部GXM所需的亲和层析基质-抗体交联复合物。
本发明还提供一种上述方法制备得到的新型隐球菌荚膜多糖GXM免疫亲和层析柱。
本发明还提供一种新型隐球菌荚膜多糖GXM的制备方法，所述制备方法包括利用上述GXM免疫亲和层析柱进行GXM的纯化的步骤；
优选的，所述制备方法包括如下步骤：
(1)新型隐球菌荚膜多糖GXM的粗提；
(2)利用新型隐球菌荚膜多糖GXM免疫亲和层析柱进行新型隐球菌荚膜多糖GXM粗提物的纯化。
所述的新型隐球菌荚膜多糖GXM的粗提，包括如下步骤：
(1)培养新型隐球菌，至菌浓度达到对数期后期；
(2)将菌液高压灭菌，离心除去菌体，保留上清液；
(3)向上清液中边搅拌边缓慢加入醋酸钙粉末，然后加冰醋酸调pH至酸性；
(4)向步骤(3)得到的溶液中加入乙醇，4℃静置过夜，离心，弃去上清，干燥沉淀，得到新型隐球菌荚膜多糖GXM的粗提物。
优选的，所述的新型隐球菌荚膜多糖的粗提的步骤(1)中的新型隐球菌的培养条件为：培养基采用YM肉汤培养基，培养温度为30℃，震荡速度为200rpm，培养时间为32-40小时，所述的YM肉汤培养基配方为每升培养基含：3g酵母提取物，5g蛋白胨，3g麦芽糖提取物，10g D-葡萄糖。
优选的，步骤(3)中所述的醋酸钙粉末的加入量为其在所述上清液中的终浓度达到2-8％，冰醋酸调节pH值至4.6-5.2。
优选的，步骤(4)中所述的乙醇的浓度为90-98％，加入量为步骤(3)得到的溶液体积的2-4倍。
所述的利用新型隐球菌荚膜多糖GXM免疫亲和层析柱进行新型隐球菌荚膜多糖GXM 粗提物的纯化，包括如下步骤：
(1)用与待纯化样品溶液相同的缓冲液冲洗上述新型隐球菌荚膜多糖GXM免疫亲和层析柱；
(2)使待纯化样品溶液流经步骤(1)处理后的层析柱；
(3)用结合缓冲液洗柱；
(4)用预洗脱缓冲液洗柱；
(5)采用分段洗脱，连续以洗脱缓冲液流过层析柱，分管收集每一组分；
(6)检测每管的GXM含量，将浓度高的各管合并。
优选的，(7)用10-25倍柱床体积的起始缓冲液流经层析柱使其再生，并加入硫柳汞使其终浓度为0.005-0.015％，在4℃条件下保存层析柱。
优选的，步骤(2)中所述的待纯化样品的流速为0.5-1.5mL/h，用泵控制流速，优选使用蠕动泵。
优选的，步骤(3)中所述的结合缓冲液选自pH6.0-8.0的PBS缓冲液、pH6.0-8.0的Tris-HCl缓冲液、pH6.0-8.0醋酸-醋酸钠缓冲液中的一种，优选pH6.0-8.0的PBS缓冲液；步骤(3)中所述的结合缓冲液的用量为10-25倍柱床体积，优选为20倍柱床体积。
优选的，步骤(4)中所述的预洗脱缓冲液为pH8.5-9.5碳酸盐缓冲液，用量为10-25倍柱床体积，进一步优选为20倍柱床体积。
优选的，步骤(5)中所述的洗脱缓冲液为pH3.0的缓冲液，优选pH3.0的0.1M甘氨酸缓冲液、pH3.0的柠檬酸-磷酸盐缓冲液、pH3.0的柠檬酸-柠檬酸钠缓冲液、pH3.0的醋酸-醋酸钠缓冲液，用量为0.4-0.8柱床体积。
优选的，步骤(6)得到的GXM洗脱液进行透析去盐处理或用缓冲液调节其pH值。
本发明提供一种上述方法制备得到的新型隐球菌荚膜多糖GXM。
本发明还提供一种新型隐球菌荚膜多糖GXM免疫亲和层析柱在纯化新型隐球菌荚膜多糖GXM和新型隐球菌性脑膜炎的早期诊断中的应用。
本发明还提供一种新型隐球菌荚膜多糖GXM在新型隐球菌性脑膜炎的早期诊断试剂中的应用。
本发明还提供一种抗新型隐球菌荚膜多糖GXM的多克隆抗体，其由下述方法制备得到的：用GXM作为免疫原免疫动物得到的血清进行分离纯化，得到抗体；所述纯化方法为饱和硫酸铵盐析沉淀法和/或亲和层析法，所述免疫方法可以为皮下注射、脾内注射、静脉注射或腹腔注射，免疫剂量为0.01-1mg/只，免疫的动物为大鼠、小鼠、豚鼠、兔、鸡、羊、马、猪或驴，优选兔。
上述抗GXM多克隆抗体的制备方法，具体步骤为：
(1)用GXM抗原免疫动物；
(2)测定免疫后动物的血清效价，从免疫后的动物体内取血；
(3)用饱和硫酸铵盐析沉淀法和/或亲和层析法进行初步纯化。
由上述步骤制备得到的抗GXM多克隆抗体可与GXM特异性结合，用SDS-PAGE显示为均一抗体产物，间接法ELISA检测显示其效价大于1:1×106。
本发明还提供一种新型隐球菌荚膜多糖GXM抗原免疫检测试剂盒，所述试剂盒包括：GXM包被的酶标载体、抗GXM多克隆酶标抗体、GXM标准品。
所述的GXM标准品由一系列不同浓度的GXM溶液组成，所述的GXM溶液是将GXM溶于合适的溶剂中形成具有特定浓度的GXM的溶液。
所述的GXM包被的酶标载体，由以下方法制备得到：
①用包被缓冲液稀释GXM，得到GXM包被液，加入酶标板孔中进行包被；
②向步骤①得到的酶标板孔中加入封闭液进行封闭；
③将步骤②得到的酶标板弃去封闭液，孵育，得到GXM包被的酶标载体。
优选的，所述的酶标载体为微孔板、塑料管；更优选的，所述的酶标载体为微孔板；
优选的，所述的用于标记的酶为辣根过氧化物酶(HRP)、碱性磷酸酶(AP)；更优选的， 所述的用于标记的酶为辣根过氧化物酶；
优选的，所述的GXM标准品至少有0-100ng/mL范围内的3种不同浓度；
在本发明的一个具体实施方式中，上述新型隐球菌荚膜多糖抗原免疫检测试剂盒中，所述GXM标准品有5种不同浓度，分别记为a、b、c、d和e，其浓度分别为100、32、10、6.4、3.2ng/mL。
优选的，上述新型隐球菌荚膜多糖抗原免疫检测试剂盒，还包括样本处理液、浓缩洗液、样本稀释液、底物溶液和终止液；
优选的，所述的浓缩洗液、样本稀释液、样本处理液、底物溶液和终止液的组分及配比如下：
浓缩洗液：含0.4-1.0％吐温的磷酸盐缓冲液；
样本稀释液：人工脑脊液或人工血清；
样本处理液选自以下溶液：pH2.0-10.0的蛋白变性溶液；pH4.0-4.8的0.05-0.2mol/L的EDTA(乙二胺四乙酸二钠)溶液；pH2.2-2.8的0.07-0.2mol/L的甘氨酸-HCL(甘氨酸-盐酸)溶液；pH8.0-9.0的0.05-15mg/mL的链酶蛋白酶；pH8.5-1.0的0.01-0.1mol/L的SDS(十二烷基磺酸钠)溶液或pH7.2-8.0的1-8mol/L的尿素；
底物溶液：TMB(四甲基联苯胺)、OPD(邻苯二胺)、OT(邻联甲苯胺)、ABTS(2,2'-连氮-双(3-乙基苯并噻唑-6-磺酸))或p-NPP(对硝基苯磷酸酯)；优选TMB；
终止液：1-20mol/L的硫酸溶液，优选2mol/L的硫酸溶液。
在本发明的一个具体实施方式中，所述的浓缩洗液的组分及配比为：按重量份数计，氯化钠96.0份、氯化钾2.40份、十二水合磷酸氢二钠42.96份、磷酸二氢钾2.88份、吐温-20 0.05份、超纯水1000份。
本发明还提供一种新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备方法，其具体步骤如下：
(1)制备酶标载体
①用包被缓冲液稀释GXM，得到GXM包被液，加入酶标板孔中进行包被；
②向步骤①得到的酶标板孔中加入封闭液进行封闭；
③将步骤②得到的酶标板弃去封闭液，孵育，得到GXM包被的酶标载体；
(2)GXM标准品的制备
稀释GXM抗原，制备GXM标准品；
(3)抗GXM多克隆酶标抗体溶液的配制
用于标记的酶为辣根过氧化物酶，采用过碘酸盐氧化法进行抗GXM多克隆酶标抗体的制备；或用于标记的酶为碱性磷酸酶，采用戊二醛交联法进行抗GXM多克隆酶标记抗体的制备；
将抗GXM多克隆酶标抗体用酶结合物稳定剂稀释制得抗GXM多克隆酶标抗体溶液。优选的，所述的新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备方法包括如下具体步骤：
(1)制备酶标载体
①用包被缓冲液将GXM稀释至25-2000ng/100μL，得到GXM包被液并加入酶标板孔中，每孔分别加入60-200μL包被液，酶标板置于2-8℃下包被8-16h；
②向步骤①得到的酶标板孔中加入封闭液，每孔分别加入60-200μL封闭液，置于37℃下封闭30-90min；
③将步骤②得到的酶标板弃去封闭液，置于37℃恒温下30-90min，得到GXM包被的酶标载体；
(2)GXM标准品的制备
将GXM抗原稀释成0-100ng/mL范围内的至少3种不同浓度；
(3)抗GXM多克隆酶标抗体溶液的配制
用于标记的酶为辣根过氧化物酶，采用过碘酸盐氧化法进行抗GXM多克隆酶标抗体的制备；或，用于标记的酶为碱性磷酸酶，采用戊二醛交联法进行抗GXM多克隆酶标记抗体的制备；
将抗GXM多克隆酶标抗体用酶结合物稳定剂以1:2000-1:20000的比例稀释制得抗GXM多克隆酶标抗体溶液；
(4)配制样本处理液、浓缩洗液、样本稀释液、底物溶液和终止液；
更优选的，所述的新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备方法还包括：
(5)将上述抗GXM多克隆酶标抗体、GXM标准品、样本处理液、浓缩洗液、样本稀释液、底物溶液和终止液封装并同GXM包被的酶标载体和封膜一同置于试剂盒盒体内。
优选的，所述的包被缓冲液选自：pH7.0-8.0的0.01-0.20mol/L的PBS(磷酸盐)缓冲溶液、pH9.0-9.6的0.05-0.20mol/L的CBS(碳酸盐)缓冲溶液或pH10.0-10.6的0.05mol/L的Tris(三羟甲基氨基甲烷)缓冲液。
优选的，所述的封闭液的组分及配比为：含3-5％的脱脂奶粉或1-4％BSA(牛血清白蛋白)的pH7.0-8.0的0.01-0.20mol/L的PBS缓冲溶液。
在本发明的一个具体实施方式中，所述GXM标准品由样本稀释液稀释GXM得到，有5种不同浓度，分别记为a、b、c、d和e，其浓度分别为100、32、10、6.4、3.2ng/mL。
本发明的另一个目的是提供一种新型隐球菌荚膜多糖抗原免疫检测试剂盒在检测GXM浓度方面的应用。
优选的，上述新型隐球菌荚膜多糖抗原免疫检测试剂盒检测GXM浓度的应用为竞争ELISA方法(一步法)，具体步骤如下：
(1)取待检样本与样本处理液以1:1-5:1的体积比混合，并煮沸1-10min后离心得到待检测物；
(2)将步骤(1)的待检测物与抗GXM多克隆酶标抗体等体积混匀并加入GXM包被的酶标板中，孵育20-60min，孵育完后洗板；
(3)向步骤(2)的酶标板中加入50-100μL的底物溶液显色，加入终止液后进行吸光度的检测。
优选的，上述新型隐球菌荚膜多糖抗原免疫检测试剂盒检测GXM浓度的应用为竞争ELISA方法(两步法)，其具体步骤如下：
(1)取待检样本与样本处理液以1:1-5:1的体积比混合，并煮沸1-10min后离心得到待检测物；
(2)将步骤(1)的待检测物与抗GXM多克隆酶标抗体等体积混匀，并孵育20-60min；
(3)将步骤(2)的混合物加入GXM包被的酶标板中并孵育20-60min，孵育完后洗板；
(4)向步骤(3)的酶标板中加入50-100μL的底物溶液显色，加入终止液后进行吸光度的检测。
上述新型隐球菌荚膜多糖抗原免疫检测试剂盒的应用原理如下：
1)将GXM包被在固相载体上，制成固相抗原；
2)将待检样本处理后与抗GXM多克隆酶标抗体混合并恒温反应；
3)将2)中的混合液加入1)的固相上，使待检抗原与包被抗原竞争结合有限的抗体结合位点；
4)经过恒温反应并彻底洗涤，再加入酶的反应底物TMB显色，颜色的深浅和待检样本中GXM浓度呈负相关；
5)用酶标仪在一定波长下测定吸光度(A值)，通过标准曲线实现对抗原的检测。
本发明所采用的新型隐球菌购自中国医学微生物菌种保藏管理中心。
本发明所采用的荚膜多糖单克隆抗体由丹娜(天津)生物科技有限公司提供。
本发明提供的新型隐球菌荚膜多糖GXM的制备方法，使用免疫亲和层析对新型隐球菌荚膜多糖GXM粗提物进行纯化，有效地避免了有毒化学试剂的使用，保证了操作人员的安全，同时避免了环境污染，且特异性高，在简化制备工艺的同时能制备出高纯度的新型隐球菌荚膜多糖。利用制备得到的GXM作为免疫原产生多克隆抗体，制得的多克隆抗体具有效价高，特异性好的特点，且多克隆抗体性质稳定，有很强的应用前景。利用上述GXM和抗GXM多克隆抗体制备得到GXM抗原免疫检测试剂盒，该试剂盒采用竞争 法，先将GXM包被在酶标板上，再将待检样本或标准抗原与包被抗原竞争结合有限的抗体结合位点，再经酶与底物发生显色反应，进行检测和计算得到抗原浓度。该检测试剂盒具有较好的敏感性、特异性、重复性和稳定性，对目标化合物的回收率较高，能提供更准确可靠的检验结果，所述试剂盒使用操作简便易行，检测快速灵敏，价格低廉，为GXM的临床检测提供了一种有效工具。
附图简要说明
图1为本发明提供的新型隐球菌荚膜多糖GXM定量检测的结果。
图2为本发明提供的新型隐球菌荚膜多糖GXM的HPLC纯度分析的结果。
图3所示为本发明提供的新型隐球菌荚膜多糖GXM的SDS-PAGE杂蛋白含量分析的结果。
图4所示为本发明提供的兔IgG型多克隆抗体的SDS-PAGE检测的结果。
图5所示为本发明提供的兔IgG型多克隆抗体的效价测定的结果。
图6所示为本发明提供的GXM免疫检测试剂盒的标准曲线图。
实施本发明的方式
为使本发明的目的、技术手段和优点更加清楚明白，以下结合附图对本发明作进一步详细说明。
以上仅为本发明的较佳实施例而已，并非用于限定本发明的保护范围。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
实施例1：
新型隐球菌荚膜多糖GXM的提取，其具体步骤如下：
(1)配制YM肉汤培养基0.6L，其成分为1.8g酵母提取物，3g蛋白胨，1.8g麦芽糖提取物，6g D-葡萄糖。培养新型隐球菌，培养温度为30℃，震荡速度为200rpm，培养时间为36h；
(2)菌液在121℃下灭菌25min，16,000g离心30分钟去除菌体；
(3)上清液中边搅拌边缓慢加入醋酸钙粉末至终浓度5％。再加冰醋酸调pH值至5.0左右；
(4)向上述溶液中加入三倍体积95％的乙醇，马上会有沉淀产生，4℃静置过夜。倒掉大部分上清，剩余物质转移至50mL圆底离心管中。12,000g离心5分钟。弃上清，沉淀干燥，得新型隐球菌荚膜多糖GXM粗提物。
实施例2
新型隐球菌荚膜多糖GXM免疫亲和层析柱的制备，其具体步骤如下：
(1)将新型隐球菌荚膜多糖GXM单克隆抗体结合到蛋白A微珠上。按每毫升湿的微珠大约可以结合2mg单克隆抗体将抗体和蛋白A混合成为稀薄的匀浆，在总量为10mL的溶液中加入大约1mL微珠，室温孵育1h，轻轻摇动混匀；
(2)用10倍体积的0.2mol/L硼酸钠(pH9.0)洗涤微珠2次，每次以3000g离心2min，或10000g离心30s；
(3)用10倍体积的0.2mol/L硼酸钠(pH9.0)重悬微珠，留取相当于10mL湿微珠的样品。加入足量的二甲基庚二酸酯(固体)至微珠匀浆中，使终浓度为20mmol/L，室温孵育30min，使结合在蛋白A微珠上的抗体与微珠基质发生交联，并轻轻混匀，得到微珠-抗体交联复合物。留取相当于10mL交联复合物的样品；
(4)用0.2mol/L乙醇胺(pH8.0)洗涤微珠-抗体交联复合物1次以终止交联反应；
(5)将步骤(4)得到的微珠-抗体交联复合物重悬于0.2mol/L乙醇胺溶液中，室温孵育2h，轻轻混匀；
(6)将步骤(5)得到的微珠-抗体交联复合物用PBS洗涤后，重悬于PBS，加入硫柳汞至其终浓度为0.01％保存；
(7)将步骤(6)得到的微珠-抗体交联复合物填经检测交联成功后，填充入层析柱中，制得新型隐球菌荚膜多糖免疫亲和层析柱，用PBS冲洗容器，收集残留的微珠。如果可能，仅使用待纯化制品中全部GXM所需的抗体微珠基质。
实施例3
实施例1制备的新型隐球菌荚膜多糖粗提物GXM的纯化，其具体步骤如下：
(1)取实施例2制备的新型隐球菌荚膜多糖GXM免疫亲和层析柱，用20倍柱床体积的起始缓冲液洗柱；
(2)将待纯化样品溶液加入层析柱，按每毫升柱体积大约1mL/h的流速，使样品溶液流过层析柱，用蠕动泵控制流速；
(3)用20倍柱床体积的结合缓冲液洗柱；
(4)用20倍柱床体积的预洗脱缓冲液洗柱；
(5)采用分段洗脱法，连续以0.5倍柱床体积的洗脱缓冲液通过层析柱，分管收集每一组分；
(6)检测每管的GXM含量，将浓度高的各管合并。根据抗原的用途，对收集的抗原洗脱液透析；
(7)用20倍柱床体积的起始缓冲液流经基质，使层析柱再生，向缓冲液中加入0.01％硫柳汞，然后将层析柱保存于4℃的环境中；
其中，起始缓冲液、结合缓冲液、预洗脱缓冲液、洗脱缓冲液分别为高纯水、pH7的PBS缓冲液、pH9的碳酸盐缓冲液、pH3.0的0.1M的甘氨酸缓冲液。
实施例4
将实施例3制备的新型隐球菌荚膜多糖GXM的检测：
A.Dubois-硫酸苯酚法测定糖含量，其具体步骤如下：
(1)称取1g葡萄糖溶于100mL dH2O中，配成1％葡萄糖溶液；
(2)取7支10mL洁净试管，每支试管分别加入1mL dH2O和25μL苯酚溶液；
(3)各支试管分别加入0、2.5、5、10、15μL葡萄糖溶液和10μL待测液；
(4)每管缓慢加入2.5mL浓硫酸，都加入浓硫酸后再统一混匀；
(5)试管水浴20分钟左右，冷却至室温；
(6)将各管中液体小心倒入玻璃比色杯中；
(7)用紫外分光光度计于485nm下测定吸光度值；
(8)绘制标准曲线；
(9)根据标准曲线计算出待测液多糖浓度。
标准曲线如图1所示，线性非常好，可用于计算待测样品浓度。
B.HPLC纯度分析，其具体步骤如下：
取新型隐球菌荚膜多糖GXM样品1mL(3.65mg/mL)
色谱柱：Sugar—ParkT72011A09P/N85188Waters
流动相：超纯水
流速：0.5mL/min
柱温：85℃
检测器：示差折光检测器
灵敏度设定范围：1.0×10-6-5.0×10-4RIU
示差范围：1.00～1.75RIU
线性动态范围：5×10-3～5×10-9RIU
噪声：＜2×10-8RIUATTN
HPLC进样，用示差折光检测器检测，结果见图2所示，出现单一吸收峰，表明样品的纯度较好。
C.SDS-PAGE杂蛋白含量分析，其具体步骤如下：
(1)配制浓度8％的SDS-PAGE凝胶，制胶；
(2)每孔加10μL实施例3制得的新型隐球菌荚膜多糖GXM样品；
(3)60V恒压电泳10分钟后120V恒压电泳大约1小时，至溴酚蓝条带距胶板下边缘1厘米时停止电泳；
(4)考马斯亮蓝染色1小时；
(5)4℃脱色过夜；
(6)拍照并观察。
胶图如图3所示，由对比可知，实施例3制得的新型隐球菌荚膜多糖样品纯度较高，无杂蛋白。
实施例5
新型隐球菌荚膜多糖GXM免疫亲和层析柱的重复性、回收率、稳定性验证：
A.重复性验证，具体步骤如下：
(1)按实施例2的步骤制备5个新型隐球菌荚膜多糖GXM免疫亲和层析柱，分别标号为001、002、003、004和005；
(2)取实施例1制备的新型隐球菌荚膜多糖粗品为待纯化样品，均匀分成5份；
(3)将5份2mL待纯化样品分别加入5个平行试验的层析柱中，按实施例3的操作进行纯化；
(4)按实施例4的操作进行纯化后的多糖GXM的鉴定，结果如下：
硫酸苯酚法定量，结果如表1所示，纯化后得到的糖总量CV＜10％；
HPLC纯度分析均出现单一吸收峰；
SDS-PAGE杂蛋白含量分析均无杂蛋白条带。
表1新型隐球菌荚膜多糖GXM免疫亲和层析柱的重复性验证结果
Figure PCTCN2017074700-appb-000001
B.回收率验证，具体步骤如下：
(1)按实施例2步骤制备5个新型隐球菌荚膜多糖GXM免疫亲和层析柱；
(2)取实施例3中制备的新型隐球菌荚膜多糖GXM做为纯化标样，浓度为3.65mg/mL；
(3)将2mL纯化标样溶液加入5个平行试验的层析柱中，按实施例3的操作进行纯化；
(4)检测纯化后标样的糖总量，结果如表2所示，计算回收率在96.5％-100.5％之间。
表2新型隐球菌荚膜多糖GXM免疫亲和层析柱的回收率验证结果
层析柱编号 001 002 003 004 005 平均值
糖总量 7.18 7.34 7.09 7,05 7.13 7.16
回收率 98.36％ 100.5％ 97.12％ 96.57％ 97.67％ 98.08％
C.稳定性验证，具体步骤如下：
(1)按实施例2的步骤制备1个新型隐球菌荚膜多糖GXM免疫亲和层析柱；
(2)取实施例3中制备的新型隐球菌荚膜多糖GXM做为纯化标样，浓度为3.65mg/mL；
(3)将1mL纯化标样溶液加入层析柱中，按实施例3的操作进行纯化，同一柱子重复进样10次(每次洗脱后再生)；
(4)检测10次纯化后标样的糖总量，结果如表3所示，计算回收率在78.08％-105.80％间。
表3新型隐球菌荚膜多糖GXM免疫亲和层析柱的稳定性验证结果
Figure PCTCN2017074700-appb-000002
由上述结果可知，按实施例2步骤制备的新型隐球菌荚膜多糖GXM免疫亲和层析柱对目标化合物的特异性和回收率非常高，且重复性和稳定性较好，用于新型隐球菌荚膜多糖GXM的纯化制备可有效降低纯化制备成本、简化制备步骤和提高制备效率。
实施例6新型隐球菌荚膜多糖GXM生物活性对比
样品：实施例3制备的新型隐球菌荚膜多糖GXM，
对照品：按照专利文献CN201110240065.X公开的方法制备的新型隐球菌荚膜多糖。
进行新型隐球菌荚膜多糖GXM生物活性对比验证，其具体步骤如下：
A.效价验证，具体步骤如下：
(1)将两种方式纯化后的荚膜多糖按10ng/well，25ng/well，50ng/well，100ng/well包被到酶标板上；
(2)将新型隐球菌荚膜多糖GXM单克隆抗体HRP标记抗体按1：2000、1：4000、1：8000、1：16000、1：32000、1：64000、1：128000稀释后加入荚膜多糖包被的酶标板上，37℃孵育60min；
(3)洗涤：移除酶标板孔中的液体，每孔每次加入不少于300μL的工作洗液，静置40s后拍干，重复上述洗涤操作，共洗涤5次；
(4)显色：洗涤结束后，每孔加入底物溶液TMB 50μL，在37℃孵育15min，避光；
(5)终止：每孔内加入50μL终止液，混匀后，读取450nm处的吸光度值。
表4新型隐球菌荚膜多糖GXM效价验证结果
Figure PCTCN2017074700-appb-000003
由上述结果可知，样品表现的效价明显高于后者，表明按本申请的方法制备的GXM抗原与抗体的结合能力远高于现有技术。
B.生物活性验证，具体步骤如下：
(1)准备一盒GXM免疫检测试剂盒；
(2)将样品和对照品分别稀释至1000ng/mL、500ng/mL、200ng/mL、100ng/mL、50ng/mL、20ng/mL、10ng/mL；
(3)使用GXM免疫检测试剂盒进行检测。
表5新型隐球菌荚膜多糖GXM生物活性验证结果
Figure PCTCN2017074700-appb-000004
由表5结果可知，样品的回收率远远高于对照品。
实施例7抗GXM多克隆抗体的制备
1.免疫动物
将GXM抗原与弗氏完全佐剂等体积混合至合适体积。充分乳化后对新西兰大耳兔进行皮下多点注射，每只兔免疫剂量控制在0.01-1mg。免疫前3天取耳血，分离血清做阴性对照。初次免疫后每2周免疫1次，方法与第1次相同。
2.多克隆抗体的获得
1)效价测定：免疫过程中，免疫后每隔几天采血测效价1次，免疫次数不少于3次；
2)分离抗血清：血清效价达到最高时，用颈动脉放血的方法大量采血。待血液凝固，血清分离出后，高速离心，取上清，-20℃保存。
3.用饱和硫酸铵盐析法进行初步纯化
(1)取2mL抗血清样本，加等体积的生理盐水，再加入4mL饱和硫酸铵溶液，4℃沉淀过夜；
(2)10000g低温离心10分钟，弃上清，将沉淀用2mLPBS溶解，缓慢滴加1mL饱和硫酸铵溶液，4℃静置1小时；
(3)10000g低温离心10分钟，弃上清，将沉淀用1mLPBS溶解，用PBS溶液4℃透析过夜。
4.用亲和层析的方法进一步纯化
(1)用5-10倍柱床体积的洗脱缓冲液(pH7.4的0.01mol/L的磷酸缓冲液)洗柱；
(2)用5-10倍柱床体积的偶联缓冲液(pH7.4的0.01mol/L的磷酸盐缓冲液)洗柱；
(3)将用饱和硫酸铵盐析法初步纯化过的样品上样；
(4)用5-10倍柱床体积的偶联缓冲液(pH7.4的0.01mol/L磷酸盐缓冲液)洗柱；
(5)用2-5倍柱床体积的洗脱缓冲液(pH2.8的0.1mol/L的甘氨酸-盐酸缓冲液)洗脱，得到抗新型隐球菌荚膜多糖GXM抗原的多克隆抗体。
实施例8抗GXM多克隆抗体的检测
1.SDS-PAGE电泳检测
对实施例7制得的抗GXM多克隆抗体进行SDS-PAGE电泳，对得到的凝胶进行考马斯亮蓝染色。实验结果见图4(pAb泳道为实施例7制得的抗GXM多克隆抗体，M泳道为蛋白Marker)。由图中可看出，在25kD和50kD分子量区分别有清晰明显的条带，分别为抗体蛋白的氢链和重链，无杂蛋白条带，说明实施例7制备的抗GXM多克隆抗体的纯度很高。
2.效价测定
用间接ELISA法对抗体效价进行测定。所用酶标二抗为辣根过氧化物酶标记的羊抗兔IgG，阴性对照为PBS溶液。检测结果见图5。从结果可看出，该抗体效价很高，大于1:1×106。
实施例9新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
1、制备酶标载体
①用包被缓冲液将GXM稀释至25ng/100μL，得到GXM包被液并加入酶标板孔中，每孔分别加入200μL包被液，酶标板置于2-8℃下包被8h；
②向酶标板孔中加入封闭液，每孔分别加入200μL封闭液，置于37℃下封闭30min；
③弃去封闭液，置于37℃恒温下30min，得到GXM包被的酶标载体；
所述的包被缓冲液为pH7.0-7.4的0.01mol/L的PBS缓冲溶液；
所述的封闭液的配制：含3％的脱脂奶粉的pH7.0-7.4的0.01mol/L的PBS缓冲溶液；
2、GXM标准品的制备
用样本稀释液将GXM抗原稀释至浓度分别为100、32、10、6.4和3.2ng/mL；
3、抗GXM多克隆酶标抗体溶液的配制：
用于标记的酶为AP，采用戊二醛交联法进行抗GXM多克隆酶标记抗体的制备；
将AP标记的抗GXM的多克隆酶标抗体用AP偶联物稳定剂以1:2000的比例稀释而成；
4、配制样本处理液、浓缩洗液、样本稀释液、底物溶液和终止液：
样本处理液：用超纯水溶解乙二胺四乙酸二钠，配制成0.05mol/L的EDTA溶液，其pH值为4.0-4.8；
浓缩洗液：按重量份数计，氯化钠96.0份，氯化钾2.40份，十二水合磷酸氢二钠42.96份，磷酸二氢钾2.88份，吐温-20 0.05份，超纯水1000份；
样本稀释液：人工血清；
底物溶液：p-NPP溶液；
终止液：将54.7mL浓硫酸溶于高纯水并稀释至100mL，得到10mol/L的硫酸溶液；
5、将上述抗GXM多克隆酶标抗体、GXM标准品、样本处理液、浓缩洗液、样本稀释液、底物溶液和终止液分别装入相应试剂瓶中，并将上述试剂瓶由海绵托架固定，并同GXM包被的酶标载体和封膜一同置于试剂盒盒体内。
实施例10新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
所述的GXM包被液，采用pH7.6-8.0的0.1mol/L的PBS缓冲溶液将GXM稀释至25ng/100μL，其余步骤同实施例9。
实施例11新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
所述的GXM包被液，采用pH7.6-8.0的0.2mol/L的PBS缓冲溶液将GXM稀释至25ng/100μL，其余步骤同实施例9。
实施例12新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
所述的GXM包被液，采用pH9.0-9.6的0.05mol/L的CBS缓冲溶液将GXM稀释至25ng/100μL，其余步骤同实施例9。
实施例13新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
所述的GXM包被液，采用pH9.0-9.6的0.1mol/L的CBS缓冲溶液将GXM稀释至25ng/100μL，其余步骤同实施例9。
实施例14新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
所述的GXM包被液，采用pH9.0-9.6的0.2mol/L的CBS缓冲溶液将GXM稀释至25ng/100μL，其余步骤同实施例9。
实施例15新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
所述的GXM包被液，采用pH10.0-10.6的0.05mol/L的Tris缓冲溶液将GXM稀释至25ng/100μL，其余步骤同实施例9。
实施例16新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
1、制备酶标载体
①用包被缓冲液将GXM稀释至500ng/100μL，得到GXM包被液并加入酶标板孔中，每孔分别加入100μL包被液，酶标板置于2-8℃下包被12h；
②向酶标板孔中加入封闭液，每孔分别加入100μL封闭液，置于37℃下封闭60min；
③弃去封闭液，置于37℃恒温下60min，得到GXM包被的酶标载体；
所述的包被缓冲液为pH7.2-7.4的0.01mol/L的PBS缓冲溶液；
所述的封闭液的配制：含4％的脱脂奶粉的pH7.2-7.4的0.1mol/L的PBS缓冲溶液；
2、GXM标准品的制备
用样本稀释液将GXM抗原稀释至浓度分别为100、32、10、6.4和3.2ng/mL；
3、抗GXM多克隆酶标抗体溶液的配制：
用于标记的酶为HRP，采用过碘酸盐氧化法进行抗GXM多克隆酶标抗体的制备；
将HRP标记的抗GXM的多克隆酶标抗体用HRP偶联物稳定剂以1:10000的比例稀释而成；
4、配制样本处理液、浓缩洗液、样本稀释液、底物溶液和终止液：
样本处理液：用超纯水溶解乙二胺四乙酸二钠，配制成0.1mol/L的EDTA溶液，其pH值为4.0-4.8；
浓缩洗液：按重量份数计，氯化钠96.0份，氯化钾2.40份，十二水合磷酸氢二钠42.96份，磷酸二氢钾2.88份，吐温-20 0.05份，超纯水1000份；
样本稀释液：人工脑脊液；
底物溶液：TMB溶液；
终止液：将浓硫酸与超纯水以1:8的体积比进行稀释，得到2mol/L的硫酸溶液；
5、将上述抗GXM多克隆酶标抗体、GXM标准品、样本处理液、浓缩洗液、样本稀释液、底物溶液和终止液分别装入相应试剂瓶中，并将上述试剂瓶由海绵托架固定，并同GXM包被的酶标载体和封膜一同置于试剂盒盒体内。
实施例17新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
所述的封闭液为含5％脱脂奶粉的pH7.6-8.0的0.2mol/L的PBS缓冲溶液，其余步骤同实施例16。
实施例18新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
所述的封闭液为含1％BSA的pH7.6-8.0的0.2mol/L的PBS缓冲溶液，其余步骤同实施例16。
实施例19新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
所述的封闭液为含2％BSA的pH7.6-8.0的0.2mol/L的PBS缓冲溶液，其余步骤同实施例16。
实施例20新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
所述的封闭液为含4％BSA的pH7.6-8.0的0.2mol/L的PBS缓冲溶液，其余步骤同实施例16。
实施例21新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
1、制备酶标载体
①用包被缓冲液将GXM稀释至2μg/100μL，得到GXM包被液并加入酶标板孔中，每孔分别加入60μL包被液，酶标板置于2-8℃下包被16h；
②向酶标板孔中加入封闭液，每孔分别加入60μL封闭液，置于37℃下封闭90min；
③弃去封闭液，置于37℃恒温下90min，得到GXM包被的酶标载体；
所述的包被缓冲液为pH7.4的0.1mol/L的PBS缓冲溶液(按重量份数计，氯化钠4.25份、十二水合磷酸氢二钠15.40份、磷酸二氢钾0.95份、超纯水500份)；
所述的封闭液的配制：含4％的BSA的pH7.2-7.4的0.1mol/L的PBS缓冲溶液；
2、GXM标准品的制备
用样本稀释液将GXM抗原稀释至浓度分别为100、32、10、6.4和3.2ng/mL；
3、抗GXM多克隆酶标抗体溶液的配制
用于标记的酶为HRP，采用过碘酸盐氧化法进行抗GXM多克隆酶标抗体的制备；
将HRP标记的抗GXM的多克隆酶标抗体用HRP偶联物稳定剂以1:20000的比例稀释而成；
4、配制样本处理液、浓缩洗液、样本稀释液、底物溶液和终止液：
样本处理液：用超纯水溶解乙二胺四乙酸二钠，配制成0.2mol/L的EDTA溶液，其pH值为4.0-4.8；
浓缩洗液：按重量份数计，氯化钠96.0份，氯化钾2.40份，十二水合磷酸氢二钠42.96份，磷酸二氢钾2.88份，吐温-20 0.05份，超纯水1000份；
样本稀释液：人工血清；
底物溶液：TMB溶液；
终止液：98％的浓硫酸；
5、将上述抗GXM多克隆酶标抗体、GXM标准品、样本处理液、浓缩洗液、样本稀释液、底物溶液和终止液分别装入相应试剂瓶中，并将上述试剂瓶由海绵托架固定，并同GXM包被的酶标载体和封膜一同置于试剂盒盒体内。
实施例22新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
样本处理液的配制方法为：用超纯水溶解十二烷基磺酸钠，配制成pH8.5-10.0的0.01mol/L的SDS溶液，其余步骤同实施例21。
实施例23新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
样本处理液的配制方法为：用超纯水溶解十二烷基磺酸钠，配制成pH8.5-10.0的0.05mol/L的SDS溶液，其余步骤同实施例21。
实施例24新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
样本处理液的配制方法为：用超纯水溶解十二烷基磺酸钠，配制成pH8.5-10.0的0.1mol/L的SDS溶液，其余步骤同实施例21。
实施例25新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
样本处理液的配制方法为：用超纯水溶解甘氨酸，配制成0.07mol/L的甘氨酸溶液，再用浓HCL溶液调节pH至pH2.2-2.8其余步骤同实施例21。
实施例26新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
样本处理液的配制方法为：用超纯水溶解甘氨酸，配制成0.13mol/L的甘氨酸，再用浓HCL溶液调节pH至pH2.2-2.8其余步骤同实施例21。
实施例27新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
样本处理液的配制方法为：用超纯水溶解甘氨酸，配制成0.2mol/L的甘氨酸，再用浓HCL溶液调节pH至pH2.2-2.8其余步骤同实施例21。
实施例28新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
样本处理液的配制方法为：用超纯水溶解链酶蛋白酶，配制成0.05mg/mL的链酶蛋白酶溶液，其pH值为pH8.0-9.0，其余步骤同实施例21。
实施例29新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
样本处理液的配制方法为：用超纯水溶解链酶蛋白酶，配制成5mg/mL的链酶蛋白酶溶液，其pH值为pH8.0-9.0，其余步骤同实施例21。
实施例30新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
样本处理液的配制方法为：用超纯水溶解链酶蛋白酶，配制成15mg/mL的链酶蛋白酶溶液，其pH值为pH8.0-9.0，其余步骤同实施例21。
实施例31新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
样本处理液的配制方法为：用超纯水溶解尿素，配制成1mol/L的尿素，其pH值为pH7.2-8.0，其余步骤同实施例21。
实施例32新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
样本处理液的配制方法为：用超纯水溶解尿素，配制成4mol/L的尿素，其pH值为pH7.2-8.0，其余步骤同实施例21。
实施例33新型隐球菌荚膜多糖抗原免疫检测试剂盒的制备
样本处理液的配制方法为：用超纯水溶解尿素，配制成8mol/L的尿素，其pH值为 pH7.2-8.0，其余步骤同实施例21。
实施例34GXM免疫检测试剂盒的操作步骤
1、样本的处理
1)将待检样本与样本处理液以1:1的体积比混合后沸水浴1min；
2)将水浴后的混合液1000g离心1min；
3)离心后上清液用于检测。
2、检测步骤
1)取实施例9-15制备的新型隐球菌荚膜多糖抗原免疫检测试剂盒，取出已包被抗原的酶标载体；
2)配制工作洗液：将浓缩洗液稀释20倍(1份浓缩洗液加19份的无菌去离子水或超纯水)；
3)样本混合：分别设标准曲线组、待测样品组，其中
标准曲线组：各标准曲线点(3.2、6.4、10、32和100ng/mL)；
待测样本组：经过步骤1处理后的待测样本重复检测10次；
将两组分别与抗GXM酶标抗体等体积混合，在37℃下孵育20min。
4)样本转移：将步骤3)的混合液转移至酶标板孔中，每孔加入60μL，在37℃下孵育20min；
5)洗涤：移除酶标板孔中的液体，每孔每次加入不少于300μL的工作洗液，静置40s后拍干，重复上述洗涤操作，共洗涤5次；
6)显色：洗涤结束后，每孔加入底物溶液p-NPP 60μL，在37℃孵育15min，避光；
7)终止：每孔内加入50μL终止液，混匀后，读取405nm处的吸光度值；
8)结果判断：在计算机中分别输入标准液和待测样品的吸光度测定值，根据计算软件绘制的半对数标准曲线和方程以及待测样品的吸光度值，计算出各待测样品中GXM的浓度值，计算CV值，比较各试剂盒检测样本的重复性。
表6实施例9-15制备的试剂盒的样本检测结果
Figure PCTCN2017074700-appb-000006
实施例9-15制备的试剂盒对同一样本的检测结果如表4所示，由表6中数据可知，各试剂盒对样本的检测结果的CV值均小于7％，表明各试剂盒对样品的检测结果的离散程度较小，重复性较好，均可用于新型隐球菌荚膜多糖抗原的免疫检测；且实施例10制备的试剂盒的检测CV值最小，为2.6％，表明在本实施例的试验中，该试剂盒检测的重复性最好。
实施例35GXM免疫检测试剂盒操作步骤
1、样本的处理
1)将待检样本与样本处理液以3:1的体积比混合后沸水浴5min；
2)将水浴后的混合液5000g离心5min；
3)离心后上清液用于检测；
2、检测步骤
1)取实施例16-20制备的新型隐球菌荚膜多糖抗原免疫检测试剂盒，取出已包被抗原的酶标载体；
2)配制工作洗液：将浓缩洗液稀释20倍(1份浓缩洗液加19份的无菌去离子水或超纯水)；
3)样本混合：分别设标准曲线组、待测样品组，其中
标准曲线组：各标准曲线点(3.2、6.4、10、32和100ng/mL)；
待测样本组：处理后的待测样本重复检测10次；
将两组分别与抗GXM酶标抗体等体积混合，在37℃下孵育40min。
4)样本转移：将步骤3)的混合液转移至酶标板孔中，每孔加入80μL，在37℃下孵育40min；
5)洗涤：移除酶标板孔中的液体，每孔每次加入不少于300μL的工作洗液，静置40s后拍干，重复上述洗涤操作，共洗涤5次；
6)显色：洗涤结束后，每孔加入底物溶液80μL，在37℃孵育15min，避光；
7)终止：每孔内加入50μL终止液，混匀后，读取450nm处的吸光度值；
8)结果判断：在计算机中分别输入标准液和待测样品的吸光度测定值，根据计算软件绘制的半对数标准曲线和方程以及待测样品的吸光度值，计算出各待测样品中GXM的浓度值，计算CV值，比较各试剂盒检测样本的重复性。
表7实施例16-20制备的试剂盒的样本检测结果
Figure PCTCN2017074700-appb-000007
Figure PCTCN2017074700-appb-000008
实施例16-20制备的试剂盒对同一样本的检测结果如表7所示，由表7中数据可知，各试剂盒对样本的检测结果的CV值均小于6％，表明各试剂盒对样品的检测结果的离散程度较小，重复性较好，均可用于新型隐球菌荚膜多糖抗原的免疫检测；且实施例16制备的试剂盒的检测CV值最小，为2.3％，表明在本实施例的试验中，该试剂盒检测的重复性最好。
实施例36GXM免疫检测试剂盒操作步骤
1、样本的处理
1)将待检样本与样本处理液以5:1的体积比混合后沸水浴10min；
2)将水浴后的混合液10000g离心10min；
3)离心后上清液用于检测；
2、检测步骤
1)取实施例21-33制备的新型隐球菌荚膜多糖抗原免疫检测试剂盒，取出已包被抗原的酶标载体；
2)配制工作洗液：将浓缩洗液稀释20倍(1份浓缩洗液加19份的无菌去离子水或超纯水)；
3)样本混合：分别设标准曲线组、待测样品组，其中
标准曲线组：各标准曲线点(3.2、6.4、10、32和100ng/mL)；
待测样本组：处理后的待测样本重复检测10次；
将两组分别与抗GXM酶标抗体等体积混合，在37℃下孵育60min。
4)样本转移：将步骤3)的混合液转移至酶标板孔中，每孔加入100μL，在37℃下孵育60min；
5)洗涤：移除酶标板孔中的液体，每孔每次加入不少于300μL的工作洗液，静置40s后拍干，重复上述洗涤操作，共洗涤5次；
6)显色：洗涤结束后，每孔加入底物溶液100μL，在37℃孵育15min，避光；
7)终止：每孔内加入50μL终止液，混匀后，读取450nm处的吸光度值；
8)结果判断：在计算机中分别输入标准液和待测样品的吸光度测定值，根据计算软件绘制的半对数标准曲线和方程以及待测样品的吸光度值，计算出各待测样品中GXM的浓度值，计算CV值，比较各试剂盒检测样本的重复性。
表8-1实施例21-27制备的试剂盒的样本检测结果
Figure PCTCN2017074700-appb-000009
Figure PCTCN2017074700-appb-000010
表8-2实施例28-33制备的试剂盒的样本检测结果
Figure PCTCN2017074700-appb-000011
Figure PCTCN2017074700-appb-000012
实施例21-33制备的试剂盒对同一样本的检测结果如表8所示，由表8中数据可知，各试剂盒对样本的检测结果的CV值均小于7％，表明各试剂盒对样品的检测结果的离散程度较小，重复性较好，均可用于新型隐球菌荚膜多糖抗原的免疫检测；且实施例26制备的试剂盒的检测CV值最小，为2.8％，表明在本实施例的试验中，该试剂盒检测的重复性最好。
实施例37GXM免疫检测试剂盒的临床应用
1、绘制标准曲线
取实施例16制备的试剂盒，按实施例35的步骤，得到各标准曲线点(3.2、6.4、10、32和100ng/mL)的测量值如表9所示，利用表9数据，以样品中GXM抗原的浓度的对数值为横轴(x轴)，以450nm处测得的吸光度值为纵轴(y轴)，作标准曲线如图6所示，得到标准曲线方程为：
y＝A+(B-A)/[1+e^(-a*x+b)]
其中，A＝1.24414；
B＝0.14362；
a＝2.92462；
b＝4.66878。
线性相关度R^2＝0.99946，标准曲线方程拟合良好。
2、GXM免疫检测试剂盒的参考值的确定
取临床确诊为隐球菌感染阳性样本30例、正常人样本200例，将样本经过离心、稀释等处理后，利用实施例11制备的试剂盒按实施例30的步骤测定OD450值，根据标准曲线方程和样本的吸光度值计算出抗原浓度值如表10所示。
根据标准曲线的结果计算检测抗原的浓度，通过检测200例正常人样本，取95％置信区间的抗原的浓度值为Cut-off上限：
Figure PCTCN2017074700-appb-000013
通过检测30例阳性病人，取95％置信区间的抗原的浓度值为Cut-off下限：
Figure PCTCN2017074700-appb-000014
Figure PCTCN2017074700-appb-000015
抗原的浓度值在6ng/ml-10ng/ml之间为疑似病人。即得到GXM免疫检测试剂盒的判断标准参考值如表11所示。
表9检测标准曲线
标准品抗原浓度 OD450
3.2 1.196
6.4 1.157
10 1.072
32 0.768
100 0.402
表10 GXM免疫检测试剂盒的ELISA临床检测结果
Figure PCTCN2017074700-appb-000016
注：*表示与正常人比较P<0.01；
表11 GXM免疫检测试剂盒判断标准参考值
阳性 疑似 阴性
抗原浓度≥10ng/mL 6ng/mL≤抗原浓度≤10ng/mL 抗原浓度≤6ng/mL
如果样本的检测结果落在疑似区间，则需要进行第二次检测确定结果。
实施例38GXM免疫检测试剂盒的方法学考察
1、敏感性实验
取实施例19制备的试剂盒，按实施例35的步骤检测20例临床确诊样本。
诊断敏感性＝阳性样本检出例数/阳性样本总例数×100％，实验结果如表12所示，由表12中的数据得到本实验所用GXM免疫检测试剂盒的敏感性在85％以上。
表12敏感性检测实验结果
序号 OD450 计算抗原浓度 结果判断
1 0.053 1684.51 阳性
2 0.061 1290.67 阳性
3 0.09 701.97 阳性
4 0.135 408.85 阳性
5 0.227 213.34 阳性
6 0.418 95.07 阳性
7 0.319 137.96 阳性
8 1.007 13.15 阳性
9 0.103 582.55 阳性
10 0.164 319.93 阳性
11 0.313 141.46 阳性
12 0.063 1219.78 阳性
13 0.342 125.72 阳性
14 0.533 65.41 阳性
15 1.122 6.56 疑似
16 0.062 1254.21 阳性
17 1.013 12.78 阳性
18 1.049 10.62 疑似
19 0.126 446.65 阳性
20 1.08 8.85 疑似
2、特异性实验
取实施例19制备的试剂盒，按实施例35的步骤检测检测20例健康人样本。
特异性＝阴性样本检出例数/阴性样本总例数×100％，实验结果如表13所示，由表13中数据得到本实验所用GXM免疫检测试剂盒的敏感性在90％以上。
表13特异性检测实验结果
Figure PCTCN2017074700-appb-000017
Figure PCTCN2017074700-appb-000018
3、回收率实验
选择正常人血液分别添加新型隐球菌荚膜多糖抗原90μg/L、30μg/L后，利用实施例19制备的试剂盒按实施例35的步骤检测抗原浓度，计算真实值与期望值的比值，得到回收率，回收率介于80-120％之间认为合格。实验结果如表14所示，由表14中数据得到说明本实验所用GXM免疫检测试剂盒的回收率介于80％-120％之间，回收率良好。
表14回收率实验结果
Figure PCTCN2017074700-appb-000019
4、重复性实验
1)批间精密度
合格标准：利用实施例19制备的试剂盒按实施例35的步骤将同一样本每天测试一次，连续10个工作日，计算其均值M、标准差SD与变异系数CV，变异系数CV≤25％为合格。实验结果如表15所示，由表15中数据得出结论：本发明提供的GXM免疫检测试剂盒的批间精密度(即变异系数CV)为15％，小于25％，符合标准。
表15批间精密度实验结果
Figure PCTCN2017074700-appb-000020
Figure PCTCN2017074700-appb-000021
2)批内精密度
合格标准：将同一本在同一批次实验中平行测定10组数据，每次测定两次，取平均值，并计算相应的抗原浓度。计算其均值M、标准差SD与变异系数CV，变异系数CV≤15％为合格。本发明提供的GXM免疫检测试剂盒的批内精密度(即变异系数CV)为7％，小于15％，符合标准，验证合格。
表16批内精密度实验结果
Figure PCTCN2017074700-appb-000022
5、稳定性实验
取实施例18制备的GXM免疫检测试剂盒在37℃环境中放置，每天通过标准曲线检测已知浓度的标准质控品溶液(55μg/L)，连续检测5天，检测值变化率(即变异系数CV)小于20％，证明试剂盒稳定。实验结果如表17所示，由表17中数据可知本实验采用实施例9制备的GXM免疫检测试剂盒的5天的变异系数CV≤20％，说明本发明提供的GXM免疫检测试剂盒的稳定性良好。
表17稳定性实验结果
Figure PCTCN2017074700-appb-000023
Figure PCTCN2017074700-appb-000024

Claims (15)

  1. 一种新型隐球菌荚膜多糖GXM的制备方法，所述制备方法包括利用GXM免疫亲和层析柱进行GXM的纯化的步骤。
  2. 如权利要求1所述的制备方法，其特征在于，所述制备方法包括如下步骤：
    (1)新型隐球菌荚膜多糖GXM的粗提；和
    (2)利用GXM免疫亲和层析柱进行新型隐球菌荚膜多糖GXM粗提物的纯化。
  3. 如权利要求2所述的制备方法，其特征在于，所述的新型隐球菌荚膜多糖GXM的提取，包括如下步骤：
    (1)培养新型隐球菌，至菌浓度达到对数期后期；
    (2)将菌液高压灭菌，离心除去菌体，保留上清液；
    (3)向上清液中边搅拌边加入醋酸钙粉末使醋酸钙在上清液中的终浓度达到2-8％，然后加冰醋酸调pH至4.6-5.2；和
    (4)向步骤(3)得到的溶液中加入90-98％的乙醇，4℃静置过夜，离心，弃去上清，干燥沉淀，得到新型隐球菌荚膜多糖GXM的粗提物。
  4. 如权利要求2所述的制备方法，其特征在于，所述的利用GXM免疫亲和层析柱进行新型隐球菌荚膜多糖GXM粗提物的纯化，包括如下步骤：
    (1)用与待纯化样品溶液相同的缓冲液冲洗GXM免疫亲和层析柱；
    (2)使待纯化样品溶液流经步骤(1)处理后的层析柱；
    (3)用结合缓冲液洗柱；
    (4)用预洗脱缓冲液洗柱；
    (5)采用分段洗脱，连续以洗脱缓冲液流过层析柱，分管收集每一组分；和
    (6)检测每管的GXM含量，将浓度高的各管合并。
  5. 如权利要求4所述的制备方法，其特征在于，步骤(2)中所述的待纯化样品的流速为0.5-1.5mL/h。
  6. 如权利要求4所述的制备方法，其特征在于，步骤(3)中所述的结合缓冲液选自PBS缓冲液、Tris-HCl缓冲液、醋酸-醋酸钠缓冲液中的一种，用量为10-25倍柱床体积；
    和/或，步骤(4)中所述的预洗脱缓冲液为碳酸盐缓冲液，用量为10-25倍柱床体积；
    和/或，步骤(5)中所述的洗脱缓冲液选自pH3.0的0.1M甘氨酸缓冲液、pH3.0的柠檬酸-磷酸盐缓冲液、pH3.0的柠檬酸-柠檬酸钠缓冲液、pH3.0的醋酸-醋酸钠缓冲液，用量为0.4-0.8柱床体积。
  7. 如权利要求1-6任一项所述的制备方法，其特征在于，所述GXM免疫亲和层析柱的制备包括如下步骤：
    (1)将新型隐球菌荚膜多糖GXM单克隆抗体溶于溶液中，并与亲和层析基质混合成匀浆；
    (2)用交联缓冲液洗涤步骤(1)得到的匀浆，并离心，得到抗体与亲和层析基质的混合物；
    (3)用交联缓冲液重悬步骤(2)得到的抗体与亲和层析基质的混合物，向得到的混悬液中加入双功能结合剂，孵育，并混匀，液固分离，得到亲和层析基质-抗体交联复合物；
    (4)用封闭溶液洗涤步骤(3)得到的亲和层析基质-抗体交联复合物；
    (5)将步骤(4)得到的亲和层析基质-抗体交联复合物重悬于封闭溶液中，孵育，混匀；
    (6)装柱：将步骤(5)得到的亲和层析基质-抗体交联复合物填充入层析柱中，制得新型隐球菌荚膜多糖GXM免疫亲和层析柱。
  8. 如权利要求7所述的制备方法，其特征在于，步骤(1)中所述的亲和层析基质选自蛋白A微珠、蛋白G微珠、活性微珠；
    和/或，步骤(1)中所述的溶液选自碳酸盐缓冲液、醋酸-醋酸钠缓冲液；
    步骤(1)中亲和层析基质和溶液的比例为：每10mL溶液中加入0.5-2.0mL亲和层析基质，亲和层析基质与抗体的比例为：每1mL亲和层析基质结合1-4mg单克隆抗体。
  9. 如权利要求7所述的制备方法，其特征在于，步骤(2)中所述的交联缓冲液为0.1-0.3mol/L的pH8.0-9.5的硼酸钠溶液，用量为5-15倍亲和层析基质体积；
    和/或，步骤(3)中所述交联缓冲液为0.1-0.3mol/L的pH8.3-9.5的硼酸钠溶液，用量为5-15倍亲和层析基质体积。
  10. 如权利要求7所述的制备方法，其特征在于，步骤(3)所述的双功能结合剂选自二甲基庚二酸酯、羰基二咪唑、溴化氰、羟基丁二酰亚胺、乙酰基碘，用量为使其在混悬液中的终浓度为15-25mmol/L。
  11. 如权利要求7所述的制备方法，其特征在于，步骤(4)和(5)中所述的封闭溶液选自乙醇胺、氨基乙烷溶液。
  12. 一种权利要求1-11任一项所述的制备方法制备得到的GXM。
  13. 一种新型隐球菌荚膜多糖GXM抗原免疫检测试剂盒，包括：GXM包被的酶标载体、抗GXM多克隆酶标抗体、GXM标准品，所述GXM为权利要求12所述的GXM，和/或，所述抗GXM多克隆抗体以权利要求12所述的GXM作为免疫原免疫动物得到的血清进行分离纯化得到。
  14. 如权利要求13所述的试剂盒，其特征在于，所述的新型隐球菌荚膜多糖抗原免疫检测试剂盒还包括：样本处理液、浓缩洗液、样本稀释液、底物溶液和终止液，所述的浓缩洗液为：含0.4％-1.0％吐温的磷酸盐缓冲液；和/或，
    所述的样本稀释液为：人工脑脊液或人工血清；和/或，
    所述的底物溶液为：TMB、OPD、OT、ABTS或p-NPP；和/或，
    所述的终止液为：1-20mol/L的硫酸溶液。
  15. 如权利要求13所述的试剂盒，特征在于，所述的样本处理液选自以下溶液：pH2.0-10.0的蛋白变性溶液；pH4.0-4.8的0.05-0.2mol/L的EDTA溶液；pH2.2-2.8的0.07-0.20mol/L的甘氨酸-HCL溶液；pH8.0-9.0的0.05-15mg/mL的链酶蛋白酶；pH8.5-1.0的0.01-0.10mol/L的SDS溶液或pH7.2-8.0的1-8mol/L的尿素。
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