WO2019000267A1 - Gm杂交瘤细胞、单克隆抗体、试剂盒及其制备方法与应用 - Google Patents

Gm杂交瘤细胞、单克隆抗体、试剂盒及其制备方法与应用 Download PDF

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WO2019000267A1
WO2019000267A1 PCT/CN2017/090519 CN2017090519W WO2019000267A1 WO 2019000267 A1 WO2019000267 A1 WO 2019000267A1 CN 2017090519 W CN2017090519 W CN 2017090519W WO 2019000267 A1 WO2019000267 A1 WO 2019000267A1
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antigen
mol
kit
solution
monoclonal antibody
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刘春龙
彭洁
张舟
翟栓柱
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 to PCT/CN2017/090519 priority Critical patent/WO2019000267A1/zh
Priority to CN201780000714.4A priority patent/CN107466319B/zh
Priority to US16/172,175 priority patent/US10428139B2/en
Publication of WO2019000267A1 publication Critical patent/WO2019000267A1/zh

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/14Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961Plant cells or fungi
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/30Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/54F(ab')2
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55Fab or Fab'
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/60Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622Single chain antibody (scFv)
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/60Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626Diabody or triabody
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/38Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus

Definitions

  • the invention relates to the field of biotechnology, in particular to a hybridoma cell, a monoclonal antibody, a kit for producing a monoclonal antibody against an Aspergillus galactomannan (GM) antigen, and a preparation method and application thereof.
  • GM Aspergillus galactomannan
  • Aspergillus is a resident fungus that is widely found in nature and is a resident fungus of normal human skin mucosa. Aspergillus spores are small, 2-3 ⁇ m in diameter, can float in the air and enter the body through the respiratory tract. As the Aspergillus enters the body mainly through the respiratory tract, the Aspergillus infection mainly occurs in the lungs.
  • IA invasive Aspergillosis
  • Aspergillus fumigatus is the most common cause of severe deep Aspergillus infection in immunosuppressed patients.
  • Pathogenic bacteria followed by Aspergillus flavus, Aspergillus niger, Aspergillus terreus and the like.
  • IA has a mortality rate of 70%-90% in patients with hematological and hematopoietic stem cell transplants (HSCT). The main reason for this high mortality rate is that it can not effectively detect IA in the early stage of the disease, and even patients If you do not get timely and effective treatment and die, it is important to choose early detection and diagnosis methods.
  • HSCT hematological and hematopoietic stem cell transplants
  • the widely recognized Aspergillus antigen detection methods mainly include 1,3- ⁇ -D glucan detection (G test) and galactomannan test (GM test).
  • the 1,3- ⁇ -D glucan antigen is a specific cell wall component of all fungi except the bacterium and cryptococci. With the serum as the test sample, the sensitivity and specificity can reach 80%.
  • due to the negative display of colonized Candida it is often necessary to carry out a combined test with the GM test to exclude, both of which are negative to substantially rule out fungal infection.
  • the serological G test is susceptible to hematology and other factors such as fibrous substances, and the 1,3- ⁇ -D glucan antigen can form an immune complex with the antibody in the blood and is rapidly cleared by the blood, resulting in False negative.
  • Galactomannan is a highly specific and highly conserved polysaccharide present in Aspergillus cell wall, which can be used as a specific molecular marker for Aspergillus detection. Enzyme-linked immunosorbent assay (ELISA) is more common. The galactomannan assay method, Acosta J reported that the GM positive result was 4.3 days earlier than the Aspergillus culture. The positive diagnosis of invasive fungal infection by serum GM test is appropriate and applicable, and is an important reminder for patients taking early antifungal therapy, especially for some high-risk patients (such as HSCT patients). Therefore, determination of GM antigen levels in serum contributes to early diagnosis and early treatment of IA.
  • ELISA Enzyme-linked immunosorbent assay
  • the sensitivity, specificity and sensitivity of the Aspergillus GM detection kit on the market are low, the sensitivity is about 83%, the specificity is 90%, and the detection limit is about 1 ⁇ g/L.
  • the double-anti-sandwich method is adopted, that is, the specific monoclonal antibody of Aspergillus is first coated on the ELISA plate, and the same monoclonal antibody labeled with horseradish peroxidase (HRP) is added to the sample to be tested.
  • HRP horseradish peroxidase
  • the antigen in the sample will bind to the specific monoclonal antibody and form a sandwich structure, and then the color developing agent will be added to develop color, and the color depth is positively correlated with the concentration of the antigen to be detected, thereby realizing the detection of the GM antigen.
  • the detection step of the method is cumbersome, and the preparation cost of the monoclonal antibody is high, which is not conducive to the promotion and popularization of the kit in clinical detection.
  • the present invention provides a hybridoma cell or a passage cell thereof capable of producing a monoclonal antibody against an Aspergillus galactomannan (GM) antigen.
  • GM Aspergillus galactomannan
  • the hybridoma cells were deposited on April 18, 2017 at the Institute of Microbiology, Chinese Academy of Sciences (address No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with the preservation number CGMCC No.13827.
  • the present invention also provides a monoclonal antibody or a specific antigen-binding fragment thereof, wherein the monoclonal antibody is produced by the above hybridoma cells.
  • the monoclonal antibody or a specific antigen-binding fragment thereof can specifically bind to an Aspergillus GM antigen.
  • specific antigen-binding fragments include ScFv, (Fab') 2 , Fab, Fv, scFv, diabodies, linear antibodies or multispecific antibodies, and the like.
  • the present invention also provides a method for preparing the above monoclonal antibody, which comprises preparation of ascites, purification of monoclonal antibodies, and purification methods thereof include saturated ammonium sulfate salting precipitation method and affinity chromatography.
  • the present invention also provides a detection reagent for detecting a GM antigen and a detection method thereof, the detection reagent comprising the above monoclonal antibody or a specific antigen-binding fragment thereof.
  • a competitive ELISA method can be used, namely:
  • the anti-GM antigen monoclonal antibody with the unlabeled enzyme is added to the solid phase carrier after the sample to be tested, so that the antigen in the sample to be tested competes with the coated antigen for binding to a limited antibody binding site, and the reaction is thermostatically and thoroughly. Washing, adding enzyme-labeled secondary antibody;
  • the absorbance (A value) is measured at a certain wavelength using a microplate reader, and the antigen is detected by a standard curve.
  • the invention also provides a detection kit and a preparation method thereof, the kit comprising the above monoclonal antibody or a specific antigen-binding fragment thereof.
  • the kit detects the Aspergillus GM antigen system using a competition ELISA method.
  • the kit includes a GM antigen-coated solid phase carrier, an anti-GM antigen monoclonal antibody, and a GM antigen standard.
  • the solid phase carrier is an ELISA plate, a microplate, a test tube or a microporous membrane.
  • the solid phase carrier is an ELISA plate; and the material of the solid phase carrier is, for example, It is polystyrene, nitrocellulose, nylon, and the like.
  • the anti-GM antigen monoclonal antibody is an anti-GM antigen monoclonal antibody of a labeling enzyme; preferably, the anti-GM antigen monoclonal antibody is a rabbit anti-GM antigen monoclonal antibody of a labeling enzyme. antibody.
  • the anti-GM antigen monoclonal antibody is an anti-GM antigen monoclonal antibody of an unlabeled enzyme
  • the kit further comprises an enzyme-labeled secondary antibody, and the enzyme-labeled secondary antibody can be combined with the anti-GM antigen monoclonal antibody
  • the anti-GM antigen monoclonal antibody is a rabbit-derived anti-GM antigen monoclonal antibody of an unlabeled enzyme
  • the kit further comprises an enzyme-labeled goat anti-rabbit secondary antibody.
  • the enzyme is Horseradish Peroxidase (HRP), Alkaline Phosphatase (AP) or Glucose Oxidase (GO); preferably, the enzyme is horseradish. Oxidase.
  • HRP Horseradish Peroxidase
  • AP Alkaline Phosphatase
  • GO Glucose Oxidase
  • the GM antigen standard comprises at least three known concentrations of GM antigen solution, the concentration of the GM antigen standard being in the range of 0-50 ng/mL.
  • the concentration of the GM antigen standard is in the range of 0-10 ng/mL, and most preferably in the range of 0-5 ng/mL.
  • the kit further comprises one or more of a sample treatment solution, a concentrated wash solution, a sample diluent, a substrate solution, and a stop solution.
  • the sample treatment liquid is selected from the group consisting of 0.03 mol/L EDTA, 0.1 mol/L EDTA, 0.12 mol/L EDTA, 0.05 mol/L proteinase K, 0.1 mol/L proteinase K, 0.2 mol/L proteinase K, 5% DMSO. , 15% DMSO or 30% DMSO.
  • the sample treatment solution is 0.12 mol/L EDTA.
  • the preparation method of the kit includes:
  • the step 1) further comprises preparing a GM antigen coating solution, preparing a blocking solution and coating the ELISA plate.
  • the buffer of the GM antigen coating solution is selected from the group consisting of 0.1 mol/L Tris-HCl, 0.1 mol/L PBS, 0.05 mol/L CBS, 0.1 mol/L CBS, 0.2 mol/L CBS, and physiological saline.
  • the buffer of the GM antigen coating solution is 0.1 mol/L Tris-HCl, and its pH is 6.0-pH 9.0;
  • the blocking solution is selected from the group consisting of 2% newborn calf serum, 5% newborn calf serum, and 8% newborn calf serum.
  • the blocking solution is 8% of newborn calf serum, which is added to physiological saline to prepare a blocking solution;
  • the present invention also provides the use of a monoclonal antibody or a specific antigen-binding fragment thereof for preparing a detection reagent or a detection kit for detecting an Aspergillus infection, which is a hybridoma cell having the accession number CGMCC No. 13827 Or its passage cells are produced.
  • Exemplary, specific antigen-binding fragments include ScFv, (Fab') 2 , Fab, Fv, scFv, diabodies, linear antibodies or multispecific antibodies, and the like.
  • the hybridoma cells or the passage cells thereof having the accession number of CGMCC No. 13827 provided by the present invention are capable of producing a monoclonal antibody which can specifically bind to the Aspergillus GM antigen, and has high specificity. Moreover, a large number of monoclonal antibodies can be obtained by culturing hybridoma cells or their passage cells to solve the cost problem of preparing them into a kit for clinical promotion and popularization.
  • the detection kit provided by the present application is capable of specifically binding to the GM antigen, and the sensitivity and specificity thereof are all above 95%, and the detection limit is reduced from 1 ng/mL of the existing product to 0.85 ng/mL, and the detection result and the reference are
  • the high compliance rate of the reagents provides more accurate and reliable test results, enabling the detection of IA early in the course of the disease, enabling patients to receive treatment in a timely and effective manner, improving patient survival and reducing unnecessary experience. Antifungal treatment.
  • the kit provided by the invention has the advantages of simple and convenient operation, rapid and sensitive detection, simple and popular microplate reader, and low price.
  • the test kit provides an effective tool for quantitative detection of GM antigen of Aspergillus.
  • Figure 1 is a graph showing the standard curve of polysaccharide content determination in the pure GM antigen of Example 1;
  • Figure 2 shows the results of HPLC detection of the pure GM antigen in Example 1;
  • Figure 3 is a graph showing the standard curve of the GM antigen immunodetection kit in Example 21.
  • the GM antigen was prepared using Aspergillus, and the Aspergillus strain used in the present invention was purchased from the American Model Culture Collection (ATCC) under the accession number ATCC 1022.
  • the Aspergillus is cultured in a solid medium until the medium is covered with green spores; the hyphae are removed by filtration, the cells and spores are inactivated; after centrifugation, the spores are collected and washed; the spores are crushed and filtered to remove spore fragments; the filtrate is subjected to alcohol precipitation. After washing, a crude extract of GM antigen is obtained; the crude extract of GM antigen is decolorized and ultrafiltered to obtain a GM antigen.
  • the solid medium is selected from the group consisting of PDA medium, Sabour's medium or Chadian medium; preferably PDA medium. Specific steps are as follows:
  • PDA solid medium 2.0L the composition is 600g potato supernatant after boiling, D-glucose 80.0g, agar powder 30.0g, Aspergillus strain is placed in the medium, cultured at 25 ° C for 3 days, until the medium is long Full of green spores.
  • the spores were washed with sterile physiological saline, and the spore suspension was filtered through 8 layers of sterile gauze for 3 times to remove hyphae.
  • the final concentration of 3.7% formaldehyde was added to the spore suspension at 4 ° C for 24 h to inactivate the cells and spores.
  • the spores were collected by centrifugation at 12,000 g for 30 min at 4 ° C, and washed 6 times with sterile physiological saline to remove formaldehyde which may be present.
  • the frozen spores were repeatedly ground with liquid nitrogen, then sterile physiological saline was added, and the spores were disrupted by an ultrasonic cell disrupter.
  • the resulting spore disrupted solution was filtered through a qualitative filter paper, and the filtrate was filtered through a 0.45 ⁇ m filter to remove spore fragments.
  • the resulting filtrate was transferred to a clean container, 2.5 volumes of absolute ethanol was added, and the mixture was allowed to stand at 4 ° C overnight.
  • the crude GM is decolorized by the method of activated carbon adsorption.
  • the specific steps are as follows:
  • GM Dissolve GM in 200 mL of deionized water, slowly add 3.0 g of activated carbon powder while stirring, and decolorize at 4 ° C for 4 hours. The solution was brownish yellow, filtered through a Buchner funnel, and filtered repeatedly until the solution was clarified to obtain a GM extract from which the pigment was removed.
  • the activated carbon particles were removed by suction filtration at room temperature, and the resulting filtrate was filtered through a 0.22 ⁇ m filter.
  • the filtrate was transferred to a 10 KD centrifugal ultrafiltration tube and centrifuged at 4000 g for 20 min to obtain a high purity GM.
  • the GM antigen pure sample obtained by the preparation method is subjected to ultraviolet absorption detection.
  • the samples were detected at UV 260 nm, UV 280 nm, and UV 320 nm, respectively, and the results are shown in Table 2 below. It can be seen from the test results that the total content of nucleic acids and proteins does not exceed 4% of the total mass of the sample.
  • Test items concentration content(%) DNA 3.641 ⁇ g/mL 0.16 protein 74.583 ⁇ g/mL 3.27 Polysaccharide 2.203mg/mL 96.57
  • the purified sample of the GM antigen obtained by the preparation method was subjected to HPLC detection.
  • the detector is a refractive index detector.
  • the test results are shown in Figure 2. It can be seen from the figure that the sample has a single peak with a narrow tip and no obvious peaks, indicating that the material contained is uniform in size and high in purity.
  • the GM antigen was purified by the preparation method, and the obtained GM antigen pure sample was identified by Bio-Rad Aspergillus fumigatus antigen detection kit.
  • the GM antigen is usually detected by the Aspergillus fumigatus antigen detection kit of the American Bio-Rad Company.
  • the GM antigen sample was tested with the kit at a sample concentration of 1 ng/mL.
  • the test results are shown in Table 3 below. From the results, it was found that the GM antigen was positive and the OD value was greater than the positive control, indicating that the galactomannan antigen of Aspergillus fumigatus can be obtained by the method of the present invention.
  • Table 3 uses Bio-Rad Aspergillus fumigatus antigen detection kit for antigen identification test results
  • molecular size can affect the immunogenicity of substances.
  • the molecular weight of an effective immunogen is mostly above 10kD; the larger the molecular weight, the stronger the immunogenicity. This may be because the high molecular substance is easy to form a colloid in an aqueous solution, stays in the body for a long time, and has a greater chance of contact with the immune cells, which is beneficial to stimulate the body to generate an immune response.
  • the chemical structure of macromolecular substances is relatively complicated, and the types and quantities of effective antigen genes are relatively large.
  • the GM antigen obtained in Example 1 has a small molecular weight and poor immunogenicity, and needs to be coupled with a related macromolecular substance to enhance its immunogenicity.
  • the macromolecule is one or more of latex microspheres, KLH (keyhole limpet hemocyanin), BSA (bovine serum albumin), GST, and partial coupling requires the addition of a crosslinking agent to promote the crosslinking rate thereof.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • GST bovine serum albumin
  • partial coupling requires the addition of a crosslinking agent to promote the crosslinking rate thereof.
  • the obtained antigenic polypeptide fragment can be used to immunize an animal.
  • aminated latex microspheres are taken as an example, and the specific coupling method is as follows:
  • the GM antigen is immunized to the animal.
  • the immunization may be performed by subcutaneous injection, footpad injection, intrasplenic injection, intravenous injection or intraperitoneal injection; the animal may be selected from rats, mice, guinea pigs, rabbits, chickens, sheep, horses, pigs or baboons.
  • the example uses rabbit as an immunized animal. The specific steps are as follows:
  • the GM antigen and the Freund's complete adjuvant were mixed in an equal volume to a suitable volume.
  • New Zealand big ears rabbits were injected subcutaneously, and the immune dose of each rabbit was controlled at 0.01-0.1 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, and the serum titer of the rabbit after immunization was measured.
  • An immune spleen cell refers to a B lymphoblastoid-plasma cell that is in an immune state in the spleen.
  • the spleen after the last booster immunization is taken to prepare a cell suspension. Since the proportion of B lymphocytes is large at this time, the success rate of cell fusion is relatively high.
  • the spleen is taken and the myeloma cells are activated.
  • Myeloma cells were fused with spleen cells at a ratio of 1:10.
  • the PEG fusion method was employed.
  • the cell culture supernatant can be detected by indirect ELISA and the positive hybridoma cells can be screened for cloning culture.
  • Positive hybridoma cells were cloned and cultured by limiting dilution method.
  • the positive hybridoma cells with the strongest titer of the test results were expanded to a cell when the cell positive rate reached 100%.
  • the titer of the culture supernatant of the hybridoma cells was measured by ELISA, and the monoclonal hybridoma cells expanded in the expanded culture were frozen in liquid nitrogen.
  • the hybridoma cells were deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee under the accession number CGMCC No.13827.
  • New Zealand white rabbits were injected intraperitoneally with 0.5 mL of paraffin oil. After 2 weeks, the hybridoma cells were collected and suspended in physiological saline, and 1 to 5 ⁇ 10 6 cells were suspended per 1 mL of physiological saline. Each rabbit was then intraperitoneally injected with 0.5 mL of cell suspension and injected with an equal amount of paraffin and Freund's incomplete adjuvant mixture. After about 7-10 days, after the ascites was obviously produced, the rabbits were sacrificed, the abdominal cavity was cut, the ascites was aspirated, and the ascites was collected by centrifugation at 12000 r/min for 15 min. The appropriate amount of preservative was added and stored at 4 °C.
  • the elution buffer is selected from the group consisting of 0.1 mol/L glycine buffer, PB buffer, citrate-phosphate buffer, citric acid-sodium citrate buffer or acetic acid-sodium acetate buffer, pH 3.0;
  • the buffer is selected from one of PBS buffer, Tris-HCl buffer, and acetic acid-sodium acetate buffer, preferably PBS buffer.
  • the specific preparation method is as follows:
  • the GM antigen is diluted to 100 ng/mL-10 ⁇ g/mL with a buffer solution; the buffer solution is selected from the group consisting of: 0.1 mol/L Tris-HCl buffer, pH 6.0-pH 9.0; 0.1 mol/L PBS buffer, pH 6.0-pH 9.0; 0.05-0.2 mol/L CBS buffer, pH 6.0-pH 9.0; or physiological saline.
  • a buffer solution 2%-8% of newborn calf serum is added to a buffer solution to prepare a blocking solution;
  • the buffer solution is selected from the group consisting of: 0.1 mol/L Tris-HCl buffer, pH 6.0-pH 9.0; 0.1 mol/L PBS buffer, pH 6.0-pH 9.0; 0.05-0.2 mol/L CBS buffer, pH 6.0-pH 9.0; or physiological saline.
  • Coating the ELISA plate Add the prepared GM antigen coating solution into the wells of the ELISA plate, and add 50-150 ⁇ L (preferably 100 ⁇ L) coating solution to each well; the ELISA plate is placed at 12-18 ° C (preferred) 15 ° C) environment coating for 6-8h (preferably 7h); the prepared blocking solution was added to the wells of the microplate, each well was added 50-150 ⁇ L (preferably 100 ⁇ L) blocking solution, placed at 12-18 ° C (preferably 15 °C) incubator, 2-4h (preferably 3h); remove the blocking solution from the incubator, discard the blocking solution, and maintain a constant temperature of 2-4h (preferably 3h) at 20-25 ° C (preferably 22 ° C).
  • the plate can be modified prior to coating the plate.
  • the method comprises the steps of placing the microplate on a medical purification operation table equipped with an ultraviolet lamp, fixing the vertical distance between the ultraviolet lamp and the microplate base, and selecting the different time periods to perform ultraviolet treatment on the microplate.
  • the standard preparation was prepared by diluting GM antigen with 0.1 mol/L PBS.
  • concentrations of GM antigen were 5 ng/mL, 2.5 ng/mL, 1 ng/mL, 0.5 ng/mL, and 0.25 ng/mL, respectively.
  • the anti-GM antigen monoclonal antibody solution is prepared by diluting the anti-GM antigen monoclonal antibody with an enzyme conjugate stabilizer at a ratio of 1:20000-1:40000 (preferably 1:30000).
  • the enzyme conjugate stabilizer is an agent capable of maintaining the stability between the antibody and the enzyme conjugate, and is capable of maintaining the activity of the antibody and the enzyme.
  • it may be an HRP enzyme conjugate stabilizer
  • the enzyme conjugate stabilizer in the present invention may be a commercially available product.
  • the enzyme-labeled secondary antibody solution is prepared by diluting horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody with HRP enzyme conjugate stabilizer at a ratio of 1:5000-1:20000 (preferably 1:10000). Made.
  • HRP horseradish peroxidase
  • the sample treatment solution is a protein denaturation solution, preferably one or more selected from the following protein denaturation solutions: 0.05-0.2 mg/mL proteinase K; pH 2-pH 10 0.03-0.18 mol/L EDTA (ethylenediamine four A solution of disodium acetate); a solution of 5-30% DMSO (dimethyl sulfoxide); a urea of 1-8 mol/L of pH 7.0-pH 8.0.
  • protein denaturation solution preferably one or more selected from the following protein denaturation solutions: 0.05-0.2 mg/mL proteinase K; pH 2-pH 10 0.03-0.18 mol/L EDTA (ethylenediamine four A solution of disodium acetate); a solution of 5-30% DMSO (dimethyl sulfoxide); a urea of 1-8 mol/L of pH 7.0-pH 8.0.
  • the concentrated washing solution is a Tween-20-containing PBS solution (abbreviated as PBST solution), wherein the PBST solution may contain a biological liquid preservative such as ProClin300.
  • the concentrated washing liquid is selected as follows:
  • the sample diluent can be a CBS dilution containing 5-15% skim milk powder, BSA or bovine serum.
  • a CBS dilution containing 10% skim milk powder was selected.
  • the substrate solution may be OPD (o-phenylenediamine), OT (o-toluidine), ABTS (2,2'-azine-bis(3-ethylbenzothiazole-6-sulfonic acid)) or p- NPP (p-nitrophenyl phosphate), preferably tetramethylbenzidine (3,3',5,5'-Tetramethylbenzidine, TMB).
  • OPD o-phenylenediamine
  • OT o-toluidine
  • ABTS 2,2'-azine-bis(3-ethylbenzothiazole-6-sulfonic acid)
  • p- NPP p-nitrophenyl phosphate
  • the stop solution may be a 1-10 mol/L sulfuric acid solution. It is preferably prepared by diluting concentrated sulfuric acid and ultrapure water in a ratio of 1:8 by using a 2 mol/L sulfuric acid solution.
  • Table 4 Types and concentrations of coating liquid, blocking solution and sample treatment liquid in various examples
  • Example Buffer in the coating solution Final concentration of GM antigen Blocking solution
  • Sample treatment solution Example 4 0.1 mol/L Tris-HCl 100ng/mL 2% newborn bovine serum 0.03mol/L EDTA
  • Example 5 0.1mol/L PBS 100ng/mL 2% newborn bovine serum 0.03mol/L EDTA
  • Example 6 0.05mol/L CBS 100ng/mL 2% newborn bovine serum 0.03mol/L EDTA
  • Example 7 0.1mol/L CBS 100ng/mL 2% newborn bovine serum 0.03mol/L EDTA
  • Example 9 Saline 100ng/mL 2% newborn bovine serum 0.03mol/L EDTA
  • Example 10 0.1 mol/L Tris-HCl 5 ⁇ g/mL 8% newborn bovine serum 0.1mol/L EDTA
  • Example 11 0.1 mol/L Tris-HCl 5 ⁇ g/mL 5% newborn
  • the horseradish peroxidase-labeled anti-GM antigen monoclonal antibody was diluted with a HRP enzyme conjugate stabilizer at a ratio of 1:20000.
  • step b) mixing the GM antigen standard and the analyte of step a) with an anti-GM antigen monoclonal antibody in equal volumes and incubating for 60 min - 120 min;
  • step b) adding the mixture of step b) to the GM antigen-coated plate and incubating for 60 min to 120 min, and washing the plate after incubation;
  • step d) adding the enzyme-labeled secondary antibody to the plate of step c) and incubating for 20-60 minutes, washing the plate after incubation;
  • step e) adding the substrate solution to the microplate of step d) for 10-15 min, adding the stop solution, detecting, and reading the absorbance at 450 nm on the microplate reader, and realizing the antigen by standard curve Detection.
  • Specific steps are as follows:
  • Standard curve group each standard curve point (GM antigen standard concentration is 5, 2.5, 1, 0.5, 0.25 ng / mL)
  • Sample group to be tested sample to be tested after processing
  • the two groups of samples were mixed with the rabbit anti-GM antigen monoclonal antibody in equal volumes, transferred to the wells of the microtiter plate, 60 ⁇ L per well, and incubated at 37 ° C for 60 min;
  • Termination Add 50 ⁇ L of stop solution to each well, mix and read at OD450nm;
  • step b) Mix the GM antigen standard and the test substance of step a) with the labeled anti-GM antigen monoclonal antibody in equal volume and add to the GM antigen-coated ELISA plate for 60-120 min, and wash after incubation. board;
  • step c) adding the substrate solution to the microplate of step b) for 10-15 min, adding the stop solution, detecting, and reading the absorbance at 450 nm on the microplate reader, and realizing the antigen by standard curve Detection.
  • the specific steps are:
  • Standard curve group each standard curve point (GM antigen standard concentration is 5, 2.5, 1, 0.5, 0.25 ng / mL)
  • Sample group to be tested sample to be tested after processing
  • the two groups of samples were separately mixed with the labeled anti-GM antigen monoclonal antibody in an equal volume, transferred to the wells of the microtiter plate, 80 ⁇ L per well, and incubated at 37 ° C for 90 min;
  • Example 10 The kit of Example 10 was taken and the clinical application test of the kit was carried out in accordance with the two-step detection procedure in Example 20.
  • the measured values of the respective standard curve points (5, 2.5, 1, 0.5, and 0.25 ng/mL) are shown in Table 5, using Table 5 data,
  • the logarithm of the concentration of GM antigen in the sample 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. 3.
  • the concentration of the detected GM antigen was calculated according to the results of the standard curve.
  • the antigen concentration value between 0.65 ng / mL - 0.85 ng / mL is a suspected patient. That is, the judgment standard reference values of the GM antigen immunoassay kit are shown in Table 6.
  • test result of the sample falls within the suspicious interval, a second test is required.
  • the kit of Example 10 was subjected to the kit methodological examination according to the detection procedure of Example 20 (sensitivity experiment, specificity experiment, recovery experiment, repetitive experiment, stability experiment).
  • the normal human blood was added to the Aspergillus galactomannan antigen 2 ⁇ g/L, 1 ⁇ g/L, and the ratio of the true value to the expected value was calculated to obtain the recovery rate, as shown in Table 10.
  • the recovery rate is between 80-120% and is considered acceptable.
  • the experimental results show that the recovery rate of this experiment is between 80% and 120%, and the recovery rate is good. And when the concentration of the added antigen is 2 ⁇ g / L, the recovery rate is between 92% and 102%, which is closer to 100%.
  • Eligibility Criteria The same specimen was tested in parallel for 10 sets of data in the same batch of experiments. Calculate the mean value M, the standard deviation SD and the coefficient of variation CV, and the coefficient of variation CV ⁇ 15% is qualified, see Table 12.
  • the intra-assay precision ie, coefficient of variation CV
  • the intra-assay precision is 1%, far less than 15%. It not only meets the standard, but also passes the verification. It also shows that the dispersion within the batch is very small and the repeatability is very good.
  • the assembled kit was placed in an environment of 37 ° C, and a standard curve was used to detect a known concentration of the antigen solution every day for 5 days.
  • the rate of change of the detection value ie, the coefficient of variation CV
  • the kit is stable.
  • the results showed that the coefficient of variation CV of 5 days was 3.5%, which was much less than 20%, indicating that the kit provided by the present invention has very good stability and makes the detection result more reliable.
  • Example 10 The kit provided in Example 10 was tested in accordance with the one-step method provided in Example 20, and similar experimental results were also obtained. Other facts were tested in accordance with the two-step method or the one-step method provided in Example 20, and similar experimental results were obtained.
  • Example 14 Take the kits prepared in Examples 4-9, and perform the inter-assay precision test on the same sample according to the detection procedure of Example 20. The same specimen is tested once a day for 10 working days to examine different GM antigen coatings. The effect of liquid on the detection of the kit, the test results are shown in Table 14 below.
  • kits prepared in Examples 10-11 were taken separately, and the same test sample was subjected to batch-to-batch precision test according to the test procedure of Example 20. The same specimen was tested once a day for 10 working days to examine different blocking solution pairs. The box detects the effect of repeatability, and the test results are shown in Table 15 below.
  • kits prepared in Examples 12-18 were taken separately, and the same test sample (concentration is known as 1.43 ng/mL) was tested for inter-assay precision and recovery according to the detection procedure of the two-step method in Example 20.
  • the same specimen was tested once a day for 10 working days, and the effects of different sample treatment solutions on the detection repeatability and recovery rate of the kit were examined.
  • the test results are shown in Table 16 below.
  • the reference value of the kit of the present invention is: the upper limit of the reference range is 0.85 ⁇ g/L, and the lower limit is 0.65 ⁇ g/L. If the concentration is ⁇ 0.85 ⁇ g/L, it is determined to be positive; if the concentration is ⁇ 0.65 ⁇ g/L, it is negative; the concentration is between 0.65ng/mL and 0.85ng/mL is suspected.
  • the Bio-rad kit determines that the I value is ⁇ 0.5 positive and the I value ⁇ 0.5 is negative.
  • Bio-rad kit is a qualitative test product, which can only provide a positive yin determination and cannot provide a specific concentration; 2) when the antigen concentration of the test sample is 0.65 ng/mL-0.85 ng/mL, Bio The results of the -rad kit assay vary widely. As in the ninth and eighteenth cases in Table 17, the actual results of the samples were positive, while the results of the Bio-rad kit tests were all negative. The kits provided by the present invention were detected as suspected patients and needed further determination. It can be seen that the kit provided by the invention is more accurate and reliable than the detection result of the Bio-rad kit.
  • the hybridoma cell or the passage cell thereof having the accession number CGMCC No.13827 provided by the present invention has stable performance, and the monoclonal antibody or the specific antigen-binding fragment thereof produced therefrom can specifically bind to the Aspergillus GM antigen. It can be made into a detection kit with sensitivity and specificity of more than 95%, and the degree of dispersion between batches, dispersion within the batch is very small, reproducibility is good, stability is high; and buffering of different coating liquids is selected.
  • the detection kit prepared by liquid, different GM antigen concentration, different blocking solution and different sample treatment liquids has small degree of dispersion and good repeatability.
  • the kit provided by the present invention can not only reduce the cost, but also can detect IA quickly and easily. The detection limit is reduced from 1 ng/mL of the existing product to 0.85 ng/mL, which can detect IA earlier, and the detection result is more accurate and reliable, so that the patient can treat early and improve the survival rate of the patient.

Abstract

提供了一种保藏编号为CGMCC No.13827的杂交瘤细胞或其传代细胞、该杂交瘤细胞所生产的能够抗曲霉菌半乳甘露聚糖抗原的单克隆抗体或其特异性抗原结合片段、以及包含该单克隆抗体或其特异性抗原结合片段的检测试剂或试剂盒。还提供了该单克隆抗体的制备方法以及该单克隆抗体或其特异性抗原结合片段在制备用于检测曲霉菌感染的检测试剂或试剂盒中的用途。

Description

GM杂交瘤细胞、单克隆抗体、试剂盒及其制备方法与应用 技术领域
本发明涉及生物技术领域,尤其涉及一种生产抗曲霉菌半乳甘露聚糖(Galactomannan,GM)抗原之单克隆抗体的杂交瘤细胞、单克隆抗体、试剂盒以及其制备方法与应用。
背景技术
曲霉菌(Aspergillus)是广泛存在于自然界的一种腐生菌及正常人体皮肤黏膜的常驻真菌。曲霉孢子较小,直径2-3μm,可以在空气中漂浮,并且通过呼吸道进入人体。由于曲霉主要通过呼吸道进入人体,所以曲霉感染主要发生在肺部。
在免疫抑制患者中侵袭性曲霉病(Invasive Aspergillosis,IA)的发病率由于抗生素的滥用而逐年增高,并成为其死亡的主要原因,烟曲霉菌是引起免疫抑制患者严重深部曲霉菌感染的最常见病原菌,其次还有黄曲霉菌、黑曲霉菌、土曲霉菌等。IA在血液病和造血干细胞移植(hematopoietic stem cell transplants,HSCT)患者中死亡率高达70%-90%,造成这种高死亡率的主要原因在于不能在病程早期对IA进行有效检测诊断,以至患者得不到及时有效的治疗而死亡,因此选择早期检测诊断方法具有重要的意义。
目前得到广泛认可的曲霉菌抗原检测方法主要有1,3-β-D葡聚糖检测(G试验)和半乳甘露聚糖检测(GM试验)。1,3-β-D葡聚糖抗原是除结合菌和隐球菌外所有真菌的一种特异性细胞壁成分,以血清为检测样本,其灵敏度和特异性可达到80%。但由于对定植的念珠菌显示阴性,常需要和GM试验联合检测进行排除,两者皆阴性才基本可排除真菌感染。另外,血清学G试验容易受到血液学及其他因素如纤维类物质等的影响,且1,3-β-D葡聚糖抗原在血液中可与抗体形成免疫复合物,迅速被血液清除,造成假阴性。
半乳甘露聚糖是存在于曲霉菌细胞壁中的一类具有高度特异性和高度保守型的多糖,可作为曲霉菌检测的特异性分子标志物,酶联免疫吸附试验(ELISA)是较为常见的半乳甘露聚糖检测方法,Acosta J报道GM阳性结果较曲霉菌培养早4.3天。血清GM试验阳性对侵袭性真菌感染的拟诊是恰当和适用的,且对于采取早期的抗真菌治疗的患者,尤其对一些高风险患者(如HSCT患者)更是重要的提示。因此测定血清中的GM抗原水平有助于IA的早期诊断、早期治疗。
目前市面上曲霉菌GM检测试剂盒检测的敏感性、特异性、灵敏性均较低,敏感性约为83%,特异性为90%,检测限约为1μg/L。且一般采用双抗夹心法,即先将曲霉菌的特异单克隆抗体包被在酶标板上,再加入待检样本和辣根过氧化物酶(HRP)标记的同一单克隆抗体,待检样本中的抗原会与特异单克隆抗体结合并形成夹心结构,再加入显色剂显色,颜色的深浅和待检抗原的浓度呈正相关,从而实现对GM抗原的检测。该方法检测步骤繁琐,且单克隆抗体制备成本高,不利于试剂盒在临床检测中的推广和普及。
发明内容
本发明一方面提供一种能够产生抗曲霉菌半乳甘露聚糖(Galactomannan,GM)抗原之单克隆抗体的杂交瘤细胞或其传代细胞。该杂交瘤细胞于2017年4月18日提交于中国科学院微生物研究所(地址为北京市朝阳区北辰西路1号院3号)保藏,保藏编号为CGMCC No.13827。
本发明还提供单克隆抗体或其特异性抗原结合片段,其中单克隆抗体由上述杂交瘤细胞所产生。该单克隆抗体或其特异性抗原结合片段能够可以与曲霉菌GM抗原特异性结合。示例性的,特异性抗原结合片段包括ScFv、(Fab’)2、Fab、Fv、scFv、双抗体、线性抗体或 多特异性抗体等。
本发明还提供上述单克隆抗体的制备方法,其包括腹水的制备,单克隆抗体的纯化,其纯化方法包括饱和硫酸铵盐析沉淀法和亲和层析法。
本发明还提供用于检测GM抗原的检测试剂及其检测方法,该检测试剂包括上述的单克隆抗体或其特异性抗原结合片段。在其检测GM抗原时,可采用竞争ELISA法,即:
a)将GM抗原包被在固相载体上;
b)将待检样本处理后与标记酶的抗GM抗原单克隆抗体加入固相载体,使待检样本中的抗原与包被后的抗原竞争结合有限的抗体结合位点;
或者,将待检样本处理后与未标记酶的抗GM抗原单克隆抗体加入固相载体,使待检样本中的抗原与包被后的抗原竞争结合有限的抗体结合位点,恒温反应并彻底洗涤,加入酶标二抗;
c)经过恒温反应并彻底洗涤,再加入酶的底物溶液显色,颜色的深浅和待检样本中GM浓度呈负相关;
d)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。
本发明还提供一种检测试剂盒及其制备方法,所述试剂盒包括上述单克隆抗体或其特异性抗原结合片段。该试剂盒检测曲霉菌GM抗原系采用竞争ELISA法。具体的,所述试剂盒包括GM抗原包被的固相载体、抗GM抗原单克隆抗体和GM抗原标准品。
在本发明一具体实施方式中,所述固相载体为酶标板、微孔板、试管或微孔滤膜,优选的,所述固相载体为酶标板;固相载体的材质例如可为聚苯乙烯、硝酸纤维素、尼龙等。
在本发明一具体实施方式中,所述抗GM抗原单克隆抗体为标记酶的抗GM抗原单克隆抗体;优选的,所述抗GM抗原单克隆抗体为标记酶的兔源抗GM抗原单克隆抗体。
或者,所述抗GM抗原单克隆抗体为未标记酶的抗GM抗原单克隆抗体,所述试剂盒还包括酶标二抗,所述酶标二抗可与抗GM抗原单克隆抗体相结合;优选的,所述抗GM抗原单克隆抗体为未标记酶的兔源抗GM抗原单克隆抗体,所述试剂盒还包括酶标羊抗兔二抗。
其中,所述酶为辣根过氧化物酶(Horseradish Peroxidase,HRP)、碱性磷酸酶(Alkaline Phosphatase,AP)或葡萄糖氧化酶(Glucose Oxidase,GO);优选的,所述酶为辣根过氧化物酶。
在本发明一具体实施方式中,所述GM抗原标准品包括至少三种已知浓度的GM抗原溶液,所述GM抗原标准品的浓度在0-50ng/mL范围内。优选的,所述GM抗原标准品的浓度在0-10ng/mL范围内,最优选的,在0-5ng/mL范围内。
在本发明一具体实施方式中,所述试剂盒还包括样本处理液、浓缩洗液、样本稀释液、底物溶液和终止液中的一种或多种。
其中所述样本处理液选自0.03mol/L EDTA、0.1mol/L EDTA、0.12mol/L EDTA、0.05mol/L蛋白酶K、0.1mol/L蛋白酶K、0.2mol/L蛋白酶K、5%DMSO、15%DMSO或30%DMSO。优选的,所述样本处理液为0.12mol/L EDTA。
所述试剂盒的制备方法包括:
1)GM抗原包被的固相载体的制备;
2)标准品的配制;
3)抗GM抗原单克隆抗体的制备。
其中,步骤1)中又包括配制GM抗原包被液、配制封闭液和包被酶标板。
所述GM抗原包被液的缓冲液选自由0.1mol/L Tris-HCl、0.1mol/L PBS、0.05mol/L CBS、0.1mol/L CBS、0.2mol/L CBS和生理盐水组成的组。优选的,GM抗原包被液的缓冲液为0.1mol/L Tris-HCl,其pH6.0-pH9.0;
所述的封闭液选自由2%新生牛血清、5%新生牛血清和8%新生牛血清组成的组。优选的,封闭液为8%的新生牛血清,其将新生牛血清加入生理盐水中,配制成封闭液;
本发明还提供单克隆抗体或其特异性抗原结合片段在制备用于检测曲霉菌感染的检测试剂或检测试剂盒中的用途,所述单克隆抗体由保藏号为CGMCC No.13827的杂交瘤细胞或其传代细胞产生。
示例性的,特异性抗原结合片段包括ScFv、(Fab’)2、Fab、Fv、scFv、双抗体、线性抗体或多特异性抗体等。
本发明的有益效果是:
本发明提供的保藏号为CGMCC No.13827的杂交瘤细胞或其传代细胞能够产生单克隆抗体,该抗体能够特异性结合曲霉菌GM抗原,其专一性高。且通过杂交瘤细胞或其传代细胞的培养能够获得大量的单克隆抗体,以解决将其制备成试剂盒在临床检测中的推广和普及时的成本问题。而且,本申请提供的检测试剂盒能够与GM抗原特异性结合,其敏感性和特异性均在95%以上,检测限从现有产品的1ng/mL降低到了0.85ng/mL,检测结果与参比试剂符合率高,能提供更准确可靠的检验结果,使得能够在病程早期即可检测出IA,使患者能够及时有效的得到治疗,提高患者的生存率,同时也减少了非必要的经验性抗真菌治疗。且本发明提供的试剂盒操作简便易行,检测快速灵敏,所用酶标仪简单、普及,价格低廉,该检测试剂盒为曲霉菌的GM抗原定量检测提供了一种有效工具。
附图说明
图1显示了实施例1中GM抗原纯品中多糖含量测定标准曲线;
图2显示了实施例1中GM抗原纯品的HPLC检测结果;
图3显示了实施例21中GM抗原免疫检测试剂盒标准曲线图。
具体实施方式
实施例1曲霉菌半乳甘露聚糖(GM)抗原的制备
采用曲霉菌制备GM抗原,本发明所采用的曲霉菌株购买自美国模式培养物集存库(ATCC),保藏编号为ATCC 1022。
将曲霉菌采用固体培养基培养至培养基长满绿色孢子;过滤除去菌丝,灭活菌体及孢子;离心后,收集孢子并洗涤;孢子经破碎后过滤除去孢子碎片;将滤液经醇沉、洗涤后,得到GM抗原粗提物;将GM抗原粗提物经脱色、超滤后得到GM抗原。
其中,固体培养基选自PDA培养基、沙氏培养基或察氏培养基;优选为PDA培养基。具体步骤如下:
一、制备GM粗品
配制PDA固体培养基2.0L,其成分为600g土豆煮沸后的上清液,D-葡萄糖80.0g,琼脂粉30.0g,将曲霉菌株置于培养基中,25℃培养3天,至培养基长满绿色孢子。用无菌生理盐水冲洗孢子,孢子悬液经8层无菌纱布过滤3次除去菌丝,在孢子悬液中加入终浓度为3.7%的甲醛4℃静置24h,灭活菌体及孢子。4℃,12,000g离心30min收集孢子,用无菌生理盐水洗涤6次,除去可能存在的甲醛。用液氮反复研磨冷冻的孢子,再加入无菌生理盐水,再用超声细胞破碎仪破碎孢子。所得孢子破碎液经定性滤纸过滤,滤液经0.45μm滤膜过滤以除去孢子碎片。将所得滤液转移至洁净容器中,加入2.5倍体积无水乙醇,4℃静置过夜。4℃,12,000g离心30min,将沉淀溶于去离子水中,加2.5倍体积无水乙醇,静置2小时,离心分离沉淀,沉淀用无水乙醇洗涤三次。4℃,12,000g离心30min,弃上清,得到粗提的GM。
二、用活性炭吸附的方法对粗提的GM进行脱色,具体步骤如下:
将GM溶于200mL去离子水,边搅拌边缓慢加入活性炭粉末3.0g,4℃脱色4小时,待 溶液棕黄色褪去,用布氏漏斗过滤,反复过滤至溶液澄清,即得到去除色素的GM提取物。
三、用超滤的方法对GM进行纯化,具体步骤如下:
室温抽滤,除去活性炭颗粒,所得滤液经0.22μm滤膜过滤。滤液转移至10KD离心超滤管,4000g离心20min,即得到高纯度GM。
四、对GM进行纯化,对得到的GM样品进行多糖、蛋白质及核酸含量检测:
1)用Dubois-硫酸苯酚法对上述得到的GM抗原纯品进行多糖含量测定,检测结果如表1-1,表1-2,附图1所示:
表1-1 GM抗原纯品中多糖含量
Figure PCTCN2017090519-appb-000001
表1-2 GM抗原纯品中多糖含量
  体积(mL) 糖含量(mg)
检测样品 0.04 0.088
全部样品 100 220
由检测结果可知,采用该制备方法从2L烟曲霉培养基最终可得到220mg烟曲霉菌半乳甘露聚糖抗原纯品。
2)对该制备方法得到的GM抗原纯品样本进行紫外吸收检测。分别在UV260nm、UV280nm、UV320nm波长下检测样本,结果如下表2。由检测结果可知,核酸及蛋白质的总含量不超过样本总质量的4%。
表2 GM抗原纯品中DNA和蛋白质含量
检测项目 浓度 含量(%)
DNA 3.641μg/mL 0.16
蛋白质 74.583μg/mL 3.27
多糖 2.203mg/mL 96.57
五、对GM进行纯化,对得到的GM样品进行HPLC检测:
对该制备方法得到的GM抗原纯化样品进行HPLC检测。检测器为示差折光检测器。 检测结果如附图2所示。从图中可看出,样品出峰单一,尖窄,并无明显杂峰出现,说明所含物质大小均一,纯度较高。
六、用该制备方法对GM抗原进行纯化,对得到的GM抗原纯品样本用Bio-Rad公司的烟曲霉抗原检测试剂盒进行抗原鉴定。
目前国际上通常采用美国Bio-Rad公司的烟曲霉抗原检测试剂盒对GM抗原进行检测。用该试剂盒对GM抗原样品进行检测,样本浓度为1ng/mL。检测结果见下表3,由结果可知,GM抗原呈阳性,且OD值大于阳性质控,说明用本发明中的方法可以获得烟曲霉的半乳甘露聚糖抗原。
表3采用Bio-Rad烟曲霉抗原检测试剂盒进行抗原鉴定检测结果
Figure PCTCN2017090519-appb-000002
实施例2GM抗原修饰
众所周知,分子大小可影响物质的免疫原性形成,一个有效免疫原的分子量大多在10kD以上;分子量越大,免疫原性越强。这可能是因高分子物质在水溶液中易形成胶体,在体内停留的时间较长,与免疫细胞接触的机会较多,有利于刺激机体产生免疫应答。另外,大分子物质的化学结构比较复杂,所含有效抗原基因的种类和数量也相对地多。
由实施例1得到的GM抗原,分子量较小,免疫原性欠佳,需与相关大分子物质偶联以提高其免疫原性。该大分子是乳胶微球,KLH(钥孔血蓝蛋白),BSA(牛血清白蛋白),GST中的一种或多种,部分偶联需加入交联剂,以促进其交联率的提高,获得的抗原多肽片段可用于免疫动物。
本实施例以氨基化乳胶微球为例,其具体的偶联方法如下:
1.用10mL洗涤/偶联缓冲液将1mL微球(100mg/mL)清洗2次;
2.重悬在10mL戊二醛溶液中(戊二醛溶解在洗涤/偶联缓冲液中,浓度为10%),确保微球充分悬浮;
3.室温(18-25℃)反应1-2h,并不断搅拌;
4.用偶联缓冲液清洗2次,重悬在5mL偶联缓冲液中,确保充分悬浮;
5.将抗原溶解在5mL的偶联缓冲液,与悬浮微球混合;
6.室温下反应2-4h,并不断搅拌;
7.清洗,重悬在10mL的终止液中,温和搅拌30min。清洗,以合适的浓度(通常为10mg/mL)重悬在储存缓冲液中;
8.在4℃下储存,备用。
实施例3抗GM抗原单克隆抗体的制备
一、抗GM抗原的单克隆抗体的制备
1.免疫动物
将GM抗原免疫动物。其中,免疫可以采用皮下注射、足垫注射、脾内注射、静脉注射或腹腔注射等方式;动物可以选取大鼠、小鼠、豚鼠、兔、鸡、羊、马、猪或驴等。本实 施例采用兔作为免疫动物,具体步骤如下:
将GM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0.01-0.1mg。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,测定免疫后兔的血清效价。
2.细胞融合
免疫脾细胞是指脾脏中处于免疫状态的B淋巴母细胞一浆母细胞。一般取最后一次加强免疫3天后的脾脏,制备成细胞悬浮液,由于此时B淋巴细胞比例较大,因而细胞融合的成功率比较高。
首先,取脾,活化骨髓瘤细胞。骨髓瘤细胞与脾细胞按1:10的比例进行融合。采用PEG融合法。
3.杂交瘤细胞的筛选与克隆化
细胞融合之后,培养基里存在两种亲本细胞和三种随机融合的细胞,要想得到能分泌目的抗体的杂交瘤细胞,就必须把成功融合的杂交瘤细胞从众多细胞中分离出来。B淋巴细胞不能再体外长期生存,需要除去的只有骨髓瘤细胞和其自身融合细胞,因此需要将融合后的细胞通过HAT培养基培养,选择性的保留杂交瘤细胞。
融合后第5天可观察细胞的生长情况,第l0~14天,即可采用间接ELISA法检测细胞培养上清并筛选阳性杂交瘤细胞,进行克隆化培养。采用有限稀释法对阳性杂交瘤细胞进行克隆化培养。将检测结果效价最强的阳性杂交瘤细胞扩大化至细胞阳性率达100%时定株。用ELISA测取杂交瘤细胞培养上清的效价,将其扩大化培养的单克隆杂交瘤细胞冻存于液氮中。并将该杂交瘤细胞送于中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏编号为CGMCC No.13827。
4.腹水的制备
选用新西兰大耳白兔腹腔内注射石蜡油0.5mL,2周后收集杂交瘤细胞,用生理盐水悬浮,每1mL生理盐水悬浮1~5×106个细胞。然后每只兔子腹腔注射0.5mL细胞悬液,并注射等量石蜡和弗氏不完全佐剂混合物。约7-10天后,明显产生腹水后,将兔子处死,剪开腹腔,吸取腹水,12000r/min离心15min收集腹腔腹水,加入适量防腐剂于4℃保存。
5.单克隆抗体的纯化
用饱和硫酸铵盐析法进行初步纯化:
取2mL腹水样本,加等体积的生理盐水,再加入4mL饱和硫酸铵溶液,于4℃沉淀过夜;
10000g低温离心10min,弃上清,将沉淀用2mL PBS溶解,缓慢滴加1mL饱和硫酸铵溶液,在4℃静置1小时;
10000g低温离心10min,弃上清,将沉淀用1mL PBS溶解,用PBS溶液4℃透析过夜;
用亲和层析的方法进一步纯化:
用5-10倍柱床体积的洗脱缓冲液洗柱;
用5-10倍柱床体积的偶联缓冲液洗柱;
用饱和硫酸铵盐析法初步纯化过的样品上样;
用5-10倍柱床体积的偶联缓冲液洗柱;
用2-5倍柱床体积的洗脱缓冲液洗脱,得到抗GM抗原单克隆抗体;
其中,洗脱缓冲液选自0.1mol/L甘氨酸缓冲液、PB缓冲液、柠檬酸-磷酸盐缓冲液、柠檬酸-柠檬酸钠缓冲液或醋酸-醋酸钠缓冲液,pH为3.0;偶联缓冲液选自PBS缓冲液、Tris-HCl缓冲液、醋酸-醋酸钠缓冲液中的一种,优选为PBS缓冲液。
曲霉菌半乳甘露聚糖(GM)抗原免疫检测试剂盒的制备
具体制备方法如下:
一、制备GM抗原包被的酶标板
1.配制GM抗原包被液:
采用缓冲溶液将GM抗原稀释至100ng/mL-10μg/mL;所述缓冲溶液选自:0.1mol/L Tris-HCl缓冲液,其pH6.0-pH9.0;0.1mol/LPBS缓冲液,其pH6.0-pH9.0;0.05-0.2mol/L CBS缓冲液,其pH6.0-pH9.0;或生理盐水。
2.配制封闭液:
将2%-8%的新生牛血清加入缓冲溶液中,配制成封闭液;所述缓冲溶液选自:0.1mol/L Tris-HCl缓冲液,其pH6.0-pH9.0;0.1mol/L PBS缓冲液,其pH6.0-pH9.0;0.05-0.2mol/L CBS缓冲液,其pH6.0-pH9.0;或生理盐水。
3.包被酶标板:将配制的GM抗原包被液加入酶标板孔中,每孔分别加入50-150μL,(优选100μL)包被液;酶标板置于12-18℃(优选15℃)环境下包被6-8h(优选7h);将配制的封闭液加入酶标板孔中,每孔分别加入50-150μL(优选100μL)封闭液,置于12-18℃(优选15℃)恒温箱,2-4h(优选3h);从恒温箱取出酶标板后弃去封闭液,20-25℃(优选22℃)恒温2-4h(优选3h)。
在包被酶标板之前,可对酶标板进行修饰。方法为将酶标板置于装有紫外灯的医用净化操作台上,固定紫外灯与微孔板基底的垂直距离,选择不同的时间段对微孔板进行紫外处理。
二、配制标准品(定量标准曲线的建立)
标准品的制备是将GM抗原用0.1mol/L PBS稀释液配制而成,GM抗原的浓度分别是5ng/mL,2.5ng/mL,1ng/mL,0.5ng/mL,0.25ng/mL。
三、配制抗GM抗原单克隆抗体溶液
抗GM抗原单克隆抗体溶液的配制是将抗GM抗原单克隆抗体用酶偶联物稳定剂以1:20000-1:40000(优选1:30000)的比例稀释而成。
酶偶联物稳定剂能够保持抗体与酶偶联物之间稳定性的试剂,能够保持抗体及酶的活性。优选的,其可为HRP酶偶联物稳定剂,本发明中酶偶联物物稳定剂可为市售产品。
四、配制酶标二抗溶液
酶标二抗溶液的配制是将辣根过氧化物酶(HRP)标记的羊抗兔二抗用HRP酶偶联物稳定剂以1:5000-1:20000(优选1:10000)的比例稀释而成。
五、样本处理液
样本处理液为蛋白变性溶液,优选的,选自以下蛋白变性溶液中的一种或多种:0.05-0.2mg/mL蛋白酶K;pH2-pH10的0.03-0.18mol/L EDTA(乙二胺四乙酸二钠)溶液;5-30%DMSO(二甲基亚砜)溶液;pH7.0-pH8.0的1-8mol/L的尿素。
六、浓缩洗液(20×0.01M PBS)
浓缩洗液为含吐温-20的PBS溶液(简称PBST溶液),其中,PBST溶液中可包含生物液体防腐剂如ProClin300。本实施例选用浓缩洗液如下:
按重量份数计氯化钠160.0份,氯化钾4.0份,十二水合磷酸氢二钠31.6份,磷酸二氢钾2.8份,吐温-20 0.2份,ProClin300 2份,超纯水1000份,混合均匀。
七、样本稀释液
样本稀释液可为含5-15%脱脂乳粉、BSA或牛血清的的CBS稀释液。本实施例选择含10%脱脂乳粉的CBS稀释液。
八、底物溶液
底物溶液可为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),优选的为四甲基联苯胺(3,3',5,5'-Tetramethylbenzidine,TMB)。
九、终止液
终止液可为1-10mol/L的硫酸溶液。优选为2mol/L硫酸溶液,将浓硫酸与超纯水按1:8比例稀释,配制而成。
本申请对上述制备方法中的各个条件进行了试验。其中实施例4-9试验了不同GM抗原包被液对试剂盒检测重复性的影响;实施例10-11试验了不同封闭液对试剂盒检测重复性的影响;实施例12-18试验了不同样本处理液对试剂盒检测重复性和回收率的影响,具体实施方式如下表4。
表4各个实施例中包被液、封闭液及样本处理液种类及浓度
实施例 包被液中的缓冲液 GM抗原终浓度 封闭液 样本处理液
实施例4 0.1mol/L Tris-HCl 100ng/mL 2%新生牛血清 0.03mol/L EDTA
实施例5 0.1mol/L PBS 100ng/mL 2%新生牛血清 0.03mol/L EDTA
实施例6 0.05mol/L CBS 100ng/mL 2%新生牛血清 0.03mol/L EDTA
实施例7 0.1mol/L CBS 100ng/mL 2%新生牛血清 0.03mol/L EDTA
实施例8 0.2mol/L CBS 100ng/mL 2%新生牛血清 0.03mol/L EDTA
实施例9 生理盐水 100ng/mL 2%新生牛血清 0.03mol/L EDTA
实施例10 0.1mol/L Tris-HCl 5μg/mL 8%新生牛血清 0.1mol/L EDTA
实施例11 0.1mol/L Tris-HCl 5μg/mL 5%新生牛血清 0.1mol/L EDTA
实施例12 0.1mol/L Tris-HCl 10μg/mL 8%新生牛血清 0.12mol/L EDTA
实施例13 0.1mol/L Tris-HCl 10μg/mL 8%新生牛血清 0.05mol/L蛋白酶K
实施例14 0.1mol/L Tris-HCl 10μg/mL 8%新生牛血清 0.1mol/L蛋白酶K
实施例15 0.1mol/L Tris-HCl 10μg/mL 8%新生牛血清 0.2mol/L蛋白酶K
实施例16 0.1mol/L Tris-HCl 10μg/mL 8%新生牛血清 5%DMSO
实施例17 0.1mol/L Tris-HCl 10μg/mL 8%新生牛血清 15%DMSO
实施例18 0.1mol/L Tris-HCl 10μg/mL 8%新生牛血清 30%DMSO
实施例19标记酶的抗GM抗原单克隆抗体溶液的制备
将辣根过氧化物酶标记的抗GM抗原单克隆抗体用HRP酶偶联物稳定剂以1:20000的比例稀释而成。
实施例20曲霉菌半乳甘露聚糖抗原免疫检测试剂盒检测步骤
两步法:
a)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;
b)将GM抗原标准品和步骤a)的待检测物分别与抗GM抗原单克隆抗体等体积混匀并孵育60min-120min;
c)将步骤b)的混合物加入GM抗原包被的酶标板中并孵育60min-120min,孵育后洗板;
d)向步骤c)的酶标板中加入酶标二抗并孵育20-60min,孵育后洗板;
e)向步骤d)的酶标板中加入底物溶液显色10-15min后,加入终止液后进行检测,于酶标仪上读取450纳米处的吸光光度值,通过标准曲线实现对抗原的检测。具体步骤如下:
一、样本的处理
1)将待检样本与样本处理液以1:3的体积比混合后沸水浴1min。
2)将水浴后的混合液1,000g离心1min。
3)离心后上清液用于检测。
二、检测步骤
1)取出已预包被抗原的96孔酶标板;
2)配制工作洗涤液:浓缩洗液稀释20×(1份浓缩洗液(20×0.01M PBS)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(GM抗原标准品浓度分别为5,2.5,1,0.5,0.25ng/mL)
待测样本组:处理后的待测样本
将两组样本分别与兔源抗GM抗原单克隆抗体等体积混合,转移至酶标板孔中,每孔加入60μL,在37℃下孵育60min;
4)洗涤:甩掉反应液,每孔每次加入不少于300μL的洗涤液,静置40s后拍干,重复上述洗涤操作,共洗涤3次;
5)加入酶标二抗:洗涤结束后,每孔加入酶标羊抗兔二抗60μL,在37℃下孵育20min;
6)洗涤:同步骤4);
7)显色:洗涤结束后,每孔加入底物溶液60μL,在37℃孵育15min,避光;
8)终止:每孔内加入50μL终止液,混匀后,在OD450nm处读数;
9)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,即可自动计算出各待测样品中GM抗原的浓度值。
或曲霉菌半乳甘露聚糖抗原免疫检测试剂盒检测步骤(一步法)
a)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;
b)将GM抗原标准品和步骤a)的待检测物分别与标记酶的抗GM抗原单克隆抗体等体积混匀并加入GM抗原包被的酶标板中同时孵育60-120min,孵育后洗板;
c)向步骤b)的酶标板中加入底物溶液显色10-15min后,加入终止液后进行检测,于酶标仪上读取450纳米处的吸光光度值,通过标准曲线实现对抗原的检测。具体步骤为:
1)、2)同两步法中的1)、2);
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(GM抗原标准品浓度分别为5,2.5,1,0.5,0.25ng/mL)
待测样本组:处理后的待测样本
将两组样本分别与标记酶的抗GM抗原单克隆抗体等体积混合,转移至酶标板孔中,每孔加入80μL,在37℃下孵育90min;
4)洗涤:同上;
5)显色:同上;
6)终止:同上;
7)结果判断:同上。
实施例21曲霉菌半乳甘露聚糖抗原免疫检测试剂盒的临床应用
取实施例10的试剂盒,按照实施例20中的两步法检测步骤进行试剂盒的临床应用检测。
1.绘制标准曲线
取实施例10的试剂盒,按实施例20的检测步骤,得到各标准曲线点(5,2.5,1,0.5和0.25ng/mL)的测量值如表5所示,利用表5数据,以样品中GM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3所示,得到标准曲线方程为:y=-0.21ln(x)+0.755,线性相关度R2=0.999,标准曲线方程拟合良好。
表5检测标准曲线
抗原浓度(ng/mL) OD450
5 0.416
2.5 0.558
1 0.756
0.5 0.906
0.25 1.045
2.GM抗原免疫检测试剂盒参考值的确定
临床确诊为曲霉菌感染阳性样本30例,正常人样本200例,将样本处理后,取实施例4的试剂盒按照实施例20的检测步骤测定OD450值,根据标准曲线(表5,图3)计算GM抗原浓度值,如表6所示。
表6 GM抗原免疫检测试剂盒参考值的确定ELISA临床检测结果
Figure PCTCN2017090519-appb-000003
注:*表示与正常人比较P<0.01;
根据标准曲线的结果计算检测GM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下限:
Figure PCTCN2017090519-appb-000004
(平均值)+2s(标准偏差)=0.45+2*0.10=0.65,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上限:
Figure PCTCN2017090519-appb-000005
(平均值)-2s(标准偏差)=1.55-2*0.35=0.85,抗原的浓度值在0.65ng/mL-0.85ng/mL之间为疑似病人。即得到GM抗原免疫检测试剂盒的判断标准参考值如表6所示。
表7 GM抗原免疫检测试剂盒判断标准参考值
Figure PCTCN2017090519-appb-000006
如果样本的检测结果落在可疑区间,则需要进行第二次检测。
实施例22试剂盒的方法学考察
取实施例10的试剂盒按照实施例20的检测步骤进行试剂盒方法学考察(敏感性实验、特异性实验、回收率实验、重复性实验、稳定性实验)。
1.敏感性实验
收集临床确诊样本20例进行检验。
诊断敏感性=阳性样本检出例数/阳性样本总例数×100%,实验结果见表8,由结果可知,本实验的敏感性在95%以上。
表8敏感性检测实验结果
序号 OD450 计算抗原浓度(μg/L) 结果判断
1 0.695 1.33 阳性
2 0.570 2.4 阳性
3 0.625 1.85 阳性
4 0.493 3.45 阳性
5 0.720 1.18 阳性
6 0.593 2.15 阳性
7 0.502 3.31 阳性
8 0.637 1.75 阳性
9 0.576 2.33 阳性
10 0.708 1.25 阳性
11 0.819 0.74 疑似
12 0.493 3.45 阳性
13 0.634 1.77 阳性
14 0.503 3.3 阳性
15 0.674 1.47 阳性
16 0.627 1.83 阳性
17 0.612 1.97 阳性
18 0.434 4.57 阳性
19 0.494 3.44 阳性
20 0.768 0.94 阳性
2.特异性实验
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%,实验结果见表9,由结果可知,本实验的特异性在95%以上,其中只有一例检测出为疑似患者,需要进行第二次检测。
表9特异性检测实验结果
序号 OD450 计算抗原浓度(μg/L) 结果判断
1 0.915 0.47 阴性
2 0.971 0.36 阴性
3 0.919 0.46 阴性
4 0.849 0.64 阴性
5 0.910 0.48 阴性
6 0.989 0.33 阴性
7 0.996 0.32 阴性
8 0.893 0.52 阴性
9 0.938 0.42 阴性
10 1.040 0.26 阴性
11 0.902 0.5 阴性
12 0.977 0.35 阴性
13 0.989 0.33 阴性
14 0.802 0.8 疑似
15 0.885 0.54 阴性
16 0.954 0.39 阴性
17 0.954 0.39 阴性
18 0.893 0.52 阴性
19 0.906 0.49 阴性
20 0.870 0.58 阴性
3.回收率实验
选择正常人血液添加曲霉半乳甘露聚糖抗原2μg/L、1μg/L后检测,计算真实值与期望值的比值,得到回收率,见表10。回收率介于80-120%之间认为合格。由实验结果说明本实验的回收率介于80%-120%之间,回收率良好。且当添加抗原浓度为2μg/L时,回收率介于92%~102%,更加接近于100%。
表10回收率结果实验
Figure PCTCN2017090519-appb-000007
Figure PCTCN2017090519-appb-000008
4.重复性实验
1)批间精密度
合格标准:将同一标本每天一次测试,连续11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,结果见表11。结论:本产品批间精密度(即变异系数CV)为3%,远小于25%,符合标准,证明本产品批间精密度很好,批间离散程度非常小,重复性好。
表11批间精密度结果实验
Figure PCTCN2017090519-appb-000009
2)批内精密度
合格标准:将同一标本在同一批次实验中平行测定10组数据。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,见表12。本产品批内精密度(即变异系数CV)为1%,远小于15%,不仅符合标准,验证合格,而且说明其批内离散程度非常小,重复性很好。
表12批内精密度结果实验
序号 1 2 OD450 计算抗原浓度(μg/L)
1 0.634 0.638 0.636 1.76
2 0.637 0.635 0.636 1.76
3 0.632 0.641 0.637 1.75
4 0.641 0.633 0.637 1.75
5 0.633 0.638 0.636 1.76
6 0.635 0.632 0.634 1.78
7 0.637 0.643 0.640 1.72
8 0.635 0.636 0.636 1.76
9 0.631 0.633 0.632 1.79
10 0.642 0.634 0.638 1.74
      M 1.76
      SD 0.0184
      CV 1%
5.稳定性实验
将组装好的试剂盒在37℃环境中放置,每天做标准曲线检测已知浓度的抗原溶液,连续检测5天,检测值变化率(即变异系数CV)小于20%,结果参见表13,证明试剂盒稳定。其结果显示5天的变异系数CV为3.5%,远远小于20%,说明本发明提供的试剂盒稳定性非常好,使其检测结果更加可靠。
表13稳定性试验结果
Figure PCTCN2017090519-appb-000010
Figure PCTCN2017090519-appb-000011
实施例10中提供的试剂盒按照实施例20提供的一步法试验,也得出类似的实验结果。其他事实例按照实施例20提供的两步法或一步法试验,均得到类似的实验结果。
实施例23不同GM抗原包被液对试剂盒检测重复性的影响
分别取实施例4-9制备的试剂盒,按照实施例20的检测步骤对同一待测样本进行批间精密度检测,将同一标本每天一次测试,连续10个工作日,考察不同GM抗原包被液对试剂盒检测的影响,检测结果参见下表14。
表14
Figure PCTCN2017090519-appb-000012
Figure PCTCN2017090519-appb-000013
由表14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GM抗原的免疫检测;且实施例4制备的试剂盒的检测样本浓度的变异系数CV值最小,表明实施例4中包被液较优。
实施例24不同封闭液对试剂盒检测重复性的影响
分别取实施例10-11制备的试剂盒,按照实施例20的检测步骤对同一待测样本进行批间精密度检测,将同一标本每天一次测试,连续10个工作日,考察不同封闭液对试剂盒检测重复性的影响,检测结果参见下表15。
表15
Figure PCTCN2017090519-appb-000014
Figure PCTCN2017090519-appb-000015
由表15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GM抗原的免疫检测;且实施例10制备的试剂盒的检测样本浓度的变异系数CV值最小,表明实施例10中封闭液较优。
实施例25不同样本处理液对试剂盒检测重复性和回收率的影响
分别取实施例12-18制备的试剂盒,按照实施例20中两步法的检测步骤对同一待测样本(浓度已知,为1.43ng/mL)进行批间精密度和回收率检测,将同一标本每天一次测试,连续10个工作日,考察不同样本处理液对试剂盒检测重复性和回收率的影响,检测结果参见下表16。
表16
Figure PCTCN2017090519-appb-000016
Figure PCTCN2017090519-appb-000017
由表16中数据可知,各试剂盒对样本的检测结果的CV值均小于8%,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GM抗原的免疫检测;且实施例12制备的试剂盒的检测样本浓度的变异系数CV值最小,且回收率接近100%,表明实施例12中处理液较优。
实施例26本发明试剂盒与Bio-rad公司产品检测样本结果对比
分别用本发明试剂盒(以本发明实施例12提供的试剂盒为例)与Bio-rad公司的试剂盒(批号为6H0042)检测24例样本,具体结果见下表17。
其中本发明试剂盒参考值判定标准为:参考范围上限为0.85μg/L,下限为0.65μg/L。如浓度≥0.85μg/L,则定为阳性;如浓度<0.65μg/L,则定为阴性;浓度在0.65ng/mL-0.85ng/mL之间为疑似病人。Bio-rad公司试剂盒判定标准为I值≥0.5为阳性,I值<0.5为阴性。
表17本发明试剂盒与Bio-rad试剂盒比较实验结果
Figure PCTCN2017090519-appb-000018
由表17可知,1)Bio-rad试剂盒为定性检测产品,仅能提供阴阳性判定,无法提供具体浓度;2)当检测样本的抗原浓度为0.65ng/mL-0.85ng/mL时,Bio-rad试剂盒检测的结果偏差很大。如表17中的第9例和第18例,样本的实际结果为阳性,而Bio-rad试剂盒检测的结果却均为阴性,本发明提供的试剂盒检测为疑似患者,需要进一步确定。可见,本发明提供的试剂盒相较于Bio-rad试剂盒的检测结果更加准确、可靠。
本发明提供的保藏号为CGMCC No.13827的杂交瘤细胞或其传代细胞,性能稳定,由其产生的单克隆抗体或其特异性抗原结合片段,能够特异性结合曲霉菌GM抗原。将其制成检测试剂盒,敏感性、特异性均能够达到95%以上,且批间离散程度、批内离散程度非常小,重复性好,稳定性高;而且选择不同的包被液的缓冲液,不同GM抗原浓度,不同的封闭液以及不同的样本处理液所制备的检测试剂盒,检测结果的离散程度均较小,重复性好。本发明提供的试剂盒不仅能够降低成本,而且能够快速、简便地检测出IA。其检测限从现有产品的1ng/mL降低到了0.85ng/mL,能够更早的检测出IA,且检测结果更加准确可靠,使得患者能够及早治疗,提高患者的生存率。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换等,均应包含在本发明的保护范围之内。

Claims (10)

  1. 杂交瘤细胞或其传代细胞,其中所述的杂交瘤细胞的保藏号为CGMCC No.13827。
  2. 单克隆抗体或其特异性抗原结合片段,其中所述的单克隆抗体由权利要求1所述杂交瘤细胞所产生,所述特异性抗原结合片段能特异性结合曲霉菌GM抗原。
  3. 根据权利要求2所述的抗体或其特异性抗原结合片段,其中所述特异性抗原结合片段包括选自由ScFv、(Fab’)2、Fab、Fv、scFv、双抗体、线性抗体和多特异性抗体组成的组。
  4. 检测试剂,其包含权利要求2或3所述的抗体或其特异性抗原结合片段。
  5. 检测试剂盒,其包括权利要求2或3所述的抗体或其特异性抗原结合片段。
  6. 根据权利要求5所述的试剂盒,所述试剂盒还包括包被液的缓冲液、封闭液和/或样本处理液。
  7. 根据权利要求6所述的试剂盒,其中所述包被液的缓冲液选自由0.1mol/L Tris-HCl、0.1mol/L PBS、0.05mol/L CBS、0.1mol/L CBS、0.2mol/L CBS和生理盐水组成的组;所述的封闭液选自由2%新生牛血清、5%新生牛血清和8%新生牛血清组成的组;和,所述样本处理液选自由0.03mol/L EDTA、0.1mol/L EDTA、0.12mol/L EDTA、0.05mol/L蛋白酶K、0.1mol/L蛋白酶K、0.2mol/L蛋白酶K、5%DMSO、15%DMSO和30%DMSO组成的组。
  8. 根据权利要求5所述的试剂盒,所述试剂盒还包括:包被液的缓冲液:0.1mol/L Tris-HCl;封闭液:8%新生牛血清;和样本处理液:0.12mol/L EDTA。
  9. 权利要求2或3中所定义的单克隆抗体的制备方法,其包括培养权利要求1中所述的杂交瘤细胞或其传代细胞。
  10. 权利要求2或3中所定义的单克隆抗体或其特异性抗原结合片段在制备用于检测曲霉菌感染的检测试剂或检测试剂盒中的用途。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101149377A (zh) * 2007-10-29 2008-03-26 南方医科大学珠江医院 用于检测曲霉的抗体
WO2010082034A1 (en) * 2009-01-16 2010-07-22 University Of Exeter Antibody

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105866407A (zh) * 2016-04-22 2016-08-17 丹娜(天津)生物科技有限公司 一种曲霉菌半乳甘露聚糖抗原免疫检测试剂盒及其制备方法与应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101149377A (zh) * 2007-10-29 2008-03-26 南方医科大学珠江医院 用于检测曲霉的抗体
WO2010082034A1 (en) * 2009-01-16 2010-07-22 University Of Exeter Antibody

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HAO, WEI ET AL.: "Preparation of a Group of Aspergillus Monoclonal Antibodies and Study in Immunopathologic Diagnosis", GUANGDONG MEDICAL JOURNAL, vol. 29, no. 10, 10 October 2008 (2008-10-10), pages 1633 - 1636, ISSN: 1001-9448 *
THORNTON, CR: "Development of an Immunochromatographic Lateral-Flow Device for Rapid Serodiagnosis of Invasive Aspergillosis", CLINICAL AND VACCINE IMMUNOLOGY, vol. 15, no. 7, 31 July 2008 (2008-07-31), pages 1095 - 1105, XP007912963, ISSN: 1556-6811 *

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