WO2021128949A1 - 抗mSEB蛋白抗体及其应用和包含其的试剂盒 - Google Patents

抗mSEB蛋白抗体及其应用和包含其的试剂盒 Download PDF

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WO2021128949A1
WO2021128949A1 PCT/CN2020/114286 CN2020114286W WO2021128949A1 WO 2021128949 A1 WO2021128949 A1 WO 2021128949A1 CN 2020114286 W CN2020114286 W CN 2020114286W WO 2021128949 A1 WO2021128949 A1 WO 2021128949A1
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seq
antibody
amino acid
acid sequence
heavy chain
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曾浩
邹全明
章金勇
罗萍
敬海明
李海波
夏鑫
曾妮
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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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    • 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 enterotoxin is a superantigen exotoxin, usually produced by pathogenic Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus (MRSA). SE can produce biological effects by prompting most T cells to release a large number of cytokines, causing clinical symptoms such as toxin shock syndrome.
  • SE has 9 serotypes including SEA, SEB, SEC, SED and SEE.
  • the amino acid sequence of SEB is highly conserved among various strains of Staphylococcus aureus.
  • SEB has a better humoral response advantage than other antigens, and the prepared human SEB monoclonal antibody can effectively treat the common toxic shock syndrome of Staphylococcus aureus infection.
  • the applicant constructed and obtained the three-site SEB (L45R, Y89A, Y94A) mutant mSEB as one of the antigen components of the recombinant Staphylococcus aureus vaccine by referring to the relevant research literature.
  • mSEB no longer has the toxicity and superantigen activity of wild-type SEB, and has good safety; at the same time, it retains good immunogenicity and immune protection.
  • mSEB protein as the immunogen, monoclonal antibodies against multiple active epitopes of mSEB 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 anti-mSEB protein antibody, which can effectively detect the content of mSEB protein in a sample such as a vaccine, so as to solve the above-mentioned problems in the prior art.
  • the present invention provides an anti-mSEB 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 GYTFTSYW (SEQ ID NO:1);
  • the amino acid sequence of the heavy chain CDR2 region is FDPDSSYT (SEQ ID NO: 2);
  • the amino acid sequence of the heavy chain CDR3 region is TRSYYGRGGFDC (SEQ ID NO: 3); or
  • amino acid sequence of the heavy chain CDR1 region is GYSVTSGYA (SEQ ID NO: 7);
  • the amino acid sequence of the heavy chain CDR2 region is ISYNGIT (SEQ ID NO: 8);
  • amino acid sequence of the heavy chain CDR3 region is AREYRFDGYYYAMDY (SEQ ID NO: 9);
  • amino acid sequence of the light chain CDR1 region is QSLLNSGNQKNY (SEQ ID NO: 4);
  • amino acid sequence of the light chain CDR2 region is GAS (SEQ ID NO: 5);
  • amino acid sequence of the light chain CDR3 region is QNDHSYPPT (SEQ ID NO: 6); or
  • amino acid sequence of the light chain CDR1 region is QSLVHSNGNTY (SEQ ID NO: 10);
  • the amino acid sequence of the light chain CDR2 region is KVS (SEQ ID NO: 11);
  • amino acid sequence of the light chain CDR3 region is SRTTHVPFT (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 FDPDSSYT (SEQ ID NO: 2);
  • the amino acid sequence of the heavy chain CDR3 region is TRSYYGRGGFDC (SEQ ID NO: 3);
  • amino acid sequence of the light chain CDR1 region is QSLLNSGNQKNY (SEQ ID NO: 4);
  • amino acid sequence of the light chain CDR2 region is GAS (SEQ ID NO: 5);
  • amino acid sequence of the light chain CDR3 region is QNDHSYPPT (SEQ ID NO: 6); or
  • amino acid sequence of the heavy chain CDR1 region is GYSVTSGYA (SEQ ID NO: 7);
  • the amino acid sequence of the heavy chain CDR2 region is ISYNGIT (SEQ ID NO: 8);
  • amino acid sequence of the heavy chain CDR3 region is AREYRFDGYYYAMDY (SEQ ID NO: 9);
  • amino acid sequence of the light chain CDR1 region is QSLVHSNGNTY (SEQ ID NO: 10);
  • the amino acid sequence of the light chain CDR2 region is KVS (SEQ ID NO: 11);
  • amino acid sequence of the light chain CDR3 region is SRTTHVPFT (SEQ ID NO: 12).
  • the antibody is antibody 3C7, and its heavy chain and light chain sequences are respectively:
  • the antibody is antibody 4C7, 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 mSEB protein, the kit comprising the antibody of the present invention.
  • the antibodies are antibodies 3C7 and 4C7.
  • 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 mSEB protein standards.
  • the antibody 3C7 is a detection antibody
  • the antibody 4C7 is a coating antibody
  • the present invention provides a method for detecting the content of mSEB protein, which includes using antibody 3C7 and antibody 4C7 to perform Elisa detection using the double antibody sandwich method.
  • the method includes:
  • the mSEB protein content detected by the method ranges from 0.046875 to 3 ⁇ g/ml.
  • the fourth aspect provides the application of the anti-mSEB protein antibody of the present invention in detecting the content of mSEB protein.
  • sequence of the mSEB protein is:
  • SEQ ID NO: 1 of the patent document The entire content of CN103694321A is incorporated herein by reference.
  • the anti-mSEB protein antibody of the present invention can specifically bind to mSEB protein and effectively detect the content of mSEB protein.
  • the sensitivity is high, the lowest limit can reach 0.046875 ⁇ g/mL, the detection range is wide, 0.046875-3 ⁇ g/mL, which can meet vaccine quantification Need for testing.
  • FIG. 1 Screening results of 3# mouse monoclonal cell line
  • FIG. 1 Screening results of 2# mouse monoclonal cell line
  • 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 mSEB protein as an immunogen to immunize mice. Through cell fusion and screening, multiple mouse monoclonal antibodies that specifically recognize mSEB protein are obtained, and antibodies with higher affinity for mSEB protein are selected for ascites preparation. After purification, HRP labeling was performed separately.
  • paired antibodies that can be used to detect mSEB content are obtained: coating antibody 4C7, detection antibody 3C7, and determine the detection conditions and standard curve.
  • the detection sensitivity reaches 0.046875 ⁇ g/mL, and the linear range is 0.046875-3 ⁇ g/ mL, to meet the requirements of vaccine quantitative testing.
  • 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.
  • the mSEB protein (1.4mg/ml) was mixed with the adjuvants CFA and AD11.15 to prepare the immunogen, that is, the mSEB protein was first mixed with the CFA in a volume ratio of 5:6, as the immunogen A, and the mSEB protein was then 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.
  • NC is a negative control, 5% milk-PBS; PC is 2# mouse serum.
  • NC is a negative control
  • PC is the serum of 2# mice immunized for the first time.
  • 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.
  • 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.
  • Binding sensitivity and specificity of the detection results of which 4 strains of mouse monoclonal antibodies have better results, 3C7, 4C7, 2E4 and 3G1 respectively.
  • HI protein is shown in CN103695508A
  • amino acid sequence of HI protein is shown in SEQ ID NO:1 of CN103695508A
  • MntC protein is shown in CN103694323A
  • amino acid sequence of MntC protein is shown in SEQ ID NO:1 of CN103694323A.
  • the 4 positive clones (3C7, 4C7, 2E4 and 3G1) 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.
  • the best pairing results were selected by selecting mouse monoclonal antibody 4C7 as the coating antibody and mouse monoclonal antibody 3C7 as the detection antibody.
  • the heavy chain sequence of antibody 3C7 is shown in SEQ ID NO: 13
  • the light chain sequence is shown in SEQ ID NO: 14
  • the heavy chain sequence of antibody 4C7 is shown in SEQ ID NO: 15
  • the light chain sequence is shown in SEQ ID NO: 16.
  • the 3 CDR regions of the heavy chain of antibody 3C7 are shown in SEQ ID NOs: 1 to 3, and the 3 CDR regions of the light chain are shown in SEQ ID NOs: 4 to 6, respectively; the sequence of the heavy chain of antibody 4C7 is shown in SEQ ID NO: 4 to 6 respectively.
  • 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.
  • the monoclonal antibodies 3C7 and 4C7 were selected as the antibody preparation kit.
  • the reagents and items in the kit may 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
  • mSEB 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.
  • 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 Na2CO3 solution and mix, wait for the solution Centrifuge after clearing and 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 mSEB protein content in the 20 samples detected by the kit is greater than 21.375 ⁇ g/mL, and the RSD is 6.90% less than 10%, indicating that the kit can be used for the quantitative detection of mSEB protein. And the applicability is good.

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Abstract

提供一种抗mSEB蛋白单克隆抗体及其应用，该抗体包含重链和轻链，其重链氨基酸序列如SEQ ID NO:13或15所示，轻链氨基酸序列如SEQ ID NO:14或16所示，该抗体能够与mSEB蛋白特异性结合，可用于mSEB蛋白含量的检测。

Description

抗mSEB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
金黄色葡萄球菌肠毒素(SE)是超抗原外毒素，通常由致病性金黄色葡萄球菌特别是耐甲氧西林金黄色葡萄球菌(MRSA)产生。SE可通过促使多数T细胞释放大量细胞因子而产生生物学效应，引起毒素休克综合征等临床症状。目前SE有SEA、SEB、SEC、SED和SEE等9个血清型。SEB在金黄色葡萄球菌各菌株中氨基酸序列高度保守。研究证实，SEB具有比其他抗原更好的体液应答优势，制备的人SEB单克隆抗体可有效治疗金葡菌感染常见的毒性休克综合症。申请人参照相关研究文献构建获得了SEB三位点(L45R,Y89A,Y94A)突变体mSEB作为重组金黄色葡萄球菌疫苗抗原组分之一。经体内外实验证实：mSEB不再具有野生型SEB的毒性及超抗原活性，安全性良好；同时保留了良好的免疫原性及免疫保护性。
采用重组mSEB蛋白作为免疫原，经免疫、筛选、鉴定后获得的针对mSEB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mSEB抗原含量的检测试剂盒。目前尚未有特异性mSEB抗体存在。
发明内容
基于上述原因，本发明提供一种抗mSEB蛋白的抗体，能够有效检测样品如疫苗中的mSEB蛋白的含量，以解决现有技术存在的上述问题。
第一个方面，本发明提供抗mSEB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GYTFTSYW(SEQ ID NO:1)；
所述重链CDR2区的氨基酸序列为FDPSDSYT(SEQ ID NO:2)；
所述重链CDR3区的氨基酸序列为TRSYYGRGGFDC(SEQ ID NO:3)；或者
所述重链CDR1区的氨基酸序列为GYSVTSGYA(SEQ ID NO:7)；
所述重链CDR2区的氨基酸序列为ISYNGIT(SEQ ID NO:8)；
所述重链CDR3区的氨基酸序列为AREYRFDGYYYAMDY(SEQ ID NO: 9)；以及
所述轻链CDR1区的氨基酸序列为QSLLNSGNQKNY(SEQ ID NO:4)；
所述轻链CDR2区的氨基酸序列为GAS(SEQ ID NO:5)；
所述轻链CDR3区的氨基酸序列为QNDHSYPPT(SEQ ID NO:6)；或者
所述轻链CDR1区的氨基酸序列为QSLVHSNGNTY(SEQ ID NO:10)；
所述轻链CDR2区的氨基酸序列为KVS(SEQ ID NO:11)；
所述轻链CDR3区的氨基酸序列为SRTTHVPFT(SEQ ID NO:12)。
优选地，所述抗体包含重链和轻链，其中，
所述重链CDR1区的氨基酸序列为GYTFTSYW(SEQ ID NO:1)；
所述重链CDR2区的氨基酸序列为FDPSDSYT(SEQ ID NO:2)；
所述重链CDR3区的氨基酸序列为TRSYYGRGGFDC(SEQ ID NO:3)；
所述轻链CDR1区的氨基酸序列为QSLLNSGNQKNY(SEQ ID NO:4)；
所述轻链CDR2区的氨基酸序列为GAS(SEQ ID NO:5)；
所述轻链CDR3区的氨基酸序列为QNDHSYPPT(SEQ ID NO:6)；或者
所述重链CDR1区的氨基酸序列为GYSVTSGYA(SEQ ID NO:7)；
所述重链CDR2区的氨基酸序列为ISYNGIT(SEQ ID NO:8)；
所述重链CDR3区的氨基酸序列为AREYRFDGYYYAMDY(SEQ ID NO:9)；
所述轻链CDR1区的氨基酸序列为QSLVHSNGNTY(SEQ ID NO:10)；
所述轻链CDR2区的氨基酸序列为KVS(SEQ ID NO:11)；
所述轻链CDR3区的氨基酸序列为SRTTHVPFT(SEQ ID NO:12)。
进一步优选地，所述抗体为抗体3C7，其重链和轻链序列分别为：
重链：
Figure PCTCN2020114286-appb-000001
轻链：
Figure PCTCN2020114286-appb-000002
所述抗体为抗体4C7，其重链和轻链序列分别为：
重链：
Figure PCTCN2020114286-appb-000003
轻链：
Figure PCTCN2020114286-appb-000004
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mSEB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3C7和4C7。
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mSEB蛋白标准品。
更优选地，所述抗体3C7为检测抗体，所述抗体4C7为包被抗体。
第三个方面，本发明提供一种检测mSEB蛋白含量的方法，包括使用抗体3C7和抗体4C7，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4C7包被酶联板；
2)将被测样品与抗体4C7反应；
3)使用抗体3C7进行检测和显色反应，测定450nm下吸光值；
4)利用mSEB蛋白标准品制备标准曲线，根据标准曲线计算被测样品中mSEB蛋白含量。
优选地，所述方法检测的mSEB蛋白含量范围是0.046875～3μg/ml。
第四个方面，提供本发明的抗mSEB蛋白抗体在检测mSEB蛋白含量中的应用。
本发明中，所述mSEB蛋白的序列为：
MESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSAINVKSIDQFRYFDLIYSIKDTKLGNYDNVRVEFKNKDLADKYKDKYVDVFGANAYYQCAFSKKTNDINSHQTDKRKTCMYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLVKNKKLYEFNNSPYETGYIKFIENENSF WYDMMPAPGDKFDQSKYLMMYNDNKMVDSKDVKIEVYLTTKKK(SEQ ID:17)。mSEB的具体信息，包括制备等内容，参见CN103694321A，mSEB蛋白的氨基酸序列如该专利文件的SEQ ID NO:1所示。CN103694321A的全部内容通过引用的方式结合至本文。
本发明的抗mSEB蛋白抗体能够与mSEB蛋白特异性结合，有效地检测出mSEB蛋白的含量，灵敏度高，最低限可达到0.046875μg/mL，检测范围广，0.046875-3μg/mL，能够满足疫苗定量检测的需要。
附图说明
图1：3#小鼠单克隆细胞株的筛选结果；
图2：2#小鼠单克隆细胞株的筛选结果；
图3：标准曲线拟合方程图。
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。
本发明使用mSEB蛋白作为免疫原免疫小鼠，通过细胞融合和筛选，获得多株特异识别mSEB蛋白的小鼠单克隆抗体，选取与mSEB蛋白的亲和力较高的抗体，进行腹水制备，通过G蛋白纯化后，分别进行HRP标记。
经过配对检测，条件优化后，获得可用于检测mSEB含量的配对抗体：包被抗体4C7，检测抗体3C7，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0.046875-3μg/mL，满足疫苗定量检测的要求。
主要试剂
Figure PCTCN2020114286-appb-000005
Figure PCTCN2020114286-appb-000006
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 PCTCN2020114286-appb-000007
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mSEB蛋白的小鼠单克隆抗体的制备
使用mSEB蛋白(1.4mg/ml)分别与佐剂CFA和AD11.15混合，制备免疫原，即，mSEB蛋白先与CFA按体积比5：6混合，作为免疫原A，mSEB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mSEB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；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 PCTCN2020114286-appb-000008
注：NC为阴性对照，5％牛奶-PBS；PC为2#小鼠血清。
从结果中可以看出，免疫第14天小鼠尾血识别mSEB重组蛋白的抗体滴度只有1:500，继续加强免疫小鼠一次后，再次评价小鼠尾血的抗体滴度，结果见表2。
表2：加强免疫后小鼠尾血抗体效价评价
Figure PCTCN2020114286-appb-000009
注：NC为阴性对照；PC为第一次免疫的2#小鼠血清。
从结果中看出，其中3#小鼠尾血识别mSEB重组蛋白的抗体滴度最高，达到1:5000，比上一次尾血评价有显著的提高。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，采用常规ELISA进行阳性克隆筛选，筛选标准为OD值是NC值的2.1倍，筛选结果如图1所示。
从筛选结果可以看出，没有获得阳性克隆。对剩下的1#和2#小鼠进行加强免疫一次，然后再次评价尾血的抗体滴度，见表3。
表3：加强免疫后小鼠尾血抗体效价评价
Figure PCTCN2020114286-appb-000010
注：NC为阴性对照。
从结果中看出，2#小鼠尾血识别mSEB蛋白的抗体滴度最高，达到1:10000。因此选择2#小鼠再一次与骨髓瘤细胞SP2/0进行细胞融合。融合后挑取376个单克隆杂交瘤细胞培养后，进行ELISA筛选，筛选结果如图2所示。
从筛选结果中，挑选了8个阳性克隆进行筛选的确认实验，实验过程如下：包被mSEB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4所示。
表4:阳性克隆的确认
克隆号 1D1 2A10 2A11 2C4 2E4 3C7 3G1 4C7 NC NC NC PC
OD 450 999 1.185 0.564 999 999 999 1.282 999 0.059 0.072 0.071 999
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4列出的阳性克隆复筛结果，挑选出8个阳性克隆继续进行灵敏度检测实验：分别使用mSEB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，实验结果见表5。并且进行特异性检测实验： 分别使用HI、MntC及mSEB重组蛋白包被酶标板(浓度皆为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(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，混匀后在酶标仪上读取OD 450值，实验结果见表6。
表5:灵敏度检测结果
ng/孔 1D1 2A10 2C4 2E4 3C7 3G1 4C7 2A11 NC NC NC PC
100 999 0.365 2.045 999 0.933 0.457 999 0.355 0.055 0.064 0.125 999
30 0.24 0.069 0.115 2.768 0.085 0.072 0.189 0.052 0.048 0.054 0.047 999
10 0.061 0.041 0.042 0.635 0.043 0.044 0.054 0.044 0.042 0.042 0.046 0.953
3 0.055 0.051 0.057 0.612 0.052 0.057 0.063 0.065 0.069 0.076 0.068 0.462
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。
表6：特异性检测结果
Figure PCTCN2020114286-appb-000011
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。
结合灵敏度以及特异性的检测结果，其中4株鼠单抗的结果较好，分别为3C7、4C7、2E4和3G1。
本实验中，HI蛋白的信息参见CN103695508A，HI蛋白的氨基酸序列如CN103695508A的SEQ ID NO:1所示；MntC蛋白的信息参见CN103694323A， MntC蛋白的氨基酸序列如CN103694323A的SEQ ID NO:1所示。这两份申请的内容通过引用的方式并入本申请中。
实施例2：纯化抗体的制备和筛选
1、腹水制备
对上面获得的4株阳性克隆(3C7、4C7、2E4和3G1)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，结果见表7。
表7:鼠单抗纯化抗体的评价
Figure PCTCN2020114286-appb-000012
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，3C7和4C7纯化抗体的灵敏度在0.005-0.05μg/mL之间，2E4的灵敏度较高，在0.005-0.05μg/mL之间。而3G1的亲和力太差。
实施例3：抗体的配对检测和筛选实验
使用高碘酸钠氧化法对3C7、4C7和2E4分别进行HRP标记，然后通过ELISA评价标记后抗体与mSEB重组蛋白的结合能力，结果见表8。
表8：HRP标记抗体的评价结果
Figure PCTCN2020114286-appb-000013
从表8可以看出，HRP标记后的抗体3C7和4C7与mSEB蛋白的结合能力与标记前的抗体相比，均没有显著的降低，进行配对实验(见表9)，挑选配对抗体，而2E4标记后与mSEB的结合能力显著降低，不作为酶标抗体进行配对实验。
表9：抗体的两两配对实验结果
Figure PCTCN2020114286-appb-000014
Figure PCTCN2020114286-appb-000015
根据表9的配对结果，以及综合考虑3个鼠单抗的纯化抗体得率，选择鼠单抗4C7作为包被抗体、鼠单抗3C7作为检测抗体的配对结果最佳。
经测序分析，抗体3C7的重链序列如SEQ ID NO:13所示，轻链序列如SEQ ID NO:14所示；抗体4C7重链序列如SEQ ID NO:15所示，轻链序列如SEQ ID NO:16所示。进一步分析，抗体3C7重链的3个CDR区序列分别如SEQ ID NO:1～3所示，轻链的3个CDR区序列分别如SEQ ID NO:4～6所示；抗体4C7重链的3个CDR区序列分别如SEQ ID NO:7～9所示，轻链的3个CDR区序列分别如SEQ ID NO:10～12所示。
实施例4：mSEB蛋白含量检测试剂盒的制备和使用
根据上述结果，选择单抗3C7和4C7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4C7包被酶联板，8孔×12条
2.酶结合物(抗体3C7)，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mSEB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4C7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)。
经过一系列的优化操作，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 PCTCN2020114286-appb-000016
Figure PCTCN2020114286-appb-000017
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0.046875-3μg/mL。
实施例5：检测实验
使用该试剂盒检测金葡菌疫苗20180903批成品中mSEB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Na2CO3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mSEB蛋白含量结果
Figure PCTCN2020114286-appb-000018
Figure PCTCN2020114286-appb-000019
由以上结果可以看出，用该试剂盒检测的20份样品中mSEB蛋白含量均大于标准规定21.375μg/mL，RSD为6.90％小于10％，说明该试剂盒可以用于mSEB蛋白的定量检测，且适用性良好。

Claims (10)

  1. 抗mSEB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GYTFTSYW(SEQ ID NO:1)；
    所述重链CDR2区的氨基酸序列为FDPSDSYT(SEQ ID NO:2)；
    所述重链CDR3区的氨基酸序列为TRSYYGRGGFDC(SEQ ID NO:3)；或者
    所述重链CDR1区的氨基酸序列为GYSVTSGYA(SEQ ID NO:7)；
    所述重链CDR2区的氨基酸序列为ISYNGIT(SEQ ID NO:8)；
    所述重链CDR3区的氨基酸序列为AREYRFDGYYYAMDY(SEQ ID NO:9)；以及
    所述轻链CDR1区的氨基酸序列为QSLLNSGNQKNY(SEQ ID NO:4)；
    所述轻链CDR2区的氨基酸序列为GAS(SEQ ID NO:5)；
    所述轻链CDR3区的氨基酸序列为QNDHSYPPT(SEQ ID NO:6)；或者
    所述轻链CDR1区的氨基酸序列为QSLVHSNGNTY(SEQ ID NO:10)；
    所述轻链CDR2区的氨基酸序列为KVS(SEQ ID NO:11)；
    所述轻链CDR3区的氨基酸序列为SRTTHVPFT(SEQ ID NO:12)。
  2. 根据权利要求1所述的抗体，所述抗体包含重链和轻链，其中，
    所述重链CDR1区的氨基酸序列为GYTFTSYW(SEQ ID NO:1)；
    所述重链CDR2区的氨基酸序列为FDPSDSYT(SEQ ID NO:2)；
    所述重链CDR3区的氨基酸序列为TRSYYGRGGFDC(SEQ ID NO:3)；
    所述轻链CDR1区的氨基酸序列为QSLLNSGNQKNY(SEQ ID NO:4)；
    所述轻链CDR2区的氨基酸序列为GAS(SEQ ID NO:5)；
    所述轻链CDR3区的氨基酸序列为QNDHSYPPT(SEQ ID NO:6)；或者
    所述重链CDR1区的氨基酸序列为GYSVTSGYA(SEQ ID NO:7)；
    所述重链CDR2区的氨基酸序列为ISYNGIT(SEQ ID NO:8)；
    所述重链CDR3区的氨基酸序列为AREYRFDGYYYAMDY(SEQ ID NO:9)；
    所述轻链CDR1区的氨基酸序列为QSLVHSNGNTY(SEQ ID NO:10)；
    所述轻链CDR2区的氨基酸序列为KVS(SEQ ID NO:11)；
    所述轻链CDR3区的氨基酸序列为SRTTHVPFT(SEQ ID NO:12)。
  3. 根据权利要求2所述的抗体，其中，所述抗体为抗体3C7，其重链和轻链序列分别为：
    重链：
    VKLQESGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNFDPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYFCTRSYYGRGGFDCWGQGTTVTVSS(SEQ ID NO:13)；
    轻链：
    DVLMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRLTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPPTFGGGTKLEI(SEQ ID NO:14)；或者
    所述抗体为抗体4C7，其重链和轻链序列分别为：
    重链：
    GPGLVKPSQSLSLTCTVTGYSVTSGYAWNWIRQFPGNKLEWMGYISYNGITGYNPSLKSRVSLTRDTSKNQFFLQLNSVTTEDTATYYCAREYRFDGYYYAMDYWGQGTPVTVSS(SEQ ID NO:15)；
    轻链：
    DIVMTQTTLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSRTTHVPFTFGSGTKLEIK(SEQ ID NO:16)。
  4. 根据权利要求1～3任一项所述的抗体，其中，所述抗体为IgG抗体。
  5. 一种检测mSEB蛋白含量的试剂盒，所述试剂盒包括权利要求1～4任一项所述的抗体。
  6. 根据权利要求5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。
  7. 根据权利要求6所述的试剂盒，其中，所述试剂盒还包括酶联板、封板膜和/或mSEB蛋白标准品。
  8. 根据权利要求5～7任一项所述的试剂盒，其中，所述抗体为抗体3C7和抗体4C7，所述抗体3C7为检测抗体，所述抗体4C7为包被抗体。
  9. 一种检测mSEB蛋白含量的方法，包括使用权利要求1～4任一项所述抗体，利用双抗体夹心法进行Elisa检测；优选地，所述抗体为抗体3C7和抗体4C7，所述方法包括：
    1)使用抗体4C7包被酶联板；
    2)将被测样品与抗体4C7反应；
    3)使用抗体3C7进行检测和显色反应，测定450nm下吸光值；
    4)利用mSEB蛋白标准品制备标准曲线，根据标准曲线计算被测样品中mSEB蛋白含量；
    优选地，所述方法检测的mSEB蛋白含量范围是0.046875～3μg/ml。
  10. 权利要求1～4任一项所述的抗体在检测mSEB蛋白含量中的应用。
PCT/CN2020/114286 2019-12-26 2020-09-09 抗mSEB蛋白抗体及其应用和包含其的试剂盒 WO2021128949A1 (zh)

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