WO2021128947A1 - 抗MntC蛋白抗体及其应用和包含其的试剂盒 - Google Patents

抗MntC蛋白抗体及其应用和包含其的试剂盒 Download PDF

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WO2021128947A1
WO2021128947A1 PCT/CN2020/114284 CN2020114284W WO2021128947A1 WO 2021128947 A1 WO2021128947 A1 WO 2021128947A1 CN 2020114284 W CN2020114284 W CN 2020114284W WO 2021128947 A1 WO2021128947 A1 WO 2021128947A1
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antibody
seq
light chain
heavy chain
mntc protein
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PCT/CN2020/114284
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English (en)
French (fr)
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杨峰
杨茜
蔡昌芝
徐丽敏
杜欢
邹全明
曾浩
赵安妮
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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 WO2021128947A1 publication Critical patent/WO2021128947A1/zh

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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)

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  • the invention belongs to the field of biotechnology, and relates to a monoclonal antibody and its application.
  • Manganese ion transporter C subunit is a highly conserved cell surface protein in the Staphylococcus aureus manganese ion transporter complex (MntA ⁇ B ⁇ C). It consists of 285 amino acids and has a molecular weight of 32kD.
  • MntC can be used as a vaccine antigen to quickly mediate an effective immune response to prevent Staphylococcus aureus infection.
  • anti-MntC monoclonal antibody induced strong phagocytic activity of neutrophils in mice infected with Staphylococcus aureus, and produced immune protection in the passive immune protection model of mice.
  • MntC protein as the immunogen, monoclonal antibodies against multiple active epitopes of MntC obtained after immunization, screening and identification can be used to prepare specific therapeutic antibody drugs for immune prevention and treatment of Staphylococcus aureus infection, and to detect recombinant gold
  • a kit for detecting the content of MntC antigen in Staphylococcus aureus vaccine but there is no specific monoclonal antibody.
  • the present invention provides a monoclonal antibody against MntC protein, which can effectively detect the content of MntC protein in samples such as vaccines, so as to solve the above problems.
  • the present invention provides an anti-MntC protein antibody, 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 GDSITNGY (SEQ ID NO:1);
  • amino acid sequence of the heavy chain CDR2 region is ISYSGTT (SEQ ID NO: 2);
  • amino acid sequence of the heavy chain CDR3 region is SRIRPYRHLGFDY (SEQ ID NO: 3);
  • amino acid sequence of the light chain CDR1 region is QSVDYGGDSY (SEQ ID NO: 4);
  • amino acid sequence of the light chain CDR2 region is AAS (SEQ ID NO: 5);
  • the amino acid sequence of the light chain CDR3 region is QQSNEDPWT (SEQ ID NO: 6).
  • the antibody is antibody 3C10, and its heavy chain and light chain sequences are respectively:
  • the antibody is antibody 3F6, 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 MntC protein, which includes the antibody of the present invention.
  • the antibodies are antibodies 3C10 and 3F6.
  • 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 MntC protein standards.
  • the antibody 3C10 is a detection antibody
  • the antibody 3F6 is a coating antibody.
  • the present invention provides a method for detecting the content of MntC protein, including the use of antibody 3C10 and antibody 3F6, and the double antibody sandwich method for Elisa detection.
  • the method includes:
  • the MntC protein content detected by the method ranges from 0.046875 to 0.75 ⁇ g/ml.
  • the fourth aspect provides the application of the anti-MntC protein antibody of the present invention in detecting the content of MntC protein.
  • the MntC protein sequence is:
  • CN103694323A For specific information of the MntC protein, including preparation and other content, refer to CN103694323A.
  • the amino acid sequence of the MntC protein is shown in SEQ ID NO: 1 of the patent document.
  • the entire content of CN103694323A is incorporated herein by reference.
  • the anti-MntC protein antibody of the present invention can specifically bind to MntC protein, and effectively detect the content of MntC protein.
  • the sensitivity is high, reaching 0.046875 ⁇ g/mL, and the detection range is wide, 0.046875-0.75 ⁇ g/mL, which can meet the requirements of quantitative vaccine detection. need.
  • FIG. 1 Screening results of the first batch of monoclonal cell lines
  • FIG. 1 Screening results of the second batch of monoclonal cell lines
  • 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 MntC protein as an immunogen to immunize mice, and through cell fusion and screening, two mouse monoclonal antibodies 3F6 and 3C10 that specifically recognize MntC protein are obtained, which are prepared for ascites, and after G protein purification, they are respectively labeled with HRP .
  • a pair of paired antibodies (coating antibody 3F6, detection antibody 3C10) are obtained, and the detection conditions and standard curve are determined.
  • the detection sensitivity reaches 0.046875 ⁇ g/mL, and the linear range is 0.046875-0.75 ⁇ 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 MntC protein
  • the MntC protein was mixed with the adjuvants CFA and AD11.15 to prepare the immunogen, that is, the MntC protein was first mixed with the CFA in a volume ratio of 5:6, as the immunogen A, the MntC protein was then mixed with AD11.15 in a volume ratio of 1: 1 Mix 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 the indirect ELISA method.
  • OD 450 of 999 means >3.5, which exceeds the reading range of the microplate reader; NC is a negative control.
  • 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 next step is to detect the sensitivity of the cell supernatant of all 16 positive clones: use the MntC recombinant protein to coat the ELISA plate (concentrations are 1 ⁇ g/mL, 0.3 ⁇ g/mL, 0.1 ⁇ g/mL and 0.03 ⁇ g/mL), Add 100 ⁇ L to each well and react overnight at 4°C; wash the plate 3 times with PBS solution, and block with 5% milk-PBS for 1hr at room temperature; then wash the plate with PBS solution once, add gradient dilutions of 5% milk-PBS solution Mouse tail blood, react at room temperature for 1 hr; then wash the plate with PBS solution 3 times and pat dry, add HRP-labeled goat anti-mouse IgG (Fc) secondary antibody diluted 1:2000, react at room temperature for 1 hr; PBS solution After washing the plate 5 times, pat dry, add equal volumes of solution A and
  • 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 preparation of the second batch of monoclonal antibodies was carried out according to the method described above.
  • the immunogen was prepared with MntC protein to immunize 3 mice, and the antibody titer of the tail blood of the mice on the 14th day after immunization was evaluated according to the above-mentioned indirect ELISA method. The results are shown in Table 4.
  • OD 450 of 999 means >3.5, which exceeds the reading range of the microplate reader; NC is a negative control; PC is the serum of 1# mouse for the first immunization.
  • 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.
  • 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 positive clone 3C10 can also detect a strong recognition signal.
  • the 3 positive clones (1B3, 3F6 and 3C10) 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 protein G purified mouse monoclonal antibody was evaluated using the indirect ELSIA method. The results are shown in Table 7.
  • 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.
  • 3F6 and 3C10 were respectively labeled with HRP by sodium periodate oxidation method, and then the binding ability of the labeled antibody with MntC protein was evaluated by ELISA. The results are shown in Table 8.
  • the mouse monoclonal antibody 3F6 was selected as the coating antibody and the mouse monoclonal antibody 3C10 was selected as the detection antibody, and the matching result was the best, which can be used to detect the content of MntC protein.
  • the heavy chain sequence of antibody 3C10 is shown in SEQ ID NO: 7
  • the light chain sequence is shown in SEQ ID NO: 8
  • the heavy chain sequence of antibody 3F6 is shown in SEQ ID NO: 9
  • the light chain sequence is shown in SEQ ID NO: 10.
  • Further analysis shows that the amino acid sequences of the three CDR regions of the heavy chains of antibodies 3C10 and 3F6 are shown in SEQ ID NOs: 1 to 3, and the amino acid sequences of the three CDR regions of the light chain are shown in SEQ ID NOs: 4 to 6, respectively. Shown.
  • 3C10 and 3F6 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
  • MntC protein Another single-component MntC protein is 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.
  • Sample pretreatment Take 20 finished vaccines, mix each with 600 ⁇ L 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 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 content of MntC protein in the 20 samples detected by the kit is greater than 21.375 ⁇ g/mL, and the RSD is 6.01% less than 10%, indicating that the kit can be used for the quantitative detection of MntC protein. And the applicability is good.

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Abstract

本发明提供抗MntC蛋白抗体及其应用和包含其的试剂盒，所述抗体包含重链和轻链，所述重链和轻链分别包含3个CDR区，其中所述重链CDR区氨基酸序列分别如SEQ ID NO:1～3所示，所述轻链CDR区氨基酸序列分别如SEQ ID NO:4～6所示。

Description

抗MntC蛋白抗体及其应用和包含其的试剂盒 技术领域
本发明属于生物技术领域，涉及一种单克隆抗体及其应用。
背景技术
锰离子的获取关系到金黄色葡萄球菌的代谢、存活与粘附，同时与金黄色葡萄球菌毒力作用发挥密切相关。另外，锰离子在金黄色葡萄球菌通过保护其免受中性粒细胞攻击的免疫逃逸中发挥重要作用。锰离子转运蛋白C亚单位(MntC)是金黄色葡萄球菌锰离子转运蛋白复合物(MntA\B\C)中的一个高度保守的细胞表面蛋白，由285个氨基酸组成，分子量32kD。研究表明，MntC可作为疫苗抗原快速介导有效的免疫应答阻止金黄色葡萄球菌的感染。此外，抗MntC的单克隆抗体在金黄色葡萄球菌感染的小鼠中诱导出较强的中性粒细胞吞噬活性，在小鼠的被动免疫保护模型中产生了免疫保护作用。
采用重组MntC蛋白作为免疫原，经免疫、筛选、鉴定后获得的针对MntC多个活性表位的单克隆抗体，可应用于制备免疫防治金黄色葡萄球菌感染的特异治疗性抗体药物、检测重组金黄色葡萄球菌疫苗中MntC抗原含量的检测试剂盒。但目前尚没有特异性单克隆抗体存在。
发明内容
基于上述原因，本发明提供一种抗MntC蛋白的单克隆抗体，能够有效检测样品如疫苗中的MntC蛋白的含量，以解决上述问题。
第一个方面，本发明提供抗MntC蛋白的抗体，所述抗体包含重链和轻链，所述重链和轻链分别包含3个CDR区，其中：
所述重链CDR1区的氨基酸序列为GDSITNGY(SEQ ID NO:1)；
所述重链CDR2区的氨基酸序列为ISYSGTT(SEQ ID NO:2)；
所述重链CDR3区的氨基酸序列为SRIRPYRHLGFDY(SEQ ID NO:3)；以及
所述轻链CDR1区的氨基酸序列为QSVDYGGDSY(SEQ ID NO:4)；
所述轻链CDR2区的氨基酸序列为AAS(SEQ ID NO:5)；
所述轻链CDR3区的氨基酸序列为QQSNEDPWT(SEQ ID NO:6)。
优选地，所述抗体为抗体3C10，其重链和轻链序列分别为：
重链：
Figure PCTCN2020114284-appb-000001
轻链：
DIVMTQSTASLAVSLGQRATISCKASQSVDYGGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDGATYYCQQSNEDPWTFGGGTKLEIK(SEQ ID NO:8)；或
所述抗体为抗体3F6，其重链和轻链序列分别为：
重链：
Figure PCTCN2020114284-appb-000002
轻链：
Figure PCTCN2020114284-appb-000003
优选地，所述抗体为IgG抗体。
第二个方面，本发明提供一种检测MntC蛋白含量的试剂盒，所述试剂盒包括本发明的抗体。优选地，所述抗体为抗体3C10和3F6。
优选地，所述试剂盒还包括人IgG、洗液、底物显色液A(EDTA-Na、柠檬酸、甘油、TMB)、底物显色液B(醋酸钠、柠檬酸、30％H 2O 2)、终止液(2M H 2SO 4)和BSA。
优选地，所述试剂盒还包括酶联板和封板膜。
优选地，所述试剂盒还包括MntC蛋白标准品。
更优选地，所述抗体3C10为检测抗体，所述抗体3F6为包被抗体。
第三个方面，本发明提供一种检测MntC蛋白含量的方法，包括使用抗体3C10和抗体3F6，利用双抗体夹心法进行Elisa检测。
优选地，所述方法包括：
1)使用抗体3F6包被酶联板；
2)将被测样品与抗体3F6反应；
3)使用抗体3C10进行检测和显色反应，测定450nm下吸光值；
4)利用MntC蛋白标准品制备标准曲线，根据标准曲线计算被测样品中 MntC蛋白含量。
优选地，所述方法检测的MntC蛋白含量范围是0.046875～0.75μg/ml。
第四个方面，提供本发明的抗MntC蛋白抗体在检测MntC蛋白含量中的应用。
本发明中，所述MntC蛋白序列为：
GPLGSSSDKSNGKLKVVTTNSILYDMAKNVGGDNVDIHSIVPVGQDPHEYEVKPKDIKKLTDADVILYNGLNLETGNGWFEKALEQAGKSLKDKKVIAVSKDVKPIYLNGEEGNKDKQDPHAWLSLDNGIKYVKTIQQTFIDNDKKHKADYEKQGNKYIAQLEKLNNDSKDKFNDIPKEQRAMITSEGAFKYFSKQYGITPGYIWEINTEKQGTPEQMRQAIEFVKKHKLKHLLVETSVDKKAMESLSEETKKDIFGEVYTDSIGKEGTKGDSYYKMMKSNIETVHGSMK(SEQ ID NO:11)。MntC蛋白的具体信息，包括制备等内容，参见CN103694323A，MntC蛋白的氨基酸序列如该专利文件的SEQ ID NO:1所示。CN103694323A的全部内容通过引用的方式结合至本文。
本发明的抗MntC蛋白抗体能够与MntC蛋白特异性结合，有效地检测出MntC蛋白的含量，灵敏度高，达到0.046875μg/mL，检测范围广，0.046875-0.75μg/mL，能够满足疫苗定量检测的需要。
附图说明
图1：第一批单克隆细胞株的筛选结果；
图2：第二批单克隆细胞株的筛选结果；
图3：标准品线性拟合结果。
具体实施方式
以下将结合具体实施方式说明本发明内容，但本发明范围不限于此。
如果没有特殊说明，以下实施例中使用的试剂和仪器都是本领域常规试剂和仪器，可以通过商购方式获得；所使用的方法均为常规实验方法，本领域技术人员根据实施例内容可以毫无疑问地实施所述方案并获得相应结果。
本发明使用MntC蛋白作为免疫原免疫小鼠，通过细胞融合和筛选，获得2株特异识别MntC蛋白的小鼠单克隆抗体3F6和3C10，进行腹水制备，再经过G蛋白纯化后，分别进行HRP标记。
经过配对检测，条件优化后，获得一对配对抗体(包被抗体3F6，检测抗体3C10)，并确定检测条件和标准曲线，检测灵敏度达到0.046875μg/mL，线 性范围为0.046875-0.75μg/mL，满足疫苗定量检测的要求。
主要试剂
Figure PCTCN2020114284-appb-000004
Figure PCTCN2020114284-appb-000005
主要仪器
名称 厂家 型号
移液器 Eppendorf  
移液器 Thermo Fisher Scientific 十二道
恒温培养箱 北京市永光明医疗仪器厂 DHP-420型
微孔板恒温振荡器 中仪国科有限公司 SC60-4
洗板机 北京拓普分析仪器有限公司 DEM-3型
酶标仪 Thermo Scientific Multiskan Mk3
实验动物
Balb/C小鼠：SPF(Specific Pathogen Free)级别的Balb/C小鼠购自北京维通利华实验动物技术有限公司。
缓冲液的配制
Figure PCTCN2020114284-appb-000006
底物显色液A(A液)的配制为：EDTA-Na 0.2g、柠檬酸0.95g、甘油50ml、TMB(用DMSO溶解为10mg/ml后再加)0.2g、dH 2O 500ml。
底物显色液B(B液)的配制为：醋酸钠13.6g、柠檬酸1.6g、H 2O 2(30％)0.3ml，dH 2O 500ml。
20×洗液配方为：NaCl 818g，Na 2HPO 4.12H 2O 358g，KCl 205g，H 2O定容至5L，pH值7.4～7.6。
pH 2.7的甘氨酸洗脱液配方为：甘氨酸1.9g，H 2O定容至500mL，pH值2.68～2.72。
pH 1.9的甘氨酸洗脱液配方为：甘氨酸1.9g，H 2O定容至500mL，pH值1.88～1.92。
实施例1：特异识别MntC蛋白的小鼠单克隆抗体的制备和筛选
1、第一批小鼠单克隆抗体的制备
将MntC蛋白分别与佐剂CFA和AD11.15混合，制备免疫原，即，MntC蛋白先与CFA按体积比5：6混合，作为免疫原A，MntC蛋白再与AD11.15按体积比1:1混合作为免疫原B。免疫原A是初次免疫，免疫原B是加强免疫。肌肉免疫小鼠3只，免疫后在第14天抽取小鼠尾血，使用间接ELISA方法进行尾血抗体效价的评价。
使用MntC蛋白包被酶标板(1μg/mL)，每孔加入100μL，4℃过夜反应；使用PBS溶液洗板3次，使用5％牛奶-PBS在室温封闭1hr；然后使用PBS溶液洗板1次后，加入使用5％牛奶-PBS溶液梯度稀释的小鼠尾血，室温反应1hr；然后使用PBS溶液洗板3次，并进行拍干后，加入1:2000稀释的HRP标记的羊抗小鼠IgG(Fc)二抗，室温反应1hr；PBS溶液洗板5次后，拍干，加入等体积的A液和B液，避光、室温条件下反应20min；然后加入50μL终止液(2M H 2SO 4)，混匀后在酶标仪上读取OD 450值。免疫后第14天小鼠尾血间接ELISA评价结果见表1。
表1：免疫后第14天小鼠尾血抗体效价评价
Figure PCTCN2020114284-appb-000007
注：OD 450为999表示>3.5，超出酶标仪读数范围；NC为阴性对照。
从结果中看出，其中2#小鼠尾血识别MntC重组蛋白的抗体滴度最高，达到1:50000。因此选择2#小鼠与骨髓瘤细胞SP2/0进行细胞融合。融合后挑取564个单克隆杂交瘤细胞在96孔板中进行培养，通过之前所描述的间接ELISA方法对96孔板中的细胞培养上清进行评价，筛选能够分泌识别蛋白的单克隆 抗体的单克隆细胞株，筛选结果如图1所示。
从筛选结果中，挑选了17个阳性克隆进行筛选的确认实验，实验过程如下：包被MntC蛋白100ng/孔，分别加入各克隆上清液1：1稀释作为一抗，再加入羊抗鼠二抗，保留仍为阳性的克隆，结果见表2所示。
表2:阳性克隆的确认结果
克隆号 1B12 1B3 1B7 1B8 1C8 1F1 2C12 3E10 3F6 3G3 NC PC
OD 450 0.853 999 0.719 1.001 2.599 0.886 0.108 0.691 999 3.101 0.07 999
克隆号 4F2 4H5 4H7 5A12 5B5 5F3 5H9 NC NC NC NC PC
OD 450 0.651 0.461 1.935 0.316 999 0.877 0.906 0.045 0.047 0.057 0.052 999
注：NC为阴性对照，5％牛奶-PBS；PC为阳性对照，1#血清；OD 450值为999表示OD 450值大于3.5。
从表2可以看出，经过细胞传代后，除2C12之外，其他16个阳性克隆均表现为阳性。下一步对所有16个阳性克隆进行细胞上清的灵敏度检测：分别使用MntC重组蛋白包被酶标板(浓度依次为1μg/mL、0.3μg/mL、0.1μg/mL及0.03μg/mL)，每孔加入100μL，4℃过夜反应；使用PBS溶液洗板3次，使用5％牛奶-PBS在室温封闭1hr；然后使用PBS溶液洗板1次后，加入使用5％牛奶-PBS溶液梯度稀释的小鼠尾血，室温反应1hr；然后使用PBS溶液洗板3次，并进行拍干后，加入1:2000稀释的HRP标记的羊抗小鼠IgG(Fc)二抗，室温反应1hr；PBS溶液洗板5次后，拍干，加入等体积的A液和B液，避光、室温条件下反应20min；然后加入50μL终止液(2M H 2SO 4)，混匀后在酶标仪上读取OD 450值，结果见表3。
表3：第一批单克隆细胞株的灵敏度检测
ng/孔 1B12 1B3 1B7 1B8 1C8 1F1 3E10 3F6 3G3 4F2 NC PC
100 0.105 2.601 0.132 0.098 0.327 0.089 0.107 999 0.289 0.081 0.051 999
30 0.056 0.15 0.083 0.051 0.195 0.081 0.101 999 0.244 0.084 0.051 999
10 0.041 0.047 0.042 0.04 0.054 0.042 0.039 1.961 0.066 0.041 0.044 2.023
3 0.036 0.033 0.034 0.039 0.036 0.033 0.033 0.204 0.036 0.141 0.055 0.324
ng/孔 4H5 4H7 5A12 5B5 5F3 5H9 NC NC NC NC NC PC
100 0.063 0.106 0.044 0.276 0.062 0.062 0.037 0.039 0.039 0.042 0.05 999
30 0.055 0.048 0.041 0.052 0.043 0.054 0.046 0.046 0.045 0.042 0.049 999
10 0.043 0.044 0.043 0.045 0.05 0.042 0.042 0.041 0.041 0.038 0.042 1.403
3 0.05 0.036 0.046 0.044 0.044 0.039 0.044 0.031 0.04 0.044 0.043 0.405
注：NC为阴性对照，5％牛奶-PBS；PC为阳性对照，1#血清；OD 450值为999表示OD 450值大于3.5。
从表3结果中可以看出，再经过细胞传代后，只有阳性克隆1B3和3F6还能够检测出较强的识别信号。
2、第二批小鼠单克隆抗体的制备
按照上面所描述的方法，进行第二批单克隆抗体的制备。使用MntC蛋白配制免疫原，免疫小鼠3只，并按照上述的间接ELISA方法对免疫后第14天小鼠尾血进行抗体滴度评价，结果见表4。
表4：免疫后第14天小鼠尾血抗体效价评价
Figure PCTCN2020114284-appb-000008
注：OD 450为999表示>3.5，超出酶标仪读数范围；NC为阴性对照；PC为第一次免疫的1#小鼠血清。
从结果中看出，其中3#小鼠尾血识别MntC蛋白的抗体滴度最高，达到1:50000。因此选择3#小鼠与骨髓瘤细胞SP2/0进行细胞融合。融合后挑取564个单克隆杂交瘤细胞培养后，进行ELISA筛选，筛选结果见图2。
从筛选结果中，挑选了13个阳性克隆进行筛选的确认实验，结果见表5所示。
表5:阳性克隆的确认
克隆号 1A9 2E9 2B12 3E7 3C10 4A10 4A8 4B3 4B8 4B9 NC PC
OD 450 0.369 0.059 0.147 0.288 999 0.341 0.392 0.9 0.424 0.37 0.043 999
克隆号 5A11 5B9 6A10 NC NC NC NC NC NC NC NC PC
OD 450 0.271 0.228 0.272 0.043 0.047 0.044 0.044 0.043 0.042 0.052 0.042 999
注：NC为阴性对照，5％牛奶-PBS；PC为阳性对照，1#血清；OD 450值为999表示OD 450值大于3.5。
根据表5列出的阳性克隆复筛结果，挑选出6个阳性克隆继续进行灵敏度检测实验，结果见表6。
表6:第二批单克隆细胞株的灵敏度检测
ng/孔 4A10 1A9 3C10 4A8 4B3 4B9 NC NC NC NC NC PC
100 0.091 0.058 2.063 0.063 0.053 0.055 0.055 0.067 0.066 0.061 0.075 999
30 0.056 0.06 0.054 0.055 0.056 0.059 0.062 0.065 0.068 0.072 0.075 0.996
10 0.061 0.057 0.057 0.055 0.053 0.051 0.053 0.056 0.055 0.069 0.069 0.137
3 0.115 0.066 0.049 0.052 0.065 0.065 0.084 0.076 0.082 0.096 0.11 0.125
注：NC为阴性对照，5％milk-PBS；PC为阳性对照，1#血清；OD 450值为999表示OD 450值大于3.5。
从表6结果中可以看出，再经过细胞传代后，阳性克隆3C10还能够检测出较强的识别信号。
实施例2：纯化抗体的制备和筛选
1、腹水的制备
对上面获得的3株阳性克隆(1B3、3F6和3C10)进行腹水制备。分别将约10 7个细胞注射入2只预先注射了IFA佐剂的Balb/C小鼠的腹腔，然后约10天后，分别抽取每个阳性克隆产生的腹水，然后4℃、12000rpm离心15min，收取上清进行下一步的G蛋白纯化。
2、小鼠单克隆抗体的纯化
取1mL的偶联有G蛋白的柱料加入一根空柱子中，使用PBS溶液清洗后，将2mL腹水用8mL的PBS稀释后上柱，然后将流过液重新上柱一次；然后使用pH 2.7的甘氨酸洗脱液进行洗脱，每1mL洗脱液收集一管(预先加入100μL中和液，中和液成分为1M Tris-HCl，10mM EDTA，1.5M NaCl，pH8.0-8.38)，共收集5管；紧接着使用pH 1.9的甘氨酸洗脱液进行洗脱，每1mL洗脱液收集一管(预先加入300μL中和液)，共收集3管；然后分别对每一管洗脱液进行OD 280读数，将OD 280大于0.5的洗脱液进行混合，混合后再重新测定混合液的OD 280，按照1.4的系数计算抗体浓度；抗体浓度＝OD 280/1.4。
将G蛋白纯化的鼠单抗使用间接ELSIA方法进行评价，结果见表7。
表7:鼠单抗纯化抗体的评价
Figure PCTCN2020114284-appb-000009
Figure PCTCN2020114284-appb-000010
注：OD 450值为999表示OD 450值大于3.5；NC为阴性对照；PC为各自的细胞上清。
从表7的结果中看出，3F6和3C10纯化抗体的灵敏度在0.005-0.05μg/mL之间，而1B3的亲和力太差，因此3F6和3C10可以配对使用，用于检测MntC蛋白的含量。
实施例3：抗体配对检测实验
使用高碘酸钠氧化法对3F6和3C10分别进行HRP标记，然后通过ELISA评价标记后抗体与MntC蛋白的结合能力，结果见表8。
表8：HRP标记抗体的评价结果
Figure PCTCN2020114284-appb-000011
从表8可以看出，HRP标记后的抗体与MntC蛋白的结合能力与标记前的抗体相比，均没有显著的降低。
进一步对这两个抗体进行配对实验。
表9：抗体的两两配对实验结果
Figure PCTCN2020114284-appb-000012
Figure PCTCN2020114284-appb-000013
根据表9的配对结果，选择鼠单抗3F6作为包被抗体、鼠单抗3C10作为检测抗体的配对结果最佳，可以用于检测MntC蛋白的含量。
经测序分析，抗体3C10的重链序列如SEQ ID NO:7所示，轻链序列如SEQ ID NO:8所示；抗体3F6重链序列如SEQ ID NO:9所示，轻链序列如SEQ ID NO:10所示。进一步分析得出，抗体3C10和3F6重链的三个CDR区的氨基酸序列分别如SEQ ID NO:1～3所示，轻链的三个CDR区的氨基酸序列分别如SEQ ID NO:4～6所示。
实施例4：MntC蛋白含量检测试剂盒的制备和使用
根据上述结果，选择3C10和3F6作为抗体制备试剂盒，试剂盒中的试剂和物品可以包括：
1、抗体3F6包被酶联板   8孔×12条
2.酶结合物(抗体3C10)   120μl×1管(1:100倍稀释用)
3.BSA   3g/袋×1袋
4.人IgG，10μg/ml，120μl×1管(1:100倍稀释用)
5. 20×洗液   50ml×1瓶
6.底物显色液A(EDTA-Na、柠檬酸、甘油、TMB)   7ml×1瓶
7.底物显色液B(醋酸钠、柠檬酸、30％H 2O 2)   7ml×1瓶
8.终止液(2M H 2SO 4)   7ml×1瓶
9.封板膜   2张
10.说明书   1份
另以单组份MntC蛋白作为标准品
操作步骤
1.平衡：将所需试剂移到室温(18～25℃)平衡30分钟。
2.配液：
①.1×洗涤液：取1瓶20×洗液，用去离子水稀释至1000ml，混匀后备用。
②.酶结合物稀释液(3％BSA)：将BSA(3g/袋)完全溶解到100ml①配制的1×洗涤液中，充分混匀备用。
③.酶结合物工作液：取所需酶结合物用②配制的酶结合物稀释液稀释，充分混匀备用。
④.标准品及待检品稀释液：取适量人IgG，用②稀释液将人IgG 100倍稀释后备用。
3.加样:将抗体3F6包被酶联板从密封袋中取出，将标准品及待检样本稀释后，每孔加样100μl，同时设阴性对照。用封板膜封板后置37℃温育60分钟。
4.洗涤:弃去各孔内液体，用洗涤液注满微孔，静置30秒后弃去孔内液体；重复3次，最后一次洗板完成后在面巾纸上拍干。
5.加酶:每孔加酶结合物工作液100μl，用封板膜封板后置37℃温育60分钟。
6.洗涤:弃去各孔内液体，用洗涤液注满微孔，静置30秒后弃去孔内液体；重复3次，最后一次洗板完成后在面巾纸上拍干。
7.显色:每孔加底物显色液A 50μl，底物显色液B 50μl，轻微振荡混匀后置室温暗处显色7分钟。
8.测定:每孔加终止液50μl，轻微混匀。选择酶标仪波长450nm，测定各孔吸光值(OD值)。
以标准品的结果采用logX-LogY的拟合方式进行线性拟合，最终确定了标准曲线，见表10以及双对数拟合方程图3。
表10：标准曲线
Figure PCTCN2020114284-appb-000014
Figure PCTCN2020114284-appb-000015
由以上结果可以看出，该试剂盒的检测灵敏度达到0.046875μg/mL，线性范围为0.046875-0.75μg/mL。
实施例5：试剂盒检测实验
使用该试剂盒检测金葡菌疫苗20180903批成品中MntC抗原的蛋白含量检测步骤：
1.样品预处理：取疫苗成品20支，每支混匀后吸取600μL于1.5mL EP管中，5000rpm离心10min，吸取上清300μL去掉，加入2mol/L的Na 2CO 3溶液300μL并混匀，待溶液清亮后离心取适量上清备用；
2.按照实施例4中操作步骤对样品进行检测；
3.标准品的结果采用logX-LogY的拟合方式进行线性拟合，得到标准曲线，将检测样品的吸光值(OD值)代入标准曲线计算出样品浓度，检测结果如下：
表11：试剂盒检测MntC蛋白含量结果
Figure PCTCN2020114284-appb-000016
Figure PCTCN2020114284-appb-000017
由以上结果可以看出，用该试剂盒检测的20份样品中MntC蛋白含量均大于标准规定21.375μg/mL，RSD为6.01％小于10％，说明该试剂盒可以用于MntC蛋白的定量检测，且适用性良好。

Claims (10)

  1. 抗MntC蛋白的抗体，所述抗体包含重链和轻链，所述重链和轻链分别包含3个CDR区，其中：
    所述重链CDR1区的氨基酸序列为GDSITNGY(SEQ ID NO:1)；
    所述重链CDR2区的氨基酸序列为ISYSGTT(SEQ ID NO:2)；
    所述重链CDR3区的氨基酸序列为SRIRPYRHLGFDY(SEQ ID NO:3)；以及
    所述轻链CDR1区的氨基酸序列为QSVDYGGDSY(SEQ ID NO:4)；
    所述轻链CDR2区的氨基酸序列为AAS(SEQ ID NO:5)；
    所述轻链CDR3区的氨基酸序列为QQSNEDPWT(SEQ ID NO:6)。
  2. 根据权利要求1所述的抗体，其中，
    所述抗体为抗体3C10，其重链和轻链序列分别为：
    重链：
    VKLQESGPSLVKPSQTLSLTCSVTGDSITNGYWNWIRKFPGNKLEYMGYISYSGTTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCSRIRPYRHLGFDYWGQGTTVTVSS(SEQ ID NO:7)；
    轻链：
    DIVMTQSTASLAVSLGQRATISCKASQSVDYGGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDGATYYCQQSNEDPWTFGGGTKLEIK(SEQ ID NO:8)；或
    所述抗体为抗体3F6，其重链和轻链序列分别为：
    重链：
    VQLQQSGPSLVKPSQTLSLTCSVTGDSITNGYWNWIRKFPGNKLEYMGYISYSGTTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCSRIRPYRHLGFDYWGQGTTVTVSS(SEQ ID NO:9)；
    轻链：
    DIVMTQTPASLAVSLGQRATISCKASQSVDYGGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDGATYYCQQSNEDPWTFGGGTKLEIK(SEQ ID NO:10)。
  3. 根据权利要求1或2所述的抗体，其中，所述抗体为IgG抗体。
  4. 一种检测MntC蛋白含量的试剂盒，所述试剂盒包括权利要求1～3任一项所述的抗体。
  5. 根据权利要求4所述的试剂盒，还包括人IgG、洗液、底物显色液A、底物显色液B、终止液和BSA。
  6. 根据权利要求5所述的试剂盒，还包括酶联板、封板膜和/或MntC蛋白标准品。
  7. 根据权利要求4～6任一项所述的试剂盒，其中，所述抗体为抗体3C10和抗体3F6，所述抗体3C10为检测抗体，所述抗体3F6为包被抗体。
  8. 一种检测MntC蛋白含量的方法，包括使用抗体3C10和抗体3F6，利用双抗体夹心法进行Elisa检测。
  9. 根据权利要求8所述的方法，包括：
    1)使用抗体3F6包被酶联板；
    2)将被测样品与抗体3F6反应；
    3)使用抗体3C10进行检测和显色反应，测定450nm下吸光值；
    4)利用MntC蛋白标准品制备标准曲线，根据标准曲线计算被测样品中MntC蛋白含量；
    优选地，所述方法检测的MntC蛋白含量范围是0.046875～0.75μg/ml。
  10. 权利要求1～3任一项所述的抗MntC蛋白抗体在检测MntC蛋白含量中的应用。
PCT/CN2020/114284 2019-12-26 2020-09-09 抗MntC蛋白抗体及其应用和包含其的试剂盒 WO2021128947A1 (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010151544A1 (en) * 2009-06-22 2010-12-29 Wyeth Llc Immunogenic compositions of staphylococcus aureus antigens
CN103694323A (zh) * 2013-12-09 2014-04-02 重庆原伦生物科技有限公司 金黄色葡萄球菌MntC重组蛋白及其制备方法和应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101359930B1 (ko) * 2004-09-22 2014-02-12 글락소스미스클라인 바이오로지칼즈 에스.에이. 스태필로코쿠스에 대한 예방접종에 사용하기 위한 면역원성조성물
EP3101027B1 (en) * 2013-12-09 2018-11-07 Olymvax Biopharmaceuticals Inc. Staphylococcus aureus spa5 mutant, composition comprising mutant and preparation method and use thereof
CN104211804A (zh) * 2014-08-20 2014-12-17 钱泓 一种抗金黄色葡萄球菌多克隆抗体的制备与应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010151544A1 (en) * 2009-06-22 2010-12-29 Wyeth Llc Immunogenic compositions of staphylococcus aureus antigens
CN103694323A (zh) * 2013-12-09 2014-04-02 重庆原伦生物科技有限公司 金黄色葡萄球菌MntC重组蛋白及其制备方法和应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANDERSON ANNALIESA S., SCULLY INGRID L., TIMOFEYEVA YEKATERINA, MURPHY ELLEN, MCNEIL LISA K., MININNI TERRI, NUÑEZ LORNA, CARRIERE: "Staphylococcus aureus Manganese Transport Protein C Is a Highly Conserved Cell Surface Protein That Elicits Protective Immunity Against S. aureus and Staphylococcus epidermidis", JOURNAL OF INFECTIOUS DISEASES, UNIVERSITY OF CHICAGO PRESS, US, vol. 205, no. 11, 1 June 2012 (2012-06-01), US, pages 1688 - 1696, XP055823872, ISSN: 0022-1899, DOI: 10.1093/infdis/jis272 *
BEGIER ELIZABETH; SEIDEN DAVID JOSHUA; PATTON MICHAEL; ZITO EDWARD; SEVERS JOSEPH; COOPER DAVID; EIDEN JOSEPH; GRUBER WILLIAM C.; : "SA4Ag, a 4-antigenStaphylococcus aureusvaccine, rapidly induces high levels of bacteria-killing antibodies", VACCINE, ELSEVIER, AMSTERDAM, NL, vol. 35, no. 8, 28 January 2017 (2017-01-28), AMSTERDAM, NL, pages 1132 - 1139, XP029913054, ISSN: 0264-410X, DOI: 10.1016/j.vaccine.2017.01.024 *
ZHEN SONG, WANG XIAOLI;JING HAIMING;ZHANG YI;LIU YUANYUAN;LIAO HUAXIN;ZENG HAO;ZOU QUANMING: "Sorting of MRSA Human Monoclonal Antibodies Based on Single B Cell Technology", IMMUNOLOGICAL JOURNAL, DI-3 JUNYI DAXUE, CHINA, vol. 35, no. 3, 31 March 2019 (2019-03-31), China, pages 254 - 261, XP055770478, ISSN: 1000-8861, DOI: 10.13431/j.cnki.immunol.j.20190041 *

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