WO2021128948A1 - Anticorps anti-protéine mhin2, utilisation associée et kit le contenant - Google Patents

Anticorps anti-protéine mhin2, utilisation associée et kit le contenant Download PDF

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Publication number
WO2021128948A1
WO2021128948A1 PCT/CN2020/114285 CN2020114285W WO2021128948A1 WO 2021128948 A1 WO2021128948 A1 WO 2021128948A1 CN 2020114285 W CN2020114285 W CN 2020114285W WO 2021128948 A1 WO2021128948 A1 WO 2021128948A1
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seq
amino acid
acid sequence
antibody
heavy chain
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PCT/CN2020/114285
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English (en)
Chinese (zh)
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曾浩
纪永军
邹全明
杨峰
杨茜
蔡昌芝
赵莉群
张怡
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成都欧林生物科技股份有限公司
中国人民解放军陆军军医大学
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Publication of WO2021128948A1 publication Critical patent/WO2021128948A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
    • 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
    • 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; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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]
    • 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
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
    • G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)

Definitions

  • the invention belongs to the field of biotechnology, and relates to a monoclonal antibody and its application.
  • Staphylococcus aureus ⁇ -hemolysin is an exotoxin, which is the most important pathogenic factor of Staphylococcus aureus infection. It is often caused by pathogenic Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus ( MRSA) is generated.
  • the iron surface determinant B subunit (IsdB) is an important outer membrane anchoring protein of Staphylococcus aureus, which plays an important role in the process of assisting bacteria to absorb and metabolize heme iron, as well as bacterial adhesion and colonization.
  • mHI N2 protein as the immunogen, monoclonal antibodies against multiple active epitopes of mHI N2 obtained after immunization, screening and identification can be used to prepare specific therapeutic antibody drugs for immune prevention and treatment of Staphylococcus aureus infections and detection
  • the present invention provides an anti-mHI N2 protein antibody, which can effectively detect the mHI N2 protein-containing reagent, such as the content of mHI N2 protein in a vaccine, to solve the above-mentioned problems in the prior art.
  • the present invention provides an antibody against mHI N2 protein, the antibody comprising a heavy chain and a light chain, the heavy chain and the light chain each comprising 3 CDR regions, wherein:
  • the amino acid sequence of the heavy chain CDR1 region is GYTFTSYW (SEQ ID NO:1);
  • the amino acid sequence of the heavy chain CDR2 region is INPTNGHT (SEQ ID NO: 2);
  • amino acid sequence of the heavy chain CDR3 region is ARDGYDDGSWLAY (SEQ ID NO: 3); or
  • the amino acid sequence of the heavy chain CDR1 region is GFTFSSFG (SEQ ID NO: 7);
  • amino acid sequence of the heavy chain CDR2 region is INSDSSNI (SEQ ID NO: 8);
  • the amino acid sequence of the heavy chain CDR3 region is ARSGTTVVQDH (SEQ ID NO: 9);
  • the amino acid sequence of the light chain CDR1 region is QRIGTN (SEQ ID NO: 4);
  • amino acid sequence of the light chain CDR2 region is YAS (SEQ ID NO: 5);
  • amino acid sequence of the light chain CDR3 region is QQSNSWPYS (SEQ ID NO: 6); or
  • amino acid sequence of the light chain CDR1 region is QDISNY (SEQ ID NO: 10);
  • amino acid sequence of the light chain CDR2 region is YTS (SEQ ID NO: 11);
  • the amino acid sequence of the light chain CDR3 region is QQGNTLPWT (SEQ ID NO: 12).
  • the antibody comprises a heavy chain and a light chain, wherein,
  • the amino acid sequence of the heavy chain CDR1 region is GYTFTSYW (SEQ ID NO:1);
  • the amino acid sequence of the heavy chain CDR2 region is INPTNGHT (SEQ ID NO: 2);
  • amino acid sequence of the heavy chain CDR3 region is ARDGYDDGSWLAY (SEQ ID NO: 3);
  • the amino acid sequence of the light chain CDR1 region is QRIGTN (SEQ ID NO: 4);
  • amino acid sequence of the light chain CDR2 region is YAS (SEQ ID NO: 5);
  • amino acid sequence of the light chain CDR3 region is QQSNSWPYS (SEQ ID NO: 6); or
  • the amino acid sequence of the heavy chain CDR1 region is GFTFSSFG (SEQ ID NO: 7);
  • amino acid sequence of the heavy chain CDR2 region is INSDSSNI (SEQ ID NO: 8);
  • the amino acid sequence of the heavy chain CDR3 region is ARSGTTVVQDH (SEQ ID NO: 9);
  • amino acid sequence of the light chain CDR1 region is QDISNY (SEQ ID NO: 10);
  • amino acid sequence of the light chain CDR2 region is YTS (SEQ ID NO: 11);
  • the amino acid sequence of the light chain CDR3 region is QQGNTLPWT (SEQ ID NO: 12).
  • the antibody is antibody 5A8, and its heavy chain and light chain sequences are respectively:
  • the antibody is antibody 7G4, and its heavy chain and light chain sequences are:
  • the antibody is an IgG antibody.
  • the present invention provides a kit for detecting the content of mHI N2 protein, said kit comprising the antibody of the present invention.
  • the antibodies are antibodies 5A8 and 7G4.
  • the kit also includes human IgG, lotion, substrate color development solution A (EDTA-Na, citric acid, glycerin, TMB), substrate color development solution B (sodium acetate, citric acid, 30% H 2 O 2 ), stop solution (2M H 2 SO 4 ) and BSA.
  • substrate color development solution A EDTA-Na, citric acid, glycerin, TMB
  • substrate color development solution B sodium acetate, citric acid, 30% H 2 O 2
  • the kit further includes an enzyme-linked plate and a sealing film.
  • the kit also includes mHI N2 protein standards.
  • the antibody 5A8 is a detection antibody
  • the antibody 7G4 is a coating antibody.
  • the present invention provides a method for detecting the content of mHI N2 protein, including the use of antibody 5A8 and antibody 7G4, and double antibody sandwich method for ELISA detection.
  • the method includes:
  • the mHI N2 protein content detected by the method ranges from 0.046875 to 3 ⁇ g/ml.
  • the fourth aspect provides the application of the anti-mHI N2 protein antibody of the present invention in detecting the content of mHI N2 protein.
  • sequence of the mHI N2 protein is:
  • mHI N2 protein For specific information of mHI N2 protein, including preparation and other content, refer to CN103695508A.
  • the amino acid sequence of mHI N2 protein is shown in SEQ ID NO: 1 of the patent document.
  • the entire content of CN103695508A is incorporated herein by reference.
  • the anti-mHI N2 protein antibody of the present invention can specifically bind to mHI N2 protein, effectively detecting the content of mHI N2 protein, with high sensitivity, reaching 0.046875 ⁇ g/mL, wide detection range, 0.046875-3 ⁇ g/mL, which can meet vaccine quantification Testing requirements.
  • Figure 1 1# mouse monoclonal hybridoma cell culture and screening results
  • Figure 2 2# mouse monoclonal hybridoma cell culture screening results
  • the reagents and instruments used in the following examples are all conventional reagents and instruments in the art, and can be obtained commercially; the methods used are all conventional experimental methods, and those skilled in the art can do nothing according to the content of the examples. Undoubtedly implement the program and obtain the corresponding results.
  • the present invention uses mHI N2 recombinant protein as the immunogen to immunize mice. Through multiple cell fusions and screenings, multiple strains of mouse monoclonal antibodies that specifically recognize mHI N2 protein are obtained, and high-affinity mouse monoclonal antibodies are selected for ascites preparation. After protein purification, HRP labeling was performed separately.
  • the paired antibodies (coating antibody 7G4, detection antibody 5A8) of the kit were obtained, and the detection conditions and standard curve were determined.
  • the detection sensitivity reached 0.046875 ⁇ g/mL, and the linear range was 0.046875-3 ⁇ g/mL. Meet the requirements for quantitative testing of vaccines.
  • Balb/C mice SPF (Specific Pathogen Free) Balb/C mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • the substrate color developing solution A (A solution) was prepared as follows: EDTA-Na 0.2g, citric acid 0.95g, glycerol 50ml, TMB (dissolved with DMSO to 10mg/ml and then added) 0.2g, dH 2 O 500ml.
  • the substrate color developing solution B (B solution) was prepared as follows: sodium acetate 13.6 g, citric acid 1.6 g, H 2 O 2 (30%) 0.3 ml, and dH 2 O 500 ml.
  • the formula of 20 ⁇ lotion is: NaCl 818g, Na 2 HPO 4 .12H 2 O 358g, KCl 205g, H 2 O is fixed to 5L, and the pH value is 7.4-7.6.
  • the formula of the glycine eluent with pH 2.7 is: 1.9 g of glycine, constant volume of H 2 O to 500 mL, and pH value of 2.68 ⁇ 2.72.
  • the formula of the pH 1.9 glycine eluent is: 1.9 g of glycine, constant volume of H 2 O to 500 mL, and pH value of 1.88 to 1.92.
  • Example 1 Preparation and screening of mouse monoclonal antibodies that specifically recognize mHI N2 protein
  • the mHI N2 recombinant protein (1.4mg/mL) was mixed with the adjuvants CFA and AD11.15 to prepare the immunogen, that is, the recombinant protein was first mixed with CFA in a volume ratio of 5:6, as immunogen A, and the recombinant protein was then mixed with AD11.15 was mixed in a volume ratio of 1:1 as immunogen B.
  • Immunogen A is the primary immunization
  • immunogen B is the booster immunization.
  • Three mice were immunized intramuscularly, and the tail blood of the mice was drawn on the 14th day after immunization, and the antibody titer of the tail blood was evaluated by indirect ELISA method.
  • NC is a negative control, 5% milk-PBS
  • PC is a positive control, 1# serum
  • an OD 450 value of 999 means that the OD 450 value is greater than 3.5.
  • the 4 positive clones (4D11, 4H2, 5A8, 7G4) obtained above were subjected to ascites preparation. Approximately 10 were injected into 107 cells injected intraperitoneally two previously Balb IFA adjuvant / C mice, and then about 10 days later, ascites were generated for each positive clone, then 4 °C, 12000rpm centrifuged 15min, charged The supernatant is used for the next step of protein G purification.
  • the G protein purified mouse monoclonal antibody was evaluated using the indirect ELSIA method. The results are shown in Table 3.
  • An OD 450 value of 999 means that the OD 450 value is greater than 3.5; NC is a negative control; PC is the respective cell supernatant.
  • the four purified antibodies were labeled with HRP using the sodium periodate oxidation method, and the binding ability of the labeled antibodies with the mHI N2 recombinant protein was evaluated by ELISA. The results are shown in Table 4.
  • the best matching results were selected to select mouse monoclonal antibody 7G4 as the coating antibody and mouse monoclonal antibody 5A8 as the detection antibody.
  • the heavy chain sequence of antibody 5A8 is shown in SEQ ID NO: 13
  • the light chain sequence is shown in SEQ ID NO: 14
  • the heavy chain sequence of antibody 7G4 is shown in SEQ ID NO: 15
  • the light chain sequence is shown in SEQ ID NO: 16.
  • the sequence of the 3 CDR regions of the heavy chain of antibody 5A8 are shown in SEQ ID NO: 1 to 3 respectively, and the sequences of the 3 CDR regions of the light chain are shown in SEQ ID NO: 4 to 6 respectively
  • the sequence of the heavy chain of antibody 7G4 The sequences of the three CDR regions are shown in SEQ ID NOs: 7 to 9 respectively, and the sequences of the three CDR regions of the light chain are shown in SEQ ID NOs: 10 to 12, respectively.
  • 5A8 and 7G4 are selected as antibody preparation kits.
  • the reagents and items in the kit can include:
  • Substrate color solution A (EDTA-Na, citric acid, glycerin, TMB) 7ml ⁇ 1 bottle
  • Substrate color developing solution B sodium acetate, citric acid, 30% H 2 O 2 ) 7ml ⁇ 1 bottle
  • mHI N2 protein was used as a standard product.
  • 1.1 ⁇ Washing Solution Take 1 bottle of 20 ⁇ Washing Solution, dilute to 1000ml with deionized water, mix well and set aside.
  • Enzyme conjugate diluent 3% BSA: completely dissolve BSA (3g/bag) into 100ml 1 1 ⁇ washing solution, and mix thoroughly for later use.
  • Enzyme conjugate working solution take the required enzyme conjugate and dilute it with the enzyme conjugate diluent prepared in 2, and mix thoroughly for later use.
  • Standard product and test product diluent take an appropriate amount of human IgG, dilute the human IgG 100 times with the diluent 2 and use it for later use.
  • Washing Discard the liquid in each well, fill the micropores with washing liquid, and discard the liquid in the well after standing for 30 seconds; repeat 3 times, and pat dry on facial tissue after the last wash.
  • Washing Discard the liquid in each well, fill the micropores with washing liquid, and discard the liquid in the well after standing for 30 seconds; repeat 3 times, pat dry on facial tissue after the last wash.
  • the logX-LogY fitting method is used to linearly fit the results of the standard products to obtain a standard curve.
  • the results are shown in Table 6 and Figure 3.
  • Sample pretreatment Take 20 finished vaccines, mix 600 ⁇ L of each vaccine in a 1.5mL EP tube, centrifuge at 5000rpm for 10min, remove 300 ⁇ L of supernatant, add 300 ⁇ L of 2mol/L Na 2 CO 3 solution and mix well , After the solution is clear, centrifuge to take appropriate amount of supernatant for later use;
  • test results are linearly fitted using logX-LogY fitting method to obtain a standard curve. Substitute the absorbance value (OD value) of the test sample into the standard curve to calculate the sample concentration.
  • OD value absorbance value
  • the mHI N2 protein content in the 20 samples tested by this kit is greater than 42.75 ⁇ g/mL, and the RSD is 5.66% and less than 10%, indicating that the kit can be used for mHI N2 protein detection. Quantitative detection and good applicability.

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Abstract

La présente invention concerne un anticorps anti-protéine mHIN2, une utilisation associée et un kit le contenant. L'anticorps contient une chaîne lourde et une chaîne légère, la chaîne lourde et la chaîne légère contenant trois régions CDR, respectivement, et les séquences d'acides aminés des régions CDR de la chaîne lourde étant respectivement telles que représentées dans les SEQ ID NO : 1-3 ou 7-9, et les séquences d'acides aminés des régions CDR de la chaîne légère sont respectivement telles que représentées dans les SEQ ID NO : 4-6 ou 10-12.
PCT/CN2020/114285 2019-12-26 2020-09-09 Anticorps anti-protéine mhin2, utilisation associée et kit le contenant WO2021128948A1 (fr)

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CN201911367605.3A CN111072777B (zh) 2019-12-26 2019-12-26 抗mHIN2蛋白抗体及其应用和包含其的试剂盒

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CN112920259A (zh) * 2021-02-05 2021-06-08 中国人民解放军陆军军医大学 一种用于诊断或防治金黄色葡萄球菌感染的IsdB抗原表位肽及其应用

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WO2018183475A1 (fr) * 2017-03-28 2018-10-04 Children's Medical Center Corporation Vaccin contre le staphylococcus aureus à base de système de présentation d'antigènes multiples (maps), composition immunogène et leurs utilisations
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WO2018183475A1 (fr) * 2017-03-28 2018-10-04 Children's Medical Center Corporation Vaccin contre le staphylococcus aureus à base de système de présentation d'antigènes multiples (maps), composition immunogène et leurs utilisations
EP3543255A1 (fr) * 2018-03-21 2019-09-25 Beatrix Förster Traitement de maladies apparentées au staphylocoque

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