WO2021129247A1 - 柯萨奇病毒a6型实心病毒的单克隆抗体及其应用 - Google Patents
柯萨奇病毒a6型实心病毒的单克隆抗体及其应用 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
-
- 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]
Definitions
- the present invention relates to the technical field of immunology, in particular to a monoclonal antibody of Coxsackie virus A6 type solid virus and its application.
- Hand, foot and mouth disease is an acute infectious disease caused by a variety of enterovirus infections. It is prevalent in summer and has a high incidence in preschool children. Adults can be an indirect source of infection.
- the main clinical manifestations of hand, foot and mouth disease are rashes on the mouth, hands, and feet, which can be complicated by meningitis, encephalitis, pulmonary edema, circulatory failure and other serious illnesses that lead to death.
- enterovirus 71 EV71
- coxsackievirus A16 coxsackievirus, CA16
- the ELISA rapid detection kit is the most commonly used method, which can detect the virus serotype of the patient in the shortest time. Screening a type-specific monoclonal antibody is a prerequisite for establishing a detection kit.
- the antigen content in the vaccine is a key indicator to guide the process research and evaluate the effectiveness in vitro.
- the most critical technique for establishing an antigen evaluation method is to screen suitable monoclonal antibodies, which can then be used to establish an effective ELISA evaluation system.
- Coxsackievirus A6 (coxsackievirus, CA6) and Coxsackievirus A10 (coxsackievirus, CA10) are both enteroviruses. Enteroviruses are prone to form two different structural states when cultured in vitro, one is the integrity of the nucleic acid Solid virus particles, a kind of hollow virus particles that do not contain nucleic acid, usually coexist in the two states of virus particles. According to literature records and many experimental studies, both solid virus particles and hollow virus particles are immunogenic, but solid virus particles are significantly stronger than hollow virus particles. If the virus stock solution contains a certain proportion of hollow virus particles, the total protein content of the stock solution can be effectively increased, which is of great significance to the stability of the virus stock solution and the finished vaccine.
- the present invention provides the following technical solutions:
- the present invention provides a monoclonal antibody against CA6 virus, the monoclonal antibody having the heavy chain complementarity determining region CDR1 shown in SEQ ID NO: 5, and the heavy chain complement shown in SEQ ID NO: 6
- the CA6 virus is a CA6 type solid virus.
- the present invention provides a polynucleotide sequence encoding the monoclonal antibody against CA6 virus, the polynucleotide sequence having the heavy chain complementarity determining regions CDR1 and SEQ ID shown in SEQ ID NO:1
- the heavy chain complementarity determining region CDR2 shown in NO:2 the heavy chain complementarity determining region CDR3 shown in SEQ ID NO: 3, and the light chain complementarity determining region CDR1, SEQ ID NO: 10 shown in SEQ ID NO: 9
- the light chain complementarity determining region CDR2 and the light chain complementarity determining region CDR3 shown in SEQ ID NO: 11 are shown; preferably, the polynucleotide sequence has a heavy chain with the nucleotide sequence shown in SEQ ID NO: 4; And/or, preferably, the polynucleotide sequence has a light chain of the nucleotide sequence shown in SEQ ID NO: 12;
- the CA6 virus is a CA6 type solid virus.
- the present invention provides a kit for detecting CA6 virus
- the kit includes the monoclonal antibody or the monoclonal antibody encoded by the polynucleotide sequence; preferably, the reagent
- the box also includes a polyclonal antibody; more preferably, the polyclonal antibody is a CA6 rabbit polyclonal antibody;
- the CA6 virus is a CA6 type solid virus.
- the present invention provides a kit for diagnosing hand, foot and mouth disease, the kit comprising the monoclonal antibody or the monoclonal antibody encoded by the polynucleotide sequence; preferably, the The kit also includes a polyclonal antibody; more preferably, the polyclonal antibody is a CA6 rabbit polyclonal antibody;
- the CA6 virus is a CA6 type solid virus.
- the present invention provides a monoclonal antibody against the CA6 virus or the monoclonal antibody encoded by the polynucleotide sequence in the preparation of a kit for detecting CA6 virus or diagnosing hand, foot and mouth disease application;
- the CA6 virus is a CA6 type solid virus.
- the present invention provides an application of the monoclonal antibody against the CA6 virus or the monoclonal antibody encoded by the polynucleotide sequence in the quality control of the production of a vaccine containing the CA6 virus;
- the CA6 virus is a CA6 type solid virus.
- the present invention provides a method for quality control of the production of a vaccine containing CA6 virus, the method comprising using the monoclonal antibody against the CA6 virus or the monoclonal antibody encoded by the polynucleotide sequence Steps of cloning antibody to detect CA6 virus;
- the CA6 virus is a CA6 type solid virus.
- the present invention provides a medicine for the treatment or prevention of diseases caused by CA6 virus infection, the medicine contains the monoclonal antibody against the CA6 virus or the monoclonal antibody encoded by the polynucleotide sequence Cloned antibody
- the CA6 virus is a CA6 type solid virus.
- the present invention provides a monoclonal antibody against CA6 virus or a monoclonal antibody encoded by the polynucleotide sequence for detecting the content of CA6 virus in the preparation of hand, foot and mouth vaccines or in finished vaccines Applications.
- the application is for detecting the content of CA6 solid virus in the preparation process of the hand, foot and mouth vaccine or in the finished vaccine.
- monoclonal antibodies are prepared by immunizing mice with purified CA6 virus liquid, and polyclonal antibodies are prepared by immunizing rabbits, and a cell line with strong reaction ability with CA6 type solid virus particles is selected from a large number of monoclonal cell lines, and the cells are used The strain produces large amounts of ascites. Then the monoclonal antibody and polyclonal antibody were purified separately.
- the purified monoclonal antibody is labeled with HRP enzyme, and the prepared polyclonal antibody is used to establish an antigen detection system that is more reactive with solid virus particles than hollow virus particles, which is mainly used to detect CA6 solid virus particles for antigen evaluation system.
- the most critical technique for establishing an antigen evaluation method is to screen suitable monoclonal antibodies and use the monoclonal antibodies to establish an effective ELISA evaluation system.
- the antigen evaluation system prepared by the monoclonal antibody provided by the present invention can mainly detect solid viruses and has extremely weak binding ability to hollow viruses. The detection result of the evaluation system can truly and objectively reflect the solid virus content in the virus liquid. The proportion of solid virus particles is a key indicator in the vaccine research process, and is positively related to the effectiveness of the product.
- the antigen evaluation system for detecting CA6 solid virus particles provided by the present invention is used to monitor the content of solid virus particles in the virus liquid in real time, which can provide data support for the harvest time of virus culture.
- the antigen evaluation system for detecting CA6 solid virus particles provided by the present invention can monitor the collected solid virus tubes, and provide guidance for subsequent ultracentrifugation and merger.
- the use of the antigen evaluation system of the present invention can not only provide guidance for the vaccine process research, but also quickly evaluate the effectiveness of the product.
- the prepared CA6 rapid detection kit can also be used in clinical testing to quickly determine the virus type of the patient's infection, speed up the diagnosis of the disease, and provide a strong basis for clinical treatment plans.
- the antigen detection system is of great significance for rapid clinical detection and quality evaluation of vaccine production.
- Antigen evaluation system can also be used in the preparation of rapid detection kits and the development and research of vaccines.
- Antigen evaluation system (including ELISA rapid detection kit) usually adopts double antibody sandwich method, such as polyclonal antibody (abbreviation: polyclonal antibody)-polyclonal antibody, polyclonal antibody-monoclonal antibody (abbreviation: monoclonal antibody), monoclonal antibody-polyclonal antibody , MAb-the form of mAb.
- Figure 1 is a photograph of the centrifuge tube after sucrose density gradient centrifugation in Example 1;
- Figure 2 is an electron microscope comparison of solid virus particles and hollow virus particles identified by electron microscopy in Example 1.
- the left picture in the figure is an electron microscope image of solid virus particles in tube 23, and the right image is an electron microscope image of hollow virus particles in tube 19;
- Figure 3 is an SDS-PAGE electrophoresis diagram of the monoclonal antibody in Example 3 after purification.
- 1-monoclonal antibody ascites before purification 2-purified eluent (heavy chain + light chain), 3-purified flow-through fluid;
- Figure 4 is a linear relationship diagram of the antigen evaluation system of Example 5;
- Fig. 5 is an evaluation curve diagram of samples used in the ultra-isolation segmentation process of the antigen evaluation system of Example 7.
- sucrose density gradient centrifugation is used to obtain virus particles of different properties: the sucrose density gradient centrifugation method is used to carry out hollow-solid separation and removal of impurity proteins from the preliminary purified CA6 virus liquid.
- the sucrose gradient is 15%-60%, and centrifuged at 100,000 rpm for 3-15 hours.
- the hollow virus band and the solid virus band were separated by sucrose density gradient centrifugation, and the two virus bands were taken out, and the results of the hollow and solid separation were identified by electron microscope.
- the photo of the centrifuge tube after sucrose density gradient centrifugation is shown in Figure 1. From the figure, the separated hollow virus band and solid virus band can be seen.
- the electron microscope comparison diagram of solid virus particles and hollow virus particles identified by electron microscope is shown in Figure 2.
- Hybridoma cell line preparation use CA6 solid virus purification solution (the solid virus purification solution preparation method: Vero cells culture CA6 virus, after harvesting the virus solution through clarification ultrafiltration preliminary purification and concentration, sucrose gradient density centrifugation, can obtain solid Virus particle tube and hollow virus particle tube, the solid virus particle tube is collected and then desugared treatment, and then the CA6 solid virus purified solution is obtained)
- mice without adjuvant 5 needles without adjuvant; blood sampling test: 1 week after the fourth injection of immunization, blood was collected to test the titer of indirect enzyme-linked immunosorbent assay.
- the mice with antibody titer of 10 4 or more were given the fifth injection of intraperitoneal injection for booster immunization.
- the cryopreserved CA6 solid virus murine monoclonal hybridoma cell line was removed from liquid nitrogen for resuscitation and expansion.
- the total nucleic acid was extracted when the amount was more than 10 6 and was entrusted to Beijing Liuhe Huada Gene Technology Co., Ltd. to perform PCR amplification of the monoclonal antibody.
- the chain and light chain sequences are sequenced, and then the corresponding amino acid sequence is determined by the nucleotide sequence.
- the determined polynucleotide sequence and amino acid sequence please refer to the sequence table.
- Antibody purification Centrifuge the ascites prepared by immunized mice in Example 2 at 2 ⁇ 8°C, 4000 ⁇ 8000r/min for 5 ⁇ 15 minutes, take the supernatant and filter it with qualitative filter paper, filter it with 0.45 ⁇ m filter membrane, and pass it through the affinity layer Analyze to obtain purified monoclonal antibody. After purification, the protein content was 521 ⁇ g/ml. Purified monoclonal antibodies were tested for purity and titer.
- FIG. 3 is the monoclonal antibody ascites before purification
- 2 is the purified eluate (heavy chain + light chain)
- 3 is the purified flow-through fluid.
- the proportions of the heavy chain and the light chain of the purified eluate obtained by the detection were 0.5712 for the heavy chain and 0.4288 for the light chain, respectively. From this result, the sum of the ratio of heavy chain to light chain in the purified monoclonal antibody eluate is 100%, indicating that the purification effect is very good, and there is no impurity protein.
- CA6 virus purification solution (CA6 virus is inoculated into Vero cells, and the harvested virus is obtained by clarification ultrafiltration, sucrose gradient density centrifugation, and desugar treatment) diluted with 0.01M phosphate buffer to 0.5 ⁇ 2.0 ⁇ g/ml, and coated 96-well microtiter plate at 4°C overnight or 37°C for 2 hours.
- the tested serum and negative control sera were serially diluted 108-fold gradient of 10 times according to the gradient method, were added to 10 2 10 8 dilution of the sample to the 96-well plate, 100 l added to each well, incubated for 0.5 37 °C ⁇ 2 hours, wash 2 ⁇ 5 times with 0.01M PBST20 washing solution, add anti-mouse IgG HRP (commercially available: KPL manufacturer) and incubate at 37°C for 0.5-2 hours, wash the plate 2-5 times, pat dry, and add The color substrate is developed at 37°C for 8-15 minutes, terminated by 2M H 2 SO 4 , and read at a wavelength of 450 nm.
- CA6 rabbit polyclonal antibody (manufactured by Beijing Kexing Biological Products Co., Ltd., Beijing Kexing for short) was diluted with a carbonate buffer solution according to a certain ratio, and then coated with a 96-well microtiter plate at 4°C overnight or 37°C for 2 hours.
- the technical solution involved in the present invention can be made into an enzyme-labeled dry plate in advance when the kit is made, and incubated at 2-8°C.
- Antigen dilution The CA6 virus solution is serially diluted according to a certain concentration, 80U/ml, 40U/ml, 20U/ml, 10U/ml, 5U/ml are sequentially added to the above 96-well plate, 100 ⁇ l per well, and incubated at 37°C for 0.5 After -2 hours, wash 2 to 5 times with 0.01M PBST20 washing solution, add the HRP-labeled CA6 solid virus monoclonal antibody prepared in Example 4, incubate at 37°C for 0.5-2 hours, wash the plate 2-5 times, Pat dry, add the chromogenic substrate and develop color at 37°C for 8-15 minutes, stop with 2M H 2 SO 4 , and read at the wavelength of 450-630 nm. Investigate the sensitivity and linearity of the antigen system.
- the detection sensitivity of the antigen detection system to CA6 virus is 5U/ml, and the linear correlation R 2 ⁇ 0.98.
- the results are shown in Table 2, and the linear relationship diagram of the antigen detection system is shown in Figure 4.
- CA6 solid virus particles from Beijing Kexing
- CA6 hollow virus particles from Beijing Kexing
- the pre-coated ELISA plate CA6 rabbit Multi-antibody-coated 96-well ELISA plate
- the reciprocal of the ratio of the protein concentration of the added solid virus and hollow virus is the ratio of the response ability of the antigen evaluation system to solid virus particles and hollow virus particles.
- the experimental results show that the reaction ability of the antigen evaluation system to solid virus particles is 160 times that of hollow virus particles. The results are shown in Table 3.
- the antigen evaluation system can be used to evaluate the antigen content of samples in the entire process.
- This example focuses on the system's detection of samples in each tube of sucrose density gradient centrifugation. Dilute the antigen standard and the sample to a certain concentration respectively, add to the pre-coated ELISA plate with many antibodies, follow the steps in Example 5, make a standard curve from the OD value of the reference product and the labeled amount of the antigen, and the sample OD value band Enter the standard curve to calculate the antigen content of the sample.
- Table 4 shows the evaluation curve of the antigen system used in the ultra-segmentation process.
- Tube 20 is hollow virus particles
- tube 23 is solid virus particles.
- Table 4 shows the ELISA results of antigen detection using the antigen system established by the present invention.
- Tube No. 23 is the peak value of antigen detection, indicating that the system mainly reacts with solid virus particles.
- Example 5 add EV71 virus solution, CA16 virus solution, CA10 virus solution, hepatitis A virus solution, poliovirus type I stock solution, poliovirus type II stock solution, poliovirus type III stock solution, and sample dilution.
- CA6 virus purification solution CA6 infected mouse feces samples (feces samples are diluted with PBS buffer and centrifuged to collect the supernatant) and CA6 infected mouse serum samples (eyeball blood sampling, centrifuged serum), verify that the antigen evaluation system is effective in intestinal The specificity of road virus detection. The results are shown in Table 5. The results show that the antigen evaluation system has good specificity and does not respond to other types of enteroviruses.
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Abstract
一种能与柯萨奇病毒A6型实心病毒反应的单克隆抗体,含有该单抗的试剂盒及其应用。采用纯化的CA6病毒液免疫小鼠制备单克隆抗体。所述单克隆抗体在检测CA6病毒或诊断手足口病中的应用。所述单克隆抗体对于快速检测试剂盒的制备和疫苗的开发研究,具有广泛的应用。
Description
本发明涉及免疫学技术领域,特别是涉及柯萨奇病毒A6型实心病毒的单克隆抗体及其应用。
手足口病是由多种肠道病毒感染引起的急性传染病,夏季流行,学龄前儿童高发,成人可为间接传染源。手足口病临床主要表现为口腔、手、足部位皮疹,可并发脑膜炎、脑炎、肺水肿、循环衰竭等导致死亡的重症。目前肠道病毒71型(enterovirus 71,EV71)和柯萨奇病毒A16(coxsackievirus,CA16)是中国大陆地区引起手足口病最常见的病原体。但随着检测技术及病毒分型手段的进步,近年来发现肠道病毒中的柯萨奇病毒A6(coxsackievirus,CA6)和柯萨奇病毒A10(coxsackievirus,CA10)的流行率呈现逐年增高的趋势,并成为部分地区的主要流行血清型。针对近几年病毒血清型的流行趋势,国内外众多研究机构及企业均在着力开发能够有效预防由CA6或CA10引起的手足口疾病发生的疫苗或药物。
临床治疗方面为能提供最快速的方案,通常需要一种快速的检测方法,ELISA快速检测试剂盒是最常用的方法,能够在最短的时间内检测出患者所感染的病毒血清型。筛选一株型别特异性的单克隆抗体是建立检测试剂盒的前提。此外,在疫苗的研究过程中,疫苗中抗原含量是指导工艺研究和评价体外有效性的关键指标。建立抗原评价方法最关键的技术也在于筛选到合适的单克隆抗体,继而可利用该单克隆抗体建立一套有效的ELISA评价***。
柯萨奇病毒A6(coxsackievirus,CA6)和柯萨奇病毒A10(coxsackievirus,CA10)均为肠道病毒,肠道病毒在体外培养时容易形成两种不同的结构状态,一种为包含核酸的完整实心病毒颗粒,一种为不含核酸的空心病毒颗粒,通常这两种状态的病毒颗粒是共同存在的。经文献资料记载及多次的实验研究表明,实心病毒颗粒和空 心病毒颗粒均有免疫原性,但实心病毒颗粒明显强于空心病毒颗粒。病毒原液中若含有一定比例的空心病毒颗粒,可有效提高原液总蛋白含量,对病毒原液及成品疫苗的稳定性具有重要的意义。因此,在疫苗的工艺研究和制剂研究中,能够确定产品中实心病毒颗粒和空心病毒颗粒的比例对产品的质量评价具有重要意义。分别建立能够检测实心病毒颗粒的抗原评价***及检测空心病毒颗粒的抗原评价***将对病毒原液的监测及成品疫苗的质量控制具有重要意义。
发明内容
为解决上述技术问题,本发明提供如下技术方案:
第一方面,本发明提供了一种针对CA6病毒的单克隆抗体,所述单克隆抗体具有SEQ ID NO:5所示的重链互补决定区CDR1、SEQ ID NO:6所示的重链互补决定区CDR2、SEQ ID NO:7所示的重链互补决定区CDR3,以及SEQ ID NO:13所示的轻链互补决定区CDR1、SEQ ID NO:14所示的轻链互补决定区CDR2、SEQ ID NO:15所示的轻链互补决定区CDR3;优选地,所述单克隆抗体具有SEQ ID NO:8所示的氨基酸序列的重链;和/或,优选地,所述单克隆抗体具有SEQ ID NO:16所示的氨基酸序列的轻链;
优选地,所述CA6病毒为CA6型实心病毒。
第二方面,本发明提供了一种编码所述针对CA6病毒的单克隆抗体的多核苷酸序列,所述多核苷酸序列具有SEQ ID NO:1所示的重链互补决定区CDR1、SEQ ID NO:2所示的重链互补决定区CDR2、SEQ ID NO:3所示的重链互补决定区CDR3,以及SEQ ID NO:9所示的轻链互补决定区CDR1、SEQ ID NO:10所示的轻链互补决定区CDR2、SEQ ID NO:11所示的轻链互补决定区CDR3;优选地,所述多核苷酸序列具有SEQ ID NO:4所示的核苷酸序列的重链;和/或,优选地,所述多核苷酸序列具有SEQ ID NO:12所示的核苷酸序列的轻链;
优选地,所述CA6病毒为CA6型实心病毒。
第三方面,本发明提供了一种用于检测CA6病毒的试剂盒,所 述试剂盒包括所述的单克隆抗体或所述的多核苷酸序列编码的单克隆抗体;优选地,所述试剂盒还包括多抗;更优选地,所述多抗为CA6兔多抗;
优选地,所述CA6病毒为CA6型实心病毒。
第四方面,本发明提供了一种用于诊断手足口病的试剂盒,所述试剂盒包括所述的单克隆抗体或所述的多核苷酸序列编码的单克隆抗体;优选地,所述试剂盒还包括多抗;更优选地,所述多抗为CA6兔多抗;
优选地,所述CA6病毒为CA6型实心病毒。
第五方面,本发明提供了一种所述的针对CA6病毒的单克隆抗体或所述的多核苷酸序列编码的单克隆抗体在制备用于检测CA6病毒或诊断手足口病的试剂盒中的应用;
优选地,所述CA6病毒为CA6型实心病毒。
第六方面,本发明提供了一种所述的针对CA6病毒的单克隆抗体或所述的多核苷酸序列编码的单克隆抗体在对含CA6病毒的疫苗的生产进行质量控制中的应用;
优选地,所述CA6病毒为CA6型实心病毒。
第七方面,本发明提供了一种对含CA6病毒的疫苗的生产进行质量控制的方法,所述方法包括用所述的针对CA6病毒的单克隆抗体或所述的多核苷酸序列编码的单克隆抗体检测CA6病毒的步骤;
优选地,所述CA6病毒为CA6型实心病毒。
第八方面,本发明提供了一种用于治疗或预防由CA6病毒感染引起的疾病的药物,所述药物含有所述的针对CA6病毒的单克隆抗体或所述的多核苷酸序列编码的单克隆抗体;
优选地,所述CA6病毒为CA6型实心病毒。
第九方面,本发明提供了一种所述的针对CA6病毒的单克隆抗体或所述的多核苷酸序列编码的单克隆抗体用于检测手足口疫苗制备过程中或疫苗成品中CA6病毒的含量的应用。
优选地,所述应用为用于检测手足口疫苗制备过程中或疫苗成品 中CA6实心病毒的含量。
目前还未见有文献报道一种能够主要检测CA6实心病毒颗粒的抗原评价***。在现有的工艺研究中,确定病毒液中空心和实心病毒的比例主要是依靠电镜观察的方式,电镜观察的方法对样品的浓度和缓冲体系的成分要求比较高,试验耗时长,对实验室的仪器设备要求也比较高,具有一定的局限性。若采用细胞病变法检测病毒滴度来评价样品中实心病毒颗粒的含量,耗时需要5-7天,实验操作繁琐。
本发明通过采用纯化的CA6病毒液免疫小鼠制备单克隆抗体,免疫兔制备多克隆抗体,从众多的单克隆细胞株中筛选出与CA6型实心病毒颗粒反应能力强的细胞株,利用该细胞株制备大量腹水。之后对单克隆抗体与多克隆抗体分别进行纯化。对纯化后的单克隆抗体进行HRP酶标记,与制备的多克隆抗体建立了一套与实心病毒颗粒反应能力强于空心病毒颗粒的抗原检测***,即主要用于检测CA6实心病毒颗粒的抗原评价***。
制备与CA6型实心病毒颗粒具有型别特异性结合能力的多抗和单抗,建立能够特异性检测CA6病毒的抗原评价***,并且该评价***对CA6型实心病毒颗粒的结合能力显著强于与CA6型空心病毒颗粒的结合能力。在疫苗的研究过程当中,CA6抗原含量的检测是指导工艺研究和评价体外有效性的关键指标。此外,在疫苗的工艺研究过程中,若能够客观真实的评价每一步工序段产品的实心病毒颗粒和空心病毒颗粒的含量及比例,对指导工艺参数的选择和成品的配比计量也具有重要的意义。
建立抗原评价方法最关键的技术在于筛选到合适的单克隆抗体,并利用该单克隆抗体建立一套有效的ELISA评价***。由本发明提供的单克隆抗体制备的抗原评价***,能够主要检测实心病毒,对空心病毒的结合能力极弱。该评价***的检测结果能够真实客观的反应病毒液中的实心病毒含量。实心病毒颗粒的比例是疫苗研究工艺中重点关注的指标,与产品的有效性成正相关。在病毒的培养阶段,采用本发明提供的检测CA6实心病毒颗粒的抗原评价***实时监测病毒 液中实心病毒颗粒的含量,可以为病毒培养的收获时间提供数据支持。在病毒超速离心纯化阶段,通过本发明提供的检测CA6实心病毒颗粒的抗原评价***可以监测出收集后的实心病毒管,为后续的超离合并提供指导。
采用本发明所述的抗原评价***不仅能够对疫苗的工艺研究提供指导,而且能够快速的评价产品的有效性。制备的CA6快速检测试剂盒还可应用于临床检测,快速的确定患者感染的病毒型别,加快疾病确诊,为临床的治疗方案提供强有力的依据。该抗原检测***对临床快速检测和疫苗生产的质量评价均具有重要意义。
当然,还可以将该抗原评价***运用于快速检测试剂盒的制备和疫苗的开发研究中。抗原评价***(含ELISA快速检测试剂盒)通常采用双抗夹心法,例如多克隆抗体(简称:多抗)—多抗、多抗—单克隆抗体(简称:单抗)、单抗—多抗、单抗—单抗的形式。
图1为实施例1蔗糖密度梯度离心分离后的离心管照片;
图2为实施例1电镜鉴定的实心病毒颗粒和空心病毒颗粒电镜比较图,图中左图为23号管实心病毒颗粒电镜图,右图为19号管空心病毒颗粒电镜图;
图3为实施例3单抗纯化后SDS-PAGE电泳图,图中:1-纯化前单克隆抗体腹水,2-纯化洗脱液(重链+轻链),3-纯化流穿液;
图4为实施例5抗原评价***线性关系图;
图5为实施例7抗原评价***用于超离分段工序样品的评价曲线图。
序列表说明
注:加下划线部分为CDR区,加粗字体部分为CH1端引物。
以下实施例用于说明本发明,但不用来限制本发明的范围。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的化学试剂均为常规市售试剂,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1 CA6实心病毒颗粒和空心病毒颗粒的制备
病毒的培养:采用CA6病毒毒株,培养用细胞为Vero细胞(来源于WHO世界卫生组织)。采用发酵罐微载体的模式培养细胞,将病毒按照MOI=0.0001~0.001接种至发酵罐,培养温度为36.0℃±0.5℃,病毒培养1~5天后收获得到CA6病毒液,采用100KD~300KD的膜包澄清超滤初步纯化。
蔗糖密度梯度离心分离获得不同性质的病毒颗粒:采用蔗糖密度梯度离心的方法对初步纯化后的CA6病毒液进行空实心分离和去除杂质蛋白。蔗糖梯度15%~60%,100000转离心3~15小时。蔗糖密度梯度离心分离出空心病毒条带和实心病毒条带,分别取出两条病毒带,用电镜鉴定空实心分离结果。
蔗糖密度梯度离心分离后的离心管照片见图1所示,从图中可以看出分离出的空心病毒条带和实心病毒条带。电镜鉴定的实心病毒颗粒、空心病毒颗粒电镜比较图见图2。
实施例2 CA6实心病毒单克隆抗体的制备
杂交瘤细胞株制备:采用CA6实心病毒纯化液(该实心病毒纯化液制备方法:Vero细胞培养CA6病毒,收获病毒液后经澄清超滤初步纯化和浓缩后,经蔗糖梯度密度离心,可获得实心病毒颗粒管和空心病毒颗粒管,将实心病毒颗粒管收集后脱糖处理,即得到CA6实心病毒纯化液)免疫BALB/c小鼠,每型免疫5只;在0,2,4,6,8周共5针背部皮下多点免疫;免疫剂量:0.2ml/针/只;佐剂:第1针为弗氏完全佐剂,第2、3、4针为弗氏不完全佐剂,第5针不加佐剂;采血检测:免疫第4针后1周采血检测间接酶联免疫法效价,对抗体效价达到10
4以上的小鼠进行第5针腹腔注射进行加强免疫。
细胞融合:腹腔注射加强免疫3天后,处死小鼠取脾融合,两次单克隆化获得阳性杂交瘤细胞株(细胞上清的OD450值>1.0)后,免疫小鼠制备腹水。
从液氮中取出冻存的CA6实心病毒鼠单克隆杂交瘤细胞株进行复苏、扩大培养,10
6以上数量时提取总核酸,委托北京六合华大基因科技有限公司经PCR扩增单抗的重链和轻链序列并进行测序,然后通过核苷酸序列确定对应的氨基酸序列,测定的多核苷酸序列和氨基酸序列参见序列表。
实施例3 CA6实心病毒单克隆抗体的纯化
抗体纯化:将实施例2中免疫小鼠制备的腹水2~8℃、4000~8000r/min离心5~15分钟,取上清经定性滤纸过滤后,采用0.45μm滤膜过滤,经亲和层析获得纯化的单克隆抗体。纯化后检测蛋白质含量为521μg/ml。对纯化后的单克隆抗体进行纯度检测和效价的测定。
(1)单抗纯度的检测
对纯化后的单抗进行SDS-PAGE电泳,检测IgG(免疫球蛋白型)重链和轻链所占的比例,采用凝胶成像扫描仪分析单抗的纯度,单抗纯化后SDS-PAGE电泳图见图3,图中1为纯化前单克隆抗体腹水,2为纯化洗脱液(重链+轻链),3为纯化流穿液。
图3中标记物(Marker)蛋白条带占该泳道总蛋白的比例见下表1。
表1
检测得到的纯化洗脱液的重链和轻链所占比例分别为重链占0.5712,轻链占0.4288。从该结果来看,纯化后的单抗洗脱液中重链与轻链的比例之和为100%,说明纯化的效果很好,无杂质蛋白。
(2)单抗效价的测定
预包被:CA6病毒纯化液(CA6病毒接种Vero细胞,收获病毒经澄清超滤、蔗糖梯度密度离心、脱糖处理获得)用0.01M磷酸盐缓冲液稀释至0.5~2.0μg/ml,包被96孔酶标板于4℃过夜或37℃2小时。加入终浓度0.05%吐温20的0.01M磷酸盐缓冲液洗2~5遍,再加入含有5~20%小牛血清的0.01M磷酸盐缓冲液于37℃封闭1~2小时,使用时甩去封闭液并拍板去除孔中残留封闭液。
效价测定:待检血清和阴性血清对照按照10倍梯度法进行系列梯度稀释至10
8倍,依次加入10
2至10
8稀释度样品于上述96孔板,每孔加入100μl,37℃孵育0.5~2小时,0.01M PBST20洗液洗涤2~5遍,加抗小鼠IgG HRP(市售:KPL厂家)于37℃孵育0.5-2小时,洗板2-5遍后,拍干,加入显色底物于37℃显色8~15分钟,2M H
2SO
4终止,波长450nm处读数。标准:同等稀释倍数下,样品OD450值≥阴性对照×2.1,若阴性对照吸光值<0.05,以0.05计算,判定阳性标准。采用ELISA间接法检测得效价为10
6。
实施例4 CA6实心病毒单克隆抗体的标酶
将纯化后的单克隆抗体置于透析袋内,在0.05M碳酸盐缓冲液体系中透析2~8小时,每1~2小时换透析液一次;采用高碘酸钠活化HRP,20%乙二醇终止活化。活化后的HRP加至抗体中,继续透析过夜;取出抗体,得到HRP标记的CA6实心病毒单克隆抗体,加入硼氢化钠还原;硫酸铵沉淀后采用0.01MPBS复溶,-20℃以下保存。
实施例5 抗原评价***匹配
将CA6兔多抗(北京科兴生物制品有限公司制备,简称北京科兴)采用碳酸盐缓冲溶液按照一定比例稀释后,包被96孔酶标板于4℃过夜或37℃2小时。加入终浓度0.05%吐温20的0.01M磷酸盐缓冲液洗2~5遍,再加入含有5~20%小牛血清的0.01M磷酸盐缓冲液于37℃封闭1~2小时,使用时甩去封闭液 并拍板去除孔中残留封闭液。本发明涉及的技术方案在制成试剂盒时,可提前制成酶标干板,于2-8℃培育。
抗原稀释:将CA6病毒液按照一定浓度进行系列梯度稀释,80U/ml、40U/ml、20U/ml、10U/ml、5U/ml依次加入上述96孔板,每孔加入100μl,37℃孵育0.5-2小时,0.01M PBST20洗液洗涤2~5遍,加实施例4制得的HRP标记后的CA6实心病毒单克隆抗体,于37℃孵育0.5-2小时,洗板2-5遍后,拍干,加入显色底物于37℃显色8~15分钟,2M H
2SO
4终止,波长450-630nm处读数。考察该抗原***的灵敏度、线性关系。
该抗原检测***对CA6病毒的检测灵敏度为5U/ml,线性相关性R
2≥0.98,结果具体见表2,抗原检测***线性关系图见图4。
表2 灵敏度检测结果
病毒抗原(U/ml) | OD值1 | OD值2 | OD均值 |
空白孔 | 0.0087 | 0.0092 | 0.0090 |
5 | 0.1256 | 0.1301 | 0.1279 |
10 | 0.2631 | 0.2685 | 0.2658 |
20 | 0.5511 | 0.5602 | 0.5557 |
40 | 1.0895 | 1.0785 | 1.0840 |
80 | 1.8796 | 1.9014 | 1.8905 |
实施例6 抗原评价***对实心病毒颗粒或空心病毒颗粒的结合能力评价
参照实施例5中的方法,分别将CA6实心病毒颗粒(来自北京科兴)和CA6空心病毒颗粒(来自北京科兴)按照一定的浓度系列稀释后加入预先包被好的酶标板(CA6兔多抗包被的96孔酶标板)中,检测上述抗原评价***对实心病毒颗粒和空心病毒颗粒的结合能力差异。按照达到相同OD值的条件下,加入的实心病毒、空心病毒蛋白浓度比值的倒数即为该抗原评价***对实心病毒颗粒和空心病毒颗粒的反应能力比值。实验结果显示,该抗原评价***对实 心病毒颗粒的反应能力是与空心病毒颗粒反应能力的160倍,结果见表3。
表3 对实心病毒和空心病毒反应能力的比较结果
实施例7 抗原评价***在疫苗生产中的应用
在疫苗的生产阶段,可采用该抗原评价***对整个工艺流程的样品进行抗原含量评价。本实施例重点介绍该***对蔗糖密度梯度离心各管样品的检测。将抗原标准品和样品分别稀释至一定的浓度,加入预先包被好多抗的酶标板,参照实施例5步骤执行,将参比品的OD值与抗原标示量制作标准曲线,样品OD值带入标准曲线计算样品的抗原含量,检测结果见表4,图5所示为抗原***用于超离分段工序样品的评价曲线图。
表4 抗原评价***应用于超离分段样品的评价结果
病毒液经蔗糖密度梯度离心后,实心病毒颗粒与空心病毒颗粒分别分布在不同糖度区域中,20号管为空心病毒颗粒,23号管为实心病毒颗粒。表4为采用本发明所建立的抗原***进行抗原检测的ELISA结果,23号管为抗原的检测峰值,说明本***主要与实心病毒颗粒反应。
实施例8 抗原评价***专属性的验证
参照实施例5方法,分别加入EV71病毒液、CA16病毒液、CA10病毒液、甲肝病毒液、脊髓灰质炎病毒Ⅰ型原液、脊髓灰质炎病毒Ⅱ型原液、脊髓灰质炎病毒Ⅲ型原液、样品稀释液、CA6病毒纯化液、CA6感染小鼠粪便样品(粪便样品采用PBS缓冲液稀释后离心取上清)和CA6感染小鼠血清样品(眼球采血、离心分离血清),验证该抗原评价***对肠道病毒检测的专属性。结果见表5,结果显示,该抗原评价***具有很好的专属性,对其它型别的肠道病毒均不反应。
表5 抗原评价***专属性验证结果
虽然,上文中已经用一般性说明、具体实施方式及试验对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (10)
- 针对CA6病毒的单克隆抗体,其特征在于,所述单克隆抗体具有SEQ ID NO:5所示的重链互补决定区CDR1、SEQ ID NO:6所示的重链互补决定区CDR2、SEQ ID NO:7所示的重链互补决定区CDR3,以及SEQ ID NO:13所示的轻链互补决定区CDR1、SEQ ID NO:14所示的轻链互补决定区CDR2、SEQ ID NO:15所示的轻链互补决定区CDR3;优选地,所述单克隆抗体具有SEQ ID NO:8所示的氨基酸序列的重链;和/或,优选地,所述单克隆抗体具有SEQ ID NO:16所示的氨基酸序列的轻链;优选地,所述CA6病毒为CA6型实心病毒。
- 编码权利要求1所述的针对CA6病毒的单克隆抗体的多核苷酸序列,其特征在于,所述多核苷酸序列具有SEQ ID NO:1所示的重链互补决定区CDR1、SEQ ID NO:2所示的重链互补决定区CDR2、SEQ ID NO:3所示的重链互补决定区CDR3,以及SEQ ID NO:9所示的轻链互补决定区CDR1、SEQ ID NO:10所示的轻链互补决定区CDR2、SEQ ID NO:11所示的轻链互补决定区CDR3;优选地,所述多核苷酸序列具有SEQ ID NO:4所示的核苷酸序列的重链;和/或,优选地,所述多核苷酸序列具有SEQ ID NO:12所示的核苷酸序列的轻链;优选地,所述CA6病毒为CA6型实心病毒。
- 用于检测CA6病毒的试剂盒,其特征在于,所述试剂盒包括权利要求1所述的单克隆抗体或权利要求2所述的多核苷酸序列编码的单克隆抗体;优选地,所述试剂盒还包括多抗;更优选地,所述多抗为CA6兔多抗;优选地,所述CA6病毒为CA6型实心病毒。
- 用于诊断手足口病的试剂盒,其特征在于,所述试剂盒包括权利要求1所述的单克隆抗体或权利要求2所述的多核苷酸序列编码的单克隆抗体;优选地,所述试剂盒还包括多抗;更优选地,所述多抗为CA6兔多抗;优选地,所述CA6病毒为CA6型实心病毒。
- 权利要求1所述的针对CA6病毒的单克隆抗体或权利要求2所述的多核苷酸序列编码的单克隆抗体在制备用于检测CA6病毒或诊断手足口病的试剂盒中的应用;优选地,所述CA6病毒为CA6型实心病毒。
- 权利要求1所述的针对CA6病毒的单克隆抗体或权利要求2所述的多核苷酸序列编码的单克隆抗体在对含CA6病毒的疫苗的生产进行质量控制中的应用;优选地,所述CA6病毒为CA6型实心病毒。
- 对含CA6病毒的疫苗的生产进行质量控制的方法,其特征在于,所述方法包括用权利要求1所述的针对CA6病毒的单克隆抗体或权利要求2所述的多核苷酸序列编码的单克隆抗体检测CA6病毒的步骤;优选地,所述CA6病毒为CA6型实心病毒。
- 用于治疗或预防由CA6病毒感染引起的疾病的药物,其特征在于,所述药物含有权利要求1所述的针对CA6病毒的单克隆抗体或权利要求2所述的多核苷酸序列编码的单克隆抗体;优选地,所述CA6病毒为CA6型实心病毒。
- 权利要求1所述的针对CA6病毒的单克隆抗体或权利要求2所述的多核苷酸序列编码的单克隆抗体用于检测手足口疫苗制备过程中或疫苗成品中CA6病毒的含量的应用。
- 根据权利要求9所述的应用,其特征在于,所述应用为用于检测手足口疫苗制备过程中或疫苗成品中CA6实心病毒的含量。
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