WO2021128946A1 - 抗SpA5蛋白的单克隆抗体及其应用和包含其的试剂盒 - Google Patents

抗SpA5蛋白的单克隆抗体及其应用和包含其的试剂盒 Download PDF

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WO2021128946A1
WO2021128946A1 PCT/CN2020/114283 CN2020114283W WO2021128946A1 WO 2021128946 A1 WO2021128946 A1 WO 2021128946A1 CN 2020114283 W CN2020114283 W CN 2020114283W WO 2021128946 A1 WO2021128946 A1 WO 2021128946A1
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antibody
spa5
seq
spa5 protein
protein
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PCT/CN2020/114283
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French (fr)
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邹全明
曾浩
顾江
鲁东水
杨峰
张卫军
徐丽敏
程平
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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 WO2021128946A1 publication Critical patent/WO2021128946A1/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.
  • Staphylococcus aureus protein A is present in most pathogenic Staphylococcus aureus, but not in Staphylococcus epidermidis and Staphylococcus saprophyticus. Blast comparison analysis showed that SpA is a highly conserved antigen component. Its molecular weight is 55kDa and is distributed on the surface of the cell wall. In mouse models of septic arthritis, sepsis and skin abscesses infected by Staphylococcus aureus, the virulence of Staphylococcus aureus strains with SpA deletion mutations is reduced. The reason is believed to be that the SpA bound to the IgG-Fc segment is reduced. The anti-host phagocytosis of Staphylococcus aureus.
  • the applicant referred to relevant research literature reports, through molecular design, the five domains of wild-type SpA (wSpA) were screened for multiple mutations in the active site, and finally determined [E(KKAA)-D(KKAA)-A( KKAA)-B(KKAA)-C(KKAA)]
  • the mutant is one of the components of the recombinant Staphylococcus aureus vaccine and is named SpA5.
  • the systemic biological activity test confirmed that SpA5 after the restructuring mutation no longer has the superantigen activity of wild-type wSpA to promote B cell apoptosis, and its binding activity to the IgG-Fc segment is significantly reduced.
  • SpA5 has good safety and good immunogenicity, and its single-component immunization has a BALB/c mouse immune challenge protection rate of greater than 30%. In addition, no changes in other biological functions were found.
  • SpA5 protein as the immunogen, monoclonal antibodies against multiple active epitopes of SpA5 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
  • the present invention provides an antibody against SpA5 protein, which can effectively detect the content of SpA5 protein in vaccines and other preparations containing SpA5 protein, so as to solve the above-mentioned problems in the prior art.
  • a monoclonal antibody against SpA5 protein comprising a heavy chain and a light chain, the heavy chain and the light chain each comprising 3 CDR regions, wherein,
  • amino acid sequence of the heavy chain CDR1 region is GYSITSDYG (SEQ ID NO:1);
  • the amino acid sequence of the heavy chain CDR2 region is ISYSGST (SEQ ID NO: 2);
  • amino acid sequence of the heavy chain CDR3 region is ARGNYYALDN (SEQ ID NO: 3);
  • amino acid sequence of the light chain CDR1 region is QSLLYSSNQKNY (SEQ ID NO: 4);
  • the amino acid sequence of the light chain CDR2 region is WAS (SEQ ID NO: 5);
  • the amino acid sequence of the light chain CDR3 region is QQYYSYT (SEQ ID NO: 6).
  • the antibody is antibody 5C8, wherein,
  • the sequence of the heavy chain is:
  • the sequence of the light chain is:
  • the aforementioned antibody is an IgG antibody.
  • a method for detecting the content of SpA5 protein including the detection using the monoclonal antibody of the present invention; preferably, the detection is performed by an ELISA method. More preferably, the monoclonal antibody of the present invention is used as a coating antibody, and a rabbit polyclonal antibody prepared by a conventional method is used as a detection antibody.
  • the method includes:
  • the content of SpA5 protein detected by the method ranges from 0.015625-0.5 ⁇ g/mL.
  • kits for detecting SpA5 protein comprising the anti-SpA5 protein monoclonal antibody of the present invention.
  • the kit also includes a rabbit polyclonal antibody against SpA5 protein and a HRP-labeled goat anti-rabbit IgG (Fc) secondary antibody.
  • 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, as well as enzyme-linked plates and sealing membranes and/or SpA5 protein standards.
  • 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 as well as enzyme-linked plates and sealing membranes and/or SpA5 protein standards.
  • the rabbit polyclonal antibody is prepared using conventional methods.
  • the fourth aspect provides the application of the anti-SpA5 protein monoclonal antibody of the present invention in detecting the content of SpA5 protein.
  • the SpA5 protein of the present invention is the SpA5 (KKAA) protein of CN103694322A, and its sequence is shown in SEQ ID NO:1 of CN103694322A.
  • the content of CN103694322A is incorporated into this application by reference.
  • the antibody of the present invention can specifically bind to SpA5 protein, and effectively detect the content of SpA5 protein, with high sensitivity, with a minimum limit of 0.015625 ⁇ g/mL, and a wide detection range of 0.015625-0.5 ⁇ g/mL, which can meet the requirements of quantitative vaccine detection. need.
  • FIG. 1 Screening results of monoclonal cell lines
  • Figure 2 Diagram of the double logarithmic fitting equation.
  • 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.
  • 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.
  • 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 20 ⁇ lotion formula is: NaCl 818g, Na 2 HPO 4 ⁇ 12H 2 O 358g, KCl 205g, H 2 O is fixed to 5L, and the pH 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 a pH value of 2.68 to 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.
  • the SpA5 recombinant protein (1.4mg/ml) was mixed with the adjuvant 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 then the recombinant protein was mixed with AD11. 15 Mix 1:1 by volume 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, 5% milk-PBS; 3# mouse died.
  • 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.
  • SpA5 recombinant protein was used 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, block with 5% milk-PBS at room temperature for 1hr; then wash the plate with PBS solution once, then add 5 % Milk-PBS solution gradiently diluted mouse tail blood, react at room temperature for 1 hr; then wash the plate 3 times with PBS solution and pat dry, then add 1:2000 diluted HRP-labeled goat anti-mouse IgG (Fc) two React at room temperature for 1 hr; wash the plate with PBS solution 5 times, pat dry, add equal volumes of solution A and solution B, avoid light, and react for 20 minutes at room temperature; then add 50
  • 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 G protein-purified mouse monoclonal antibody was evaluated using the indirect ELSIA method, and the evaluation method was adjusted, that is, 10 ⁇ g/mL human IgG was coated, blocked with blocking buffer, and then 2 ⁇ g/mL SpA5 protein was added.
  • This can also realize the specific detection of the mouse monoclonal antibody that recognizes SpA5, and the advantage is that the SpA5 protein that can be bound to the ELISA plate is bound to human IgG, and it must use the site that binds to the antibody non-specifically, which can be effective Avoid non-specific results due to incomplete blocking.
  • the evaluation results of purified antibodies are shown in Table 4.
  • 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.
  • Example 2 Preparation and use of a double antibody sandwich ELISA kit for detecting SpA5 protein
  • the rabbit antiserum of recombinant protein SpA5 is purified by 5HiTrap Protein A HP chromatography column. 6 The concentration of purified SpA5-pcAb immunoglobulin was measured by BCA method, and then SpA5-pcAb was adjusted to 20mg/mL. 7 The result of indirect ELISA detection of the antibody titer corresponding to the recombinant protein SpA5 was 2048000.
  • the development plan of the kit is that the coating antibody is murine monoclonal antibody, and the detection antibody is the rabbit polyclonal antibody prepared by the above method, plus HRP-labeled goat anti-rabbit IgG (Fc) secondary antibody Develop the color and complete the preparation of the kit.
  • the coating antibody is murine monoclonal antibody
  • the detection antibody is the rabbit polyclonal antibody prepared by the above method, plus HRP-labeled goat anti-rabbit IgG (Fc) secondary antibody Develop the color and complete the preparation of the kit.
  • the reagents and items in the kit can include:
  • Enzyme conjugate 120 ⁇ l ⁇ 1 tube (for 1:100 dilution)
  • 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
  • SpA5 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 heavy chain and light chain of the above-mentioned monoclonal antibody 5C8 each contain 3 CDR regions, of which,
  • amino acid sequence of the heavy chain CDR1 region is GYSITSDYG (SEQ ID NO:1);
  • the amino acid sequence of the heavy chain CDR2 region is ISYSGST (SEQ ID NO: 2);
  • amino acid sequence of the heavy chain CDR3 region is ARGNYYALDN (SEQ ID NO: 3);
  • amino acid sequence of the light chain CDR1 region is QSLLYSSNQKNY (SEQ ID NO: 4);
  • the amino acid sequence of the light chain CDR2 region is WAS (SEQ ID NO: 5);
  • the amino acid sequence of the light chain CDR3 region is QQYYSYT (SEQ ID NO: 6).
  • the test kit prepared in Example 2 was used to detect the protein content of SpA5 antigen in the 20180903 batch of Staphylococcus aureus vaccine.
  • 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

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Abstract

本发明提供一种抗SpA5蛋白的单克隆抗体及其应用和包含其的试剂盒，所述抗SpA5蛋白的单克隆抗体包含重链和轻链，重链和轻链分别包含3个CDR区，其中所述重链CDR区的氨基酸序列分别如SEQ ID NO:1～3所示，所述轻链CDR区的氨基酸序列分别如SEQ ID NO:4～6所示。

Description

抗SpA5蛋白的单克隆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。
背景技术
金黄色葡萄球菌蛋白A(SpA)存在于大多数致病性金黄色葡萄球菌中，而不存在于表皮葡萄球菌、腐生葡萄球菌中。Blast比对分析表明SpA为一种高度保守的抗原成分。其分子量为55kDa，分布在细胞壁表面。在金黄色葡萄球菌感染的脓毒性关节炎、败血症和皮肤脓肿的小鼠模型中，SpA缺失突变的金黄色葡萄球菌菌株毒力降低，其原因认为是与IgG-Fc段结合的SpA减少，降低了金葡菌的抗宿主吞噬作用。
申请人参考相关研究文献报道，通过分子设计，对野生型SpA(wSpA)的五个结构域进行了活性位点的多种突变筛选，最终确定【E(KKAA)-D(KKAA)-A(KKAA)-B(KKAA)-C(KKAA)】突变体为重组金黄色葡萄球菌疫苗组分之一，并命名为SpA5。经过系统的生物学活性检测证实，改构突变后的SpA5不再具有野生型wSpA促B细胞凋亡的超抗原活性，与IgG-Fc段的结合活性显著降低。动物安全性评价及大量的动物免疫保护实验证实，SpA5安全性好，且具有良好的免疫原性，其单组分免疫所产生的BALB/c小鼠免疫攻毒保护率大于30％。此外，未发现其他生物学功能的改变。
采用重组SpA5蛋白作为免疫原，经免疫、筛选、鉴定后获得的针对SpA5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SpA5抗原含量的检测试剂盒。目前尚未有特异性抗SpA5抗体存在。
发明内容
基于上述原因，本发明提供一种抗SpA5蛋白的抗体，能够有效检测疫苗以及其它含SpA5蛋白的制剂中SpA5蛋白的含量，以解决现有技术存在的上述问题。
第一个方面，提供一种抗SpA5蛋白的单克隆抗体，所述抗体包含重链和轻链，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GYSITSDYG(SEQ ID NO:1)；
所述重链CDR2区的氨基酸序列为ISYSGST(SEQ ID NO:2)；
所述重链CDR3区的氨基酸序列为ARGNYYALDN(SEQ ID NO:3)；
所述轻链CDR1区的氨基酸序列为QSLLYSSNQKNY(SEQ ID NO:4)；
所述轻链CDR2区的氨基酸序列为WAS(SEQ ID NO:5)；
所述轻链CDR3区的氨基酸序列为QQYYSYT(SEQ ID NO:6)。
优选地，所述抗体为抗体5C8，其中，
重链的序列为：
Figure PCTCN2020114283-appb-000001
轻链的序列为：
Figure PCTCN2020114283-appb-000002
优选地，上述抗体为IgG抗体。
第二个方面，提供一种检测SpA5蛋白含量的方法，包括使用本发明的单克隆抗体进行检测；优选地，通过ELISA方法检测。进一步优选地，本发明的单克隆抗体作为包被抗体，以常规方法制备的兔多克隆抗体作为检测抗体。
优选地，所述方法包括：
1)使用本发明的单克隆抗体包被酶联板；
2)将被测样品与本发明的单克隆抗体反应；
3)使用常规方法制备的兔多克隆抗体进行检测和显色反应，测定450nm下吸光值；
4)利用SpA5蛋白标准品制备标准曲线，根据标准曲线计算被测样品中SpA5蛋白含量；
优选地，所述方法检测的SpA5蛋白的含量范围是0.015625-0.5μg/mL。
第三个方面，提供一种检测SpA5蛋白的试剂盒，所述试剂盒包含本发明的抗SpA5蛋白的单克隆抗体。优选地，所述试剂盒还包括抗SpA5蛋白的兔多克隆抗体和HRP标记的山羊抗兔IgG(Fc)二抗。
进一步优选地，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，以及酶联板和封板膜和/或SpA5蛋白标准品。
优选地，所述兔多克隆抗体使用常规方法制备。
第四个方面，提供本发明的抗SpA5蛋白的单克隆抗体在检测SpA5蛋白含量中的应用。
在本发明中，所述SpA5蛋白的序列、制备方法等内容参考CN103694322A，本发明的SpA5蛋白为CN103694322A的SpA5(KKAA)蛋白，其序列如CN103694322A的SEQ ID NO:1所示。CN103694322A的内容通过引用的方式并入本申请中。
本发明的抗体能够与SpA5蛋白特异性结合，有效地检测出SpA5蛋白的含量，灵敏度高，最低限达到0.015625μg/mL，检测范围广，为0.015625-0.5μg/mL，能够满足疫苗定量检测的需要。
附图说明
图1：单克隆细胞株的筛选结果；
图2：双对数拟合方程图。
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。
主要试剂与仪器
主要试剂
Figure PCTCN2020114283-appb-000003
Figure PCTCN2020114283-appb-000004
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 PCTCN2020114283-appb-000005
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 2O500ml。
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SpA5蛋白小鼠单克隆抗体的制备
1、单克隆抗体的制备和筛选
将SpA5重组蛋白(1.4mg/ml)与佐剂CFA和AD11.15混合，制备免疫原，即，重组蛋白先与CFA按体积比5：6混合，作为免疫原A，重组蛋白再与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SPA5重组蛋白包被酶标板(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；用PBS溶液洗板5次后，拍干，加入等体积的A液(EDTA-Na，柠檬酸，甘油，TMB(四甲基联苯胺))和B液(醋酸钠，柠檬酸，30％H 2O 2)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 PCTCN2020114283-appb-000006
注：OD 450为999表示>3.5，超出酶标仪读数范围；NC为阴性对照，5％牛奶-PBS；3#小鼠死亡。
从结果中看出，三只小鼠尾血识别SpA5重组蛋白的抗体滴度均超过1:50000，其中1#小鼠的抗体滴度最高。因此选择1#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，其中，NC为阴性对照，5％牛奶-PBS；PC为阳性对照，1#血清。
从筛选结果中，挑选了26个阳性克隆进行筛选的确认实验，实验过程如下：包被SpA5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。
表2：阳性克隆的确认结果
克隆号 1A11 1C12 1C3 1D11 1F10 1G1 2E8 2B7 2E6 2F6 NC PC
OD 450 3.052 0.092 2.901 999 999 999 1.24 2.068 3.389 1.77 0.062 999
克隆号 2G12 2H4 3A11 3B11 3F8 5F6 3G2 3H10 3H3 4B1 NC PC
OD 450 0.275 999 0.85 0.224 999 2.608 999 0.639 999 3.132 0.054 999
克隆号 5B11 5C10 5C8 5G9 6D10 3G12 NC NC NC NC NC PC
OD 450 999 0.756 999 1.443 2.051 2.328 0.055 0.057 0.066 0.062 0.062 999
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2列出的阳性克隆复筛结果，挑选出23个阳性克隆继续进行灵敏度检测实验：分别使用SpA5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，混匀后在酶标仪上读取OD 450值，结果见表3。
表3:灵敏度检测结果
ng/孔 1A11 1C3 1D11 1F10 1G1 2E8 2B7 2E6 2F6 2H4 NC NC
100 999 999 999 999 999 999 999 999 999 999 0.056 0.052
30 2.016 0.599 2.87 3.324 2.78 0.201 0.511 0.713 0.495 2.29 0.046 0.048
10 0.085 0.062 0.182 0.202 0.088 0.039 0.042 0.051 0.147 0.083 0.039 0.039
3 0.128 0.432 0.038 0.044 0.04 0.032 0.066 0.033 0.033 0.032 0.034 0.036
ng/孔 3A11 3F8 5F6 3G2 3H10 3H3 4B1 5B11 5G10 5C8 NC PC
100 1.586 999 999 999 0.656 999 999 999 1.438 999 0.041 999
30 0.24 2.895 0.86 3.235 0.096 1.959 1.566 2.305 0.22 2.201 0.045 999
10 0.064 0.159 0.052 0.203 0.077 0.096 0.083 0.124 0.083 0.106 0.045 2.375
3 0.083 0.052 0.047 0.066 0.065 0.047 0.057 0.052 0.081 0.048 0.113 0.576
ng/孔 5G9 6D10 3G12 NC NC NC NC NC NC NC NC PC
100 999 999 999 0.051 0.046 0.051 0.051 0.061 0.06 0.061 0.054 999
30 1.045 0.734 0.61 0.044 0.041 0.044 0.043 0.043 0.048 0.049 0.048 999
10 0.069 0.055 0.055 0.058 0.041 0.039 0.037 0.038 0.04 0.039 0.039 2.712
3 0.047 0.037 0.052 0.031 0.033 0.033 0.035 0.03 0.07 0.034 0.035 0.723
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。
2、纯化抗体的制备和筛选
1)腹水制备
将第一部分细胞融合筛选获得的13株小鼠单克隆抗体进行腹水制备，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，pH 8.0-8.38)，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，评价方法进行调整，即包被 10μg/mL的人IgG，使用封闭缓冲液进行封闭后，再加入2μg/mL的SpA5蛋白。这样也能够实现特异性检测识别SpA5的鼠单抗，并且优势在于能够结合到酶标板上的SpA5蛋白都是与人IgG结合的，必然使用了与抗体非特异性结合的位点，这样能够有效的避免由于封闭不完全导致的非特异性结果的出现。纯化抗体的评价结果见表4。
表4：鼠单抗纯化抗体的评价结果
Figure PCTCN2020114283-appb-000007
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。
根据表4的结果，挑选1F10、1G1、1D11、5C8、3H3、5C10、2H4进行接下来的试剂盒的开发。
实施例2：双抗体夹心ELISA检测SpA5蛋白试剂盒的制备和使用
抗SpA5蛋白的兔多克隆抗体的制备方法：①用稀释液(10mM His，0.9％NaCl，pH＝6.0)将重组蛋白SpA5溶液调整至浓度为1mg/mL，按体积比1:1分别与弗氏佐剂(首次免疫用完全弗氏佐剂，增强免疫用不完全弗氏佐剂)乳化。②将乳化好的抗原分别在家兔四肢淋巴结附近皮下注射，每个点200μL，共0.4mg。免疫程序为0、14、21天，共免疫3次。③在首次免疫后第28天，免疫的家兔通过耳缘静脉采血100μL，分离血清，通过间接ELISA检测重组蛋白SpA5对应的抗体效价。④重组蛋白SpA5对应的抗体效价达到1：219以上之后，免疫的家兔通过心脏采血100mL。收集的血液先在37℃孵箱中放置30min，再在4℃冰箱中静置过夜。最后在低温离心机中4000g离心15min，收集的血清即为重组蛋白SpA5对应的高免血清。⑤将重组蛋白SpA5的兔抗血清经5HiTrap Protein A HP层析柱纯化。⑥用BCA法测定纯化后的SpA5-pcAb免疫球蛋白浓度，再将SpA5-pcAb调整为20mg/mL。⑦通过间接ELISA检测重组蛋白SpA5对应的抗体效价结果为2048000。
以抗SpA5蛋白的兔多克隆抗体，试剂盒的开发方案为包被抗体为鼠单抗，检测抗体为上述方法制备的兔多抗，再加上HRP标记的山羊抗兔IgG(Fc)二抗显色，完成试剂盒的制备。
试剂盒中的试剂和物品可以包括：
1、抗体5C8包被酶联板  8孔×12条
2.酶结合物(兔多克隆抗体)  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SpA5蛋白作为标准品。
试剂盒操作步骤
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:将用抗体5C8包被的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10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5以及图2。
表5：标准曲线
Figure PCTCN2020114283-appb-000008
由以上结果可以看出，该试剂盒的检测灵敏度达到0.015625μg/mL，线性范围为0.015625-0.5μg/mL。
经测序分析，鼠单抗5C8的序列为：
重链序列：
Figure PCTCN2020114283-appb-000009
轻链序列：
Figure PCTCN2020114283-appb-000010
进一步分析，上述单抗5C8的重链和轻链分别包含3个CDR区，其中，
重链CDR1区的氨基酸序列为GYSITSDYG(SEQ ID NO:1)；
重链CDR2区的氨基酸序列为ISYSGST(SEQ ID NO:2)；
重链CDR3区的氨基酸序列为ARGNYYALDN(SEQ ID NO:3)；
轻链CDR1区的氨基酸序列为QSLLYSSNQKNY(SEQ ID NO:4)；
轻链CDR2区的氨基酸序列为WAS(SEQ ID NO:5)；
轻链CDR3区的氨基酸序列为QQYYSYT(SEQ ID NO:6)。
实施例3：样品检测
使用实施例2制备的试剂盒检测金葡菌疫苗20180903批成品中SpA5抗原的蛋白含量，检测步骤如下：
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2的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6：试剂盒检测SpA5蛋白含量结果
Figure PCTCN2020114283-appb-000011
Figure PCTCN2020114283-appb-000012
由以上结果可以看出，用该试剂盒检测的20份样品中SpA5蛋白含量均大于标准规定42.75μg/mL，RSD为4.73％小于10％，说明该试剂盒可以用于SpA5蛋白的定量检测，且适用性良好。

Claims (10)

  1. 抗SpA5蛋白的单克隆抗体，所述抗体包含重链和轻链，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GYSITSDYG(SEQ ID NO:1)；
    所述重链CDR2区的氨基酸序列为ISYSGST(SEQ ID NO:2)；
    所述重链CDR3区的氨基酸序列为ARGNYYALDN(SEQ ID NO:3)；
    所述轻链CDR1区的氨基酸序列为QSLLYSSNQKNY(SEQ ID NO:4)；
    所述轻链CDR2区的氨基酸序列为WAS(SEQ ID NO:5)；
    所述轻链CDR3区的氨基酸序列为QQYYSYT(SEQ ID NO:6)。
  2. 根据权利要求1所述的抗体，其中，
    所述抗体重链的序列为：
    LQADATSTSRVALGPGLVKPSQSLSLTCTVTGYSITSDYGWNWIRQFPGNKLEWMGYISYSGSTSYNPSLKGRISITRDTSKNQFFLQLNSVTTEDTATYHCARGNYYALDNWGQGTTVTVSS(SEQ ID NO:7)；
    所述抗体轻链的序列为：
    DIQLTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYTFGGGTKLEI(SEQ ID NO:8)。
  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SpA5蛋白含量的方法，包括使用权利要求1～3任一项所述的单克隆抗体进行检测，通过ELISA方法检测，其中，所述单克隆抗体作为包被抗体，以常规方法制备的抗SpA5蛋白的兔多克隆抗体作为检测抗体。
  5. 根据权利要求4所述的方法，其中，所述方法包括：
    1)使用权利要求1～3任一项所述的单克隆抗体包被酶联板；
    2)将被测样品与所述单克隆抗体反应；
    3)使用常规方法制备的兔多克隆抗体进行检测和显色反应，测定450nm下吸光值；
    4)利用SpA5蛋白标准品制备标准曲线，根据标准曲线计算被测样品中SpA5蛋白含量。
  6. 根据权利要求4或5所述的方法，其中，所述方法检测的SpA5蛋白的含量范围是0.015625-0.5μg/mL。
  7. 一种检测SpA5蛋白含量的试剂盒，包含权利要求1～3任一项所述的抗SpA5蛋白的单克隆抗体。
  8. 根据权利要求7所述的试剂盒，还包括以常规方法制备的抗SpA5蛋白的兔多克隆 抗体和HRP标记的山羊抗兔IgG(Fc)二抗。
  9. 根据权利要求7或8所述的试剂盒，其中，所述试剂盒还包括人IgG、洗液、底物显色液A、底物显色液B、终止液和BSA，以及酶联板和封板膜和/或SpA5蛋白标准品。
  10. 权利要求1～3任一项所述的抗SpA5蛋白的单克隆抗体在检测SpA5蛋白含量中的应用。
PCT/CN2020/114283 2019-12-26 2020-09-09 抗SpA5蛋白的单克隆抗体及其应用和包含其的试剂盒 WO2021128946A1 (zh)

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