WO2022170977A1 - Anticorps monoclonal de 1,3-bêta-d-glucane antifongique et son utilisation - Google Patents

Anticorps monoclonal de 1,3-bêta-d-glucane antifongique et son utilisation Download PDF

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WO2022170977A1
WO2022170977A1 PCT/CN2022/074015 CN2022074015W WO2022170977A1 WO 2022170977 A1 WO2022170977 A1 WO 2022170977A1 CN 2022074015 W CN2022074015 W CN 2022074015W WO 2022170977 A1 WO2022170977 A1 WO 2022170977A1
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glucan
fungal
monoclonal antibody
antifungal
detection
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Chinese (zh)
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刘春龙
张舟
张玉静
周泽奇
粟艳
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Dynamiker Biotechnology Tianjin Co Ltd
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Dynamiker Biotechnology Tianjin Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
    • G01N2400/24—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar beta-D-Glucans, i.e. having beta 1,n (n=3,4,6) linkages between saccharide units, e.g. xanthan

Definitions

  • the application belongs to the technical field of fungal detection, and relates to an anti-fungal 1,3- ⁇ -D-glucan monoclonal antibody and applications thereof.
  • the detection of fungal 1,3- ⁇ -D-glucan mainly adopts the Limulus reagent method.
  • Form coagulase which catalyzes the color reaction or turbidity reaction, generates free p-nitroaniline (pNA) and causes absorbance changes, and realizes 1,3- ⁇ -D-glucan by dynamically detecting the solution absorbance change rate Quantitative detection of concentration.
  • the total detection time of this method was about 50 min, and the lower limit of linearity was 37.5 pg/mL.
  • the key raw materials for the Limulus Reagents come from the national second-class wild animal, Limulus, which has a long growth cycle and a low artificial breeding rate. Usually, it is necessary to catch wild Limulus and extract the raw materials of Limulus reagents from the blood of Limulus. Limulus is an endangered species. The detection of fungal 1,3- ⁇ -D-glucan by the Limulus reagent method can no longer meet the clinical needs, and the Limulus reagent prepared from natural Limulus blood varies greatly between batches, resulting in high production costs and poor reproducibility. Difference.
  • the Limulus reagent will also activate coagulase to form coagulase, and bacterial endotoxin is widely present in nature, so the Limulus reagent method is very susceptible to the interference of bacterial endotoxin.
  • Chemiluminescence detection has been widely used in the field of biological macromolecule detection due to its good specificity, high sensitivity, short detection time, and easy automation.
  • the detection kit adopts the sandwich method.
  • the sample, biotin-labeled antibody and ruthenium complex-labeled antibody form an antibody-antigen sandwich immune complex, and then streptavidin magnetic beads are added.
  • the interaction between biotin and streptavidin binds to the immune complex; electromagnetic action is applied to the incubation mixture, the magnetic beads and the immune complex are adsorbed on the surface of the electrode, and the substances not bound to the magnetic beads are removed by ProCell;
  • the electrode applies voltage to chemiluminesce the immune complex, and the concentration of the antigen in the sample is analyzed according to the intensity of the chemiluminescence.
  • the detection time of the detection kit is short, only 18 minutes, but the biotin in the sample will interfere with the binding of the biotin-labeled antibody to the streptavidin magnetic beads, which will affect the accuracy of the results.
  • the present application provides an anti-fungal 1,3- ⁇ -D-glucan monoclonal antibody and application thereof, the anti-fungal 1,3- ⁇ -D-glucan monoclonal antibody is effective against fungal 1,3- ⁇ -D-glucan has specific binding ability, good stability and small batch-to-batch variation.
  • the constructed chemiluminescence-based fungal 1,3- ⁇ -D-glucan detection kit achieves sensitivity, specificity and accuracy.
  • the quantitative or semi-quantitative detection of fungal 1,3- ⁇ -D-glucan has important application prospects in judging the degree of fungal infection in patients.
  • the present application provides an anti-fungal 1,3- ⁇ -D-glucan monoclonal antibody
  • the anti-fungal 1,3- ⁇ -D-glucan monoclonal antibody comprises a variable heavy chain regions and light chain variable regions
  • the heavy chain variable region includes the heavy chain CDR1 shown in SEQ ID NO:1, the heavy chain CDR2 shown in SEQ ID NO:2 and the heavy chain CDR3 shown in SEQ ID NO:3;
  • the light chain variable region includes the light chain CDR1 shown in SEQ ID NO:4, the light chain CDR2 shown in SEQ ID NO:5 and the light chain CDR3 shown in SEQ ID NO:6;
  • SEQ ID NO: 1 SGGNTNQQK
  • SEQ ID NO: 2 DSSDPPPMSLLTEV
  • SEQ ID NO: 3 NNMDRAVKL
  • SEQ ID NO: 4 GLCDETRFEC
  • SEQ ID NO: 5 VPSPLAPISKQL
  • SEQ ID NO: 6 QANIYSYCSE.
  • the anti-fungal 1,3- ⁇ -D-glucan monoclonal antibodies comprising the CDRs of SEQ ID NOs: 1 to 6 have significant specific binding to fungal 1,3- ⁇ -D-glucan It is beneficial to construct a specific, sensitive, accurate and inexpensive fungal 1,3- ⁇ -D-glucan detection kit, which is of great significance in the field of fungal 1,3- ⁇ -D-glucan detection.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7;
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8;
  • the antifungal 1,3- ⁇ -D-glucan monoclonal antibody containing the variable regions of SEQ ID NOs: 7-8 has good specificity and stability, and is compatible with fungal 1,3- ⁇ -D - Glucan has a strong affinity and can quickly combine with fungal 1,3- ⁇ -D-glucan, which is beneficial to shorten the detection time.
  • the anti-fungal 1,3- ⁇ -D-glucan monoclonal antibody further comprises a constant region.
  • the constant region comprises any one or a combination of at least two of the IgGl, IgG2, IgG3 or IgG4 constant regions.
  • the antifungal 1,3- ⁇ -D-glucan monoclonal antibody is modified with a conjugate.
  • the conjugate includes any one of a chemiluminescent group, horseradish peroxidase or a fluorescent group.
  • the present application provides a preparation method of the antifungal 1,3- ⁇ -D-glucan monoclonal antibody described in the first aspect, the preparation method comprising the following steps:
  • a cell line stably expressing anti-fungal 1,3- ⁇ -D-glucan monoclonal antibody is constructed by genetic engineering technology, and the anti-fungal 1,3- ⁇ -D-glucan monoclonal antibody thus prepared
  • the antibody has good stability, small batch-to-batch variation, and has strong specific binding to fungal 1,3- ⁇ -D-glucan.
  • the application provides a fungal 1,3- ⁇ -D-glucan chemiluminescence detection kit
  • the fungal 1,3- ⁇ -D-glucan chemiluminescence detection kit includes a detection antibody and a signal antibody
  • the detection antibody and/or the signal antibody are the anti-fungal 1,3- ⁇ -D-glucan monoclonal antibody described in the first aspect.
  • an anti-fungal 1,3- ⁇ -D-glucan monoclonal antibody with strong specific binding force to fungal 1,3- ⁇ -D-glucan is used as a detection antibody and/or a signal antibody to construct
  • the immunochemiluminescence detection kit has a wide range of raw materials, controllable, and good stability.
  • the detection method has high sensitivity, good accuracy, short time, and simple sample processing method, which solves the problem that the traditional Limulus reagent method is susceptible to endotoxin interference.
  • both ethylenediaminetetraacetic acid (EDTA) solution can be used to pretreat serum samples
  • acid-base sample release solution can be used to pretreat serum samples.
  • EDTA solution is added to serum samples, heated at 120°C for 6 minutes, 10000g Centrifuge for 10 minutes to obtain the supernatant as a test sample, or add an acidic sample release solution to the serum sample and let it stand for 5 minutes at room temperature, and then add an alkaline sample release solution for neutralization reaction to obtain a test sample.
  • EDTA solution is added to serum samples, heated at 120°C for 6 minutes, 10000g Centrifuge for 10 minutes to obtain the supernatant as a test sample, or add an acidic sample release solution to the serum sample and let it stand for 5 minutes at room temperature, and then add an alkaline sample release solution for neutralization reaction to obtain a test sample.
  • the detection antibody is coupled to a solid support, preferably a carboxylated solid support.
  • the detection antibody is modified on the solid-phase carrier by the carboxylation coupling method, which avoids relying on the interaction of biotin-streptavidin, eliminates the interference of the sample biotin on the experimental results, and significantly improves the accuracy of the results.
  • the solid phase carrier comprises any one or a combination of at least two of carboxylated magnetic particles, microtiter plates, microspheres, affinity membranes or liquid phase chips.
  • the signal antibody is modified with a chemiluminescent group.
  • the chemiluminescent group comprises any one of acridine sulfonamide, acridine ester, ruthenium complex, luminol, (diaramane)-1,2-dioxane or alkaline phosphatase one or a combination of at least two.
  • the chemiluminescence one-step sandwich method is used to detect the fungal 1,3- ⁇ -D-glucan in the sample, and the carboxylated magnetic bead-coupled detection antibody, the fungal 1,3- ⁇ -D-glucan and chemical
  • the signal antibody is modified with a luminescent group to form an antibody-antigen sandwich immune complex.
  • an excitation solution is added to emit light, and the chemiluminescence intensity is detected to analyze the fungal 1,3- ⁇ -D-
  • the content of glucan has achieved the effect of rapid and sensitive detection of fungal 1,3- ⁇ -D-glucan.
  • the fungal 1,3- ⁇ -D-glucan chemiluminescence detection kit further includes any one or at least two of a positive control substance, a negative control substance, an excitation solution, a blocking solution or a washing solution. combination.
  • the present application provides a fungal 1,3- ⁇ -D-glucan enzyme-linked immunosorbent assay kit, the fungal 1,3- ⁇ -D-glucan enzyme-linked immunosorbent assay reagent
  • the cassette includes the antifungal 1,3-beta-D-glucan monoclonal antibody of the first aspect.
  • the antifungal 1,3- ⁇ -D-glucan monoclonal antibody is modified with horseradish peroxidase.
  • the fungal 1,3- ⁇ -D-glucan enzyme-linked immunosorbent assay kit further comprises a solid phase carrier, preferably an enzyme label plate, magnetic particles, microspheres, affinity membranes or liquid phase chips. any one or a combination of at least two.
  • the fungal 1,3- ⁇ -D-glucan enzyme-linked immunosorbent assay kit further comprises any one or a combination of at least two of a blocking solution, a chromogenic solution or a stop solution.
  • the present application provides a fungal 1,3- ⁇ -D-glucan immunofluorescence detection kit
  • the fungal 1,3- ⁇ -D-glucan immunofluorescence detection kit includes the first The antifungal 1,3-beta-D-glucan monoclonal antibody of the aspect.
  • the antifungal 1,3- ⁇ -D-glucan monoclonal antibody is modified with a fluorescent group.
  • the fungal 1,3- ⁇ -D-glucan immunofluorescence detection kit further includes a blocking solution and/or a washing solution.
  • the present application provides the antifungal 1,3- ⁇ -D-glucan monoclonal antibody according to the first aspect, and the fungal 1,3- ⁇ -D-glucan chemistry according to the third aspect Luminescence detection kit, fungal 1,3- ⁇ -D-glucan enzyme-linked immunosorbent assay kit according to the fourth aspect, or fungal 1,3- ⁇ -D-glucan immunization according to the fifth aspect
  • the application of fluorescence detection kit in the preparation of fungal 1,3- ⁇ -D-glucan detection products is a method for example, and the fungal 1,3- ⁇ -D-glucan chemistry according to the third aspect.
  • Luminescence detection kit Luminescence detection kit
  • fungal 1,3- ⁇ -D-glucan enzyme-linked immunosorbent assay kit according to the fourth aspect
  • fungal 1,3- ⁇ -D-glucan immunization according to the fifth aspect
  • fluorescence detection kit in the preparation of fungal 1,3- ⁇ -D-glucan detection products.
  • the antifungal 1,3- ⁇ -D-glucan monoclonal antibody of the present application has a specific and strong affinity for fungal 1,3- ⁇ -D-glucan, and is expressed and prepared by genetic engineering methods. Good, small difference between batches, low price, suitable for mass production.
  • the fungal 1,3- ⁇ -D-glucan detection kit based on the anti-fungal 1,3- ⁇ -D-glucan monoclonal antibody of this application has stable performance, high sensitivity, and the detection time is only 10 minutes. The results were in good correlation with the detection results of the Limulus reagent method.
  • the fungal 1,3- ⁇ -D-glucan detection method of the present application is loose on the sample pretreatment conditions. It is treated with alkaline solution at 37 °C, EDTA at 100 °C or acid sample release solution at 2-40 °C After processing the samples, it does not affect the accuracy of the results, and the anti-biotin interference ability is strong, and the biotin concentration of 640ng/mL does not affect the negative and positive test results of the samples, which has important application prospects in the field of fungal detection technology.
  • Figure 1 shows the correlation between the detection results of the chemiluminescence detection method and the Limulus reagent method.
  • mice Female BALB/c mice (50 ⁇ g/mice) were immunized subcutaneously in the abdomen with fungal 1,3- ⁇ -D-glucan and every other week with fungal 1,3- ⁇ -D- emulsified in incomplete Freund’s adjuvant Dextran was used for the second, third and fourth immunization; after the fourth immunization, the tail blood of the mice was collected, and the serum titer of serial dilution was determined by ELISA. Cell fusion with myeloma cells and culture with screening medium;
  • anti-fungal 1,3- ⁇ -D-glucan monoclonal antibodies whose variable regions are shown in SEQ ID NOs: 7 to 8 are used as detection antibodies and signal antibodies to construct a chemiluminescence system to detect fungi in samples.
  • the steps are as follows:
  • the limulus reagent method was used to detect the fungal 1,3- ⁇ -D-glucan concentration in the same sample.
  • the results are shown in Table 1 and Figure 1.
  • the samples with the detection concentration ⁇ 90pg/mL are positive samples of fungal 1,3- ⁇ -D-glucan. It can be seen that based on the antifungal 1,3- ⁇ -D-glucan The chemiluminescence detection method of glycan monoclonal antibody and the detection results of the Limulus reagent method had a good correlation (R 2 >0.98).
  • Negative / Negative N5 39.27 34.74 Negative / Negative N5 40.09 34.15 Negative / Negative N6 48.16 50.17 Negative / Negative N7 57.95 52.98 Negative / Negative N8 65.16 58.89 Negative / Negative N9 71.83 50.89 Negative / Negative N10 73.32 57.54 Negative / Negative P1 132.32 116.59 positive/positive P2 154.86 137.62 positive/positive P3 167.02 149.01 positive/positive P4 184.26 206.32 positive/positive P5 205.61 176.17 positive/positive P6 253.35 258.19 positive/positive P7 276.43 247.52 positive/positive P8 302.95 274.85 positive/positive P9 386.88 328.69 positive/positive P10 517.49 452.45 positive/positive/positive
  • sample processing methods are used to pretreat the samples, and the limulus reagent method and the chemiluminescence method are respectively used to detect the concentration of fungal 1,3- ⁇ -D-glucan.
  • Example 5 Enzyme-linked immunosorbent assay based on antifungal 1,3- ⁇ -D-glucan monoclonal antibody
  • anti-fungal 1,3- ⁇ -D-glucan monoclonal antibodies whose variable regions are shown in SEQ ID NOs: 7-8 are used as coating antibodies and enzyme-labeled antibodies to construct an ELISA detection system, and the samples For the detection of 1,3- ⁇ -D-glucan in fungi, the steps are as follows:
  • the anti-fungal 1,3- ⁇ -D-glucan monoclonal antibody was prepared into a coating solution with a concentration of 1000ng/mL with 0.01M carbonate buffer (CBS), and added to the microtiter at 100 ⁇ L/well. In the well plate, coat overnight at 4°C, remove the coating solution the next day, block for 1 h, and dry for 30 min to prepare an enzyme labeling plate;
  • CBS carbonate buffer
  • the HRP-labeled anti-fungal 1,3- ⁇ -D-glucan monoclonal antibody was prepared with a conjugate stabilizer into an enzyme-labeled antibody with a concentration of 1000 ng/mL;
  • anti-fungal 1,3- ⁇ -D-glucan monoclonal antibodies whose variable regions are shown in SEQ ID NOs: 7-8 are used as binding antibodies and detection antibodies to construct an immunofluorescence detection reagent card, and the samples For the detection of 1,3- ⁇ -D-glucan in fungi, the steps are as follows:
  • the antifungal 1,3- ⁇ -D-glucan monoclonal antibody of the present application has good specificity and stability, and can rapidly bind to fungal 1,3- ⁇ -D-glucan,
  • the fungal 1,3- ⁇ -D-glucan detection method based on the anti-fungal 1,3- ⁇ -D-glucan monoclonal antibody has short detection time, high sensitivity and good accuracy, and has potential application value.
  • the present application illustrates the detailed method of the present application through the above-mentioned embodiments, but the present application is not limited to the above-mentioned detailed method, which does not mean that the present application must rely on the above-mentioned detailed method for implementation.
  • Those skilled in the art should understand that any improvement to the application, the equivalent replacement of each raw material of the product of the application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the application.

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Abstract

La présente invention concerne un anticorps monoclonal de 1,3-bêta-D-glucane antifongique et son utilisation. L'anticorps monoclonal a la capacité de liaison spécifique au 1,3-bêta-D-glucane fongique, et le kit construit peut être utilisé pour détecter le 1,3-bêta-D-glucane fongique.
PCT/CN2022/074015 2021-02-09 2022-01-26 Anticorps monoclonal de 1,3-bêta-d-glucane antifongique et son utilisation Ceased WO2022170977A1 (fr)

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CN112940114B (zh) * 2021-02-09 2022-07-12 丹娜(天津)生物科技股份有限公司 一种抗真菌1,3-β-D-葡聚糖单克隆抗体及其应用
CN113624964A (zh) * 2021-06-29 2021-11-09 上海市第十人民医院 一种侵袭性真菌的检测方法及检测试剂盒
CN113249336B (zh) * 2021-07-15 2021-10-08 天津一瑞生物科技股份有限公司 鼠抗（1,3）-β-D葡聚糖杂交瘤细胞株，单克隆抗体及应用
CN113929776B (zh) * 2021-09-07 2023-06-27 河北康立德生物科技有限公司 抗真菌(1，3)-β-D葡聚糖单克隆抗体、其编码基因及其表达和应用
CN116041499A (zh) * 2022-12-30 2023-05-02 丹娜(湖南)生物科技有限责任公司 1,3-β-D-葡聚糖结合蛋白、制备方法及应用

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CN104628861A (zh) * 2015-02-03 2015-05-20 丹娜（天津）生物科技有限公司 一种1,3-β-D-葡聚糖多克隆抗体及其制备方法
WO2020022467A1 (fr) * 2018-07-27 2020-01-30 積水メディカル株式会社 PROCÉDÉ D'ANALYSE IMMUNOLOGIQUE DE (1→3)-β-D-GLUCANE DANS UN ÉCHANTILLON BIOLOGIQUE, KIT POUR L'ANALYSE DE (1→3)- β-D-GLUCANE, ET SOLUTION DE PRÉTRAITEMENT ALCALINE POUR ÉCHANTILLON BIOLOGIQUE DESTINÉ À ÊTRE UTILISÉ DANS LE PROCÉDÉ D'ANALYSE IMMUNOLOGIQUE DE (1→3)- β-D-GLUCANE
WO2020184409A1 (fr) * 2019-03-08 2020-09-17 積水メディカル株式会社 PROCÉDÉ D'ANALYSE IMMUNOLOGIQUE DE β-D-GLUCANE DANS UN ÉCHANTILLON BIOLOGIQUE ET KIT D'ANALYSE DE β-D-GLUCANE
CN112940114A (zh) * 2021-02-09 2021-06-11 丹娜(天津)生物科技股份有限公司 一种抗真菌1,3-β-D-葡聚糖单克隆抗体及其应用

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