CN113293229A - A rotavirus titration method - Google Patents
A rotavirus titration method Download PDFInfo
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Abstract
本发明提供了一种对轮状病毒滴度进行测定的方法。具体地，本发明的方法将待测定的轮状病毒、可被所述轮状病毒感染的宿主细胞、和培养液同时置于检测孔中，使用带有可检测标记的抗轮状病毒抗体，通过直接免疫荧光法测定待测定轮状病毒的滴度。本发明的方法操作简便，灵敏度更高，准确性更好，是一种快速高效的轮状病毒滴定方法。The present invention provides a method for measuring rotavirus titer. Specifically, in the method of the present invention, the rotavirus to be determined, the host cell that can be infected by the rotavirus, and the culture medium are placed in the detection well at the same time, and an anti-rotavirus antibody with a detectable label is used, The titer of the rotavirus to be determined is determined by direct immunofluorescence. The method of the invention is easy to operate, has higher sensitivity and better accuracy, and is a fast and efficient rotavirus titration method.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a titration method of rotavirus.
Background
Rotavirus (RV) is a very broad-spectrum digestive tract virus, of which group a RV is one of the major causes of infantile diarrhea. Infant deaths worldwide each year from RV infections occur mostly in developing countries. In China, about 4000 million infants (including newborn infants) in 0-2 years old suffer from RV infectious gastroenteritis every year, and 1/4, which accounts for the number of infants, is the most main pathogen causing severe diarrhea of infants. The RV vaccine immunization can obviously reduce the proportion of severe diarrhea of infants caused by RV infection.
The virus quantification method for RV mainly comprises three methods, namely q-PCR, ELISA and cell-based titration. Q-PCR is used for indirectly determining the content of virus by quantifying the genome of the virus; ELISA is an indirect determination of virus content by quantifying major structural proteins of the virus; cell-based titration is a method of measuring the number of infectious viral particles.
For the production of RV vaccines, especially for oral administration of RV vaccines, the titer of infectious virus is the most critical indicator affecting the immune efficacy of the vaccine. The fusion protein VP4 of RV can be efficiently cleaved into VP8 with amino terminal and VP5 with carboxyl terminal under the action of pancreatin, and the two fragments play an important role in the early stage of virus invasion into host cells; therefore, pancreatin has important applications in both in vitro amplification and titration of RV.
The existing Vero cell titration method for RV mainly comprises the steps of firstly, paving Vero cells in a 96-well plate for overnight culture, washing the cells for 3-5 times by PBS (phosphate buffer solution) to ensure that no FBS exists, then, adding pancreatin with a certain concentration into virus liquid, performing gradient dilution and inoculation on the cells, and then determining the virus titer through antigen-antibody reaction, wherein the whole process is about 96 hours; the method has long detection time, uses a serum-containing culture medium, needs to thoroughly clean fetal calf serum when inoculating RV, uses two antibodies when counting positive cells, and has complex operation and higher cost.
Therefore, there is a need in the art to develop a rapid and efficient rotavirus titration method.
Disclosure of Invention
The invention aims to provide a method for quickly and efficiently measuring the titer of rotavirus.
In a first aspect of the invention, there is provided a method of determining rotavirus titre, said method comprising the steps of:
(S1) the rotavirus to be detected,Putting host cells capable of being infected by the rotavirus and a culture solution into a detection hole to form a culture system, wherein the inoculation amount of the host cells in the detection hole is 1.0 multiplied by 106-1.5×106Per cm2Detecting the bottom area of the hole;
(S2) culturing said culture system for a time t1 suitable for infection and replication of said rotavirus, whereby rotavirus infected adherent host cells are obtained, wherein t1 is 12-22 hours;
(S3) performing a fixation treatment on said rotavirus infected adherent host cell, thereby obtaining a fixed rotavirus infected adherent host cell;
(S4) contacting the immobilized rotavirus infected adherent host cell with an anti-rotavirus antibody for a time t2 to form a rotavirus-anti-rotavirus antibody complex, wherein the anti-rotavirus antibody has a detectable label and t2 is 0.5-2 hours; and
(S5) quantitatively detecting the amount of rotavirus in the detection well based on the rotavirus-anti-rotavirus antibody complex, thereby obtaining a determination result of the rotavirus titer.
In another preferred example, in step (S1), n detection holes are included, n is a positive integer ≧ 3, preferably n is 6-96.
In another preferred example, in the step (S1), the detection wells include one or more sets of detection wells, and the conditions of the detection wells are the same except for the amount of rotavirus (including the same amount of host cells, the same components and volume of culture solution, the same pancreatin and pancreatin concentrations, the same addition amount, and the like) in each detection well.
In another preferred embodiment, the amount of rotavirus in said set of test wells is diluted by a gradient, preferably at a gradient dilution ratio of 2 to 20.
In another preferred example, the detection well may be a well on a cell culture plate, such as a well on a 96-well plate; or may be a separate cell culture dish/well.
In another preferred embodiment, the dilution is performed using a virus diluent comprising the following components: DMEM medium, 1-5. mu.g/ml trypsin, and 100-400. mu.g/ml calcium chloride.
In another preferred embodiment, the virus diluent comprises the following components: DMEM medium, 4. mu.g/ml trypsin, and 200. mu.g/ml calcium chloride.
In another preferred embodiment, the host cell is selected from the group consisting of: vero, MA-104, Caco-2 or combinations thereof.
In another preferred embodiment, the amount of inoculation of said host cells in said test wells is 1.25X 106Per cm2The bottom area of the hole is detected.
In another preferred embodiment, the culture solution is a serum-free culture solution.
In another preferred embodiment, the rotavirus infected adherent host cell has a cell confluence in the test well of 90% to 150%, preferably 95% to 120%, most preferably 99% to 100%.
In another preferred example, in the step (S3), the fixing agent used in the fixing treatment is selected from the group consisting of: glacial acetone, formaldehyde, paraformaldehyde, absolute ethanol, or a combination thereof.
In another preferred embodiment, the fixing agent is 80% (v/v) of glacial acetone.
In another preferred embodiment, the time of the fixing treatment is 20 to 40 minutes, preferably 30 minutes.
In another preferred embodiment, the anti-rotavirus antibody is selected from the group consisting of: an anti-VP 6 antibody, an anti-VP 7 antibody, an anti-VP 1 antibody, an anti-VP 2 antibody, an anti-VP 4 antibody, or a combination thereof.
In another preferred embodiment, the antibody is an anti-VP 6 antibody.
In another preferred embodiment, the antibody may be murine, rabbit, human or humanized.
In another preferred embodiment, the antibody may be a monoclonal antibody or a polyclonal antibody.
In another preferred embodiment, the detectable label may be fluorescein or a chemiluminescent.
In another preferred embodiment, the detectable label is selected from the group consisting of: FITC, horseradish peroxidase (HRP), DyLight 649, or a combination thereof.
In another preferred example, in step (S4), t2 is 0.75 hours.
In another preferred example, in the step (S5), the method for quantitative determination includes:
(a) observing by a fluorescence microscope; or
(b) Detection was by AID elispot READER.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the indirect immunofluorescence assay (IFA) identification of homemade VP6 protein. The left picture shows the IFA detection result of rotavirus positive cells; the right panel is a negative control.
FIG. 2 shows the number of seeded cells in a 96-well plate as a function of cell confluence.
FIG. 3 shows the results of measuring the titer of the same virus strain at different culture times.
FIG. 4 shows the sensitivity of the method of the invention and indirect immunofluorescence titration method to rotavirus detection.
FIG. 5 shows a comparison of the titration of rotavirus by the method of the present invention and by the indirect immunofluorescence titration method.
Detailed Description
The inventors of the present invention have conducted extensive and intensive studies and, for the first time, have unexpectedly developed a rotavirus titration method by a serum-free medium direct immunofluorescence method. The inventor specifically tries to directly suspend and inoculate the virus and the Vero cells together in a 96-well plate by using a serum-free culture medium, finds that the cells can still adhere to the wall normally under the condition of 2 mu g/ml pancreatin and have a good state, and still incubate a direct standard antibody at 18h after suspension inoculation of the virus to judge the virus titer; the result shows that the detection result is similar to the titer result of the original RV indirect immunofluorescence titration method, and the method for detecting the RV titer by using the serum-free medium direct immunofluorescence method has feasibility. Therefore, systematic exploration is carried out on each key link, and finally a set of RV titration method of serum-free medium direct immunofluorescence is determined. On the basis of this, the present invention has been completed.
Term(s) for
In order that the invention may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.
The term "about" refers to a value or composition within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
As used herein, the terms "method of the invention", "the present method" and "the methods described herein" are used interchangeably and all refer to the serum-free medium direct immunofluorescence titration rotavirus method described herein.
Rotavirus (Rotavirus, RV)
RV belongs to reoviridae, genus RV, and is a non-enveloped virus; it is named because it presents a wheel shape under an electron microscope.
RV virions are regular icosahedral symmetric structures with smooth surfaces, about 70nm in diameter, composed of 3 protein capsids: an outer capsid (VP7 and VP4), a middle capsid (VP6), and an inner capsid (VP 2). The viral genome of RV consists of 11 discrete double-stranded RNAs (dsRNA) that are respectively enveloped by an inner capsid, RNA-dependent RNA polymerase (VP1) and modifying enzyme (VP 3). The 11 segments of dsRNA together encode 6 structural proteins (VP1-VP4/VP6/VP7) and 6 non-structural proteins (NSP1-NSP6), and besides 1 segment containing two open reading frames and encoding NSP5 and NSP6, each segment of dsRNA encodes one protein. In a host, co-infection of different strains of RV may cause reassortment of 11 gene segments to generate a novel type of RV.
RV can be classified into 7 groups of A, B, C, D, E, F and G, which can infect humans according to VP6 antigenicity (located in the intermediate capsid, highly conserved), wherein group A is the main pathogen causing infantile infection and severe diarrhea. RV can be further divided into 36G serotypes according to the structural protein VP7 and 51P serotypes according to the structural protein VP 4. The G-P combination serotype is determined by both VP7 and VP4 of the virus.
The invention provides a method for determining rotavirus titer. After the titer of the rotavirus to be tested is determined by the method of the invention, the rotavirus can be used for the production of RV vaccine (especially oral RV vaccine) according to the determined titer.
The methods of the invention are not particularly limited with respect to groups and serotypes of rotavirus, i.e., the methods of the invention are applicable to rotavirus of any group or serotype.
Anti-rotavirus antibody
As used herein, the term "anti-rotavirus antibody" is an antibody or polypeptide molecule capable of specifically binding to the capsid protein of rotavirus. The capsid protein may be VP1-VP4, VP6 or VP 7. The antibody can be a murine, rabbit, human or humanized antibody; also, the antibody may be a monoclonal antibody or a polyclonal antibody.
In the method of the invention, immobilized rotavirus infected adherent host cells are contacted with anti-rotavirus antibody for 0.5-2 hours to form a "rotavirus-anti-rotavirus antibody" complex.
In another preferred embodiment, the anti-rotavirus antibody is selected from the group consisting of: an anti-VP 6 antibody, an anti-VP 7 antibody, an anti-VP 1 antibody, an anti-VP 2 antibody, an anti-VP 3 antibody, an anti-VP 4 antibody, or a combination thereof.
In another preferred embodiment, the antibody is an anti-VP 6 antibody.
In a preferred embodiment of the invention, the anti-rotavirus antibody is a rabbit-derived polyclonal antibody against VP6 protein.
In addition, the anti-rotavirus antibody also carries a detectable label for detecting the formation of a "rotavirus-anti-rotavirus antibody" complex.
In another preferred embodiment, the detectable label may be fluorescein or a chemiluminescent.
In another preferred embodiment, the detectable label includes, but is not limited to, FITC, horseradish peroxidase (HRP), DyLight 649, or a combination thereof.
In a preferred embodiment of the invention, polyclonal antibodies against VP6 protein from rabbit are labeled with FITC.
As used herein, the term "VP 6 direct-labeled antibody" refers to the above-described polyclonal antibody against VP6 protein, which is of FITC-labeled rabbit origin. The antibody is prepared according to the following method:
(1) synthesizing polypeptide containing rotavirus VP6 protein antigen site;
(2) immunizing rabbit with the polypeptide once every 0, 14 and 21 days;
(3) blood is collected one week after the three-immunization, and serum is separated, so that serum containing rabbit anti-rotavirus VP6 polyclonal antibody is obtained;
(4) purifying the obtained rabbit serum by a ProteinA + G column to obtain a purified rabbit anti-rotavirus VP6 antibody;
(5) the purified rabbit anti-rotavirus VP6 antibody was chemically conjugated with fluorescein thiocyanate (FITC) to form the FITC-labeled rabbit anti-rotavirus VP6 polyclonal antibody.
Direct immunofluorescence (FA) and indirect Immunofluorescence (IFA)
Immunofluorescence technique is to combine fluorescein such as Fluorescein Isothiocyanate (FITC) with corresponding antibody (or antigen) chemically, combine the fluorescence labeled antibody with corresponding antigen in the sample to form fluorescence labeled antibody-antigen complex, and observe with fluorescence microscope. The presence of fluorescence under the mirror indicates the presence of antigen-antibody complex. The direct immunofluorescence method is to directly combine a specific antibody marked by a fluorescent pigment with a corresponding antigen so as to directly deduce the existence of the antigen; the indirect immunofluorescence method labels an antiglobulin antibody (secondary antibody) with fluorescein, and after the primary antibody binds to the corresponding antigen, the secondary antibody reacts with the bound primary antibody, thereby inferring the presence of the antigen.
In the methods of the invention, rotavirus titers are determined using direct immunofluorescence. Specifically, an anti-rotavirus antibody with a detectable label is contacted with an adherent host cell infected by rotavirus and fixed in a detection hole to form a rotavirus-anti-rotavirus antibody compound. The quantity of rotavirus in the detection hole can be quantified by directly detecting the detectable marker, so that the titer of the rotavirus to be detected is obtained. For example, in a preferred embodiment of the present invention, a "rotavirus-anti VP6 protein antibody-FITC" complex formed by contacting a rabbit-derived anti VP6 protein polyclonal antibody with FITC with immobilized rotavirus-infected adherent host cells in a detection well is used, and the amount of rotavirus in the detection well can be quantified by directly detecting FITC under a fluorescence microscope, thereby obtaining the titer of the rotavirus to be detected.
Serum-free medium
As used herein, the terms "serum-free medium" and "serum-free medium" are used interchangeably and refer to a synthetic medium that does not require the addition of serum to maintain cells in growth and proliferation in vitro for extended periods of time. In short, the cell culture medium in the in vitro cell culture process does not contain animal or human serum, and is called serum-free cell culture medium.
In the method, after the host cells are suspended by adopting a serum-free culture medium, the host cells and rotavirus diluted by a virus diluent containing pancreatin are simultaneously inoculated in a detection hole, and after the culture for about 18 hours, the fixation and the subsequent detection are directly carried out. Because the serum-free culture medium does not contain serum, the activity of pancreatin necessary for rotavirus to infect host cells is not influenced, the efficiency of rotavirus infecting the host cells is improved while the growth and the propagation of the host cells are maintained, and the time of the whole detection process is greatly shortened.
The main advantages of the invention are:
(1) compared with the original indirect immunofluorescence titration method, the method adopts the suspension inoculation method of inoculating the virus and the cells together, does not need to prepare and culture the cells in advance, and greatly saves the detection time;
(2) the method of the invention adopts a suspension inoculation method, so that the infection efficiency of the virus is higher;
(3) compared with an indirect immunofluorescence titration method, the method provided by the invention has higher detection sensitivity by adopting a serum-free direct immunofluorescence method;
(4) the method adopts FITC-coupled VP6 direct-labeled antibody, so that the experimental time of the immunoreaction part is reduced by more than 50% compared with the original indirect immunofluorescence titration method, and the operation is simpler and more convenient.
Example 1
Establishment of serum-free medium direct immunofluorescence method for titrating rotavirus
1.1 preparation of VP6 direct Standard antibody
In order to reduce the detection time of RV titer to the maximum extent, rabbit polyclonal antibody of RV VP6 protein is prepared, and the antibody can be used for indirect immunofluorescence detection (IFA) of RV by identification (FIG. 1); then purified by ProteinA/G and coupled with FITC; finally, direct immunofluorescence (FA) titers of the conjugated VP6 direct standard antibody were determined. The results showed that the prepared FITC-conjugated VP6 direct-labeled antibody had an FA use concentration of 1: 100.
1.2 determination of optimal cell usage
In virus titration, the number of cells can affect the combination of cells and virus and the display of immunofluorescence results, and finally affect the titration result. In virus titration in RV, the final titer of the virus is determined by the number of positive cells, therefore, the optimal number of cells to use is typically 100% confluency after cell attachment. This is because when the number of cells is too small, the cells may be shed after RV infection, resulting in inaccurate results; when the number of cells is too large, accumulation of positive cells may cause errors in the detection result.
To design a time-saving RV titration method, suspension vaccination was decided by diluting the virus into a cell suspension and plating it with the cells in a 96-well plate. Since the virus can be accurately detected by replicating the virus in good Vero cells for 18h, the cells are diluted to different concentrations by using a serum-free medium, inoculated into a 96-well plate (each well is inoculated with 100 mu L), the cell confluency of the cells with different concentrations after 18h is observed, and the number of the cells with the confluency reaching 100% is taken as the optimal concentration for the cell use in the method. The results show (FIG. 2), when 4X 10 of the additive was added per well5When the Vero cells are attached, the cell confluence degree reaches 100 percent, and the Vero cells are suitable for the titration of RV.
1.3 determination of the incubation time of the Virus
The detection time is closely related to the amplification speed of the virus, the detection time is too early, the expression level of the virus protein is low, and the detection result is low; the detection time is too late, so that the detection period is prolonged, unnecessary waste is caused, and meanwhile, detection result errors can be caused by cell shedding.
Therefore, diluting a RV sample to be detected by using a virus diluent (DMEM +4 mu g/ml pancreatin +200 mu g/ml calcium chloride) in a 10-fold gradient manner, paving the sample in a 96-well plate, wherein each well is 100 mu L, and each dilution is repeated for 4 times; adding Vero cell suspension cultured by serum-free medium into 96-well plate in equal volume until the cell number per well is 4 × 104. Virus titers were determined with IFA at 6h, 12h, 18h, 24h, 48h and 72h, respectively. The results are shown in FIG. 3, where the virus titer stabilized at 10 at 18h and over 18h after suspension inoculation6.5CCID50And/ml. Considering the time cost of virus titration, determination of 18h is the preferred detection time.
1.4 establishment of method for titrating rotavirus by serum-free medium direct immunofluorescence method
Based on the research and data accumulation, the related technical parameters of the direct immunofluorescence titration of rotavirus are finally determined. The method comprises the following specific steps:
(1) virus preparation: diluting virus with 10 times of virus diluent (DMEM +4 μ g/ml pancreatin +200 μ g/ml calcium chloride) in gradient, spreading in 96-well plate with each well at 100 μ L, and repeating for 4 dilutions;
(2) cell preparation: vero cells cultured in serum-free medium are digested with recombinant pancreatin and resuspended in serum-free medium to obtain cell suspension, 4X 10 cells are added per well 5100 μ L of cells;
(3) incubating the 96-well plate inoculated with the virus and the cells in a carbon dioxide incubator at 37 ℃ for 18 h;
(4) fixing: discarding the culture medium, adding 100 μ L of 80% glacial acetone into each well, and fixing at 4 deg.C for 30 min;
(5) drying: discarding the glacial acetone, and airing the plate at a ventilation position until no liquid exists in the hole;
(6) incubation of the antibody: 1:100 dilution of FITC-conjugated VP6 direct-labeled antibody, 100. mu.L of the diluted antibody per well, and incubation at 37 ℃ for 45 min;
(7) washing: discarding the antibody, washing the plate with PBS 3 times;
(8) reading and calculating: positive wells were read under a fluorescent microscope and virus titer was calculated using Reed-Muench in CCID50/ml。
Example 2
Research on detection lower limit of rotavirus titration method by serum-free medium direct immunofluorescence method
In order to determine the sensitivity of detecting the RV virus by the method established in example 1, the same RV sample is titrated by the original method (RV indirect immunofluorescence titration method) and the serum-free medium direct immunofluorescence method respectively, and the result shows that (figure 4) the detection rate of the serum-free medium direct immunofluorescence method for more than 10 infectious virus particles is 100%; the lower limit of detection of the prior RV titration method is 50 infectious virus particles. This demonstrates the higher sensitivity of the novel RV titration method described herein.
Example 3
Repeatability research of serum-free medium direct immunofluorescence titration rotavirus method
In order to detect the repeatability of the method, 3 rotaviruses with known titer are subjected to 6 parallel detections, and the coefficient of variation of the method is less than 3.7 percent (table 1), which indicates that the method has good repeatability.
TABLE 1 repeatability of the serum-free Medium direct immunofluorescence titration Rotavirus method
Example 4
Comparative study of serum-free medium direct immunofluorescence method and simple immunofluorescence method
In order to compare the results of the rotavirus titration by the method with the results of the rotavirus titration by the traditional indirect immunofluorescence method, 6 rotaviruses with different titers are selected and respectively titrated by the two methods, and the results show that the titration result of the method is 0.5-0.75lgCCID50/ml higher than that of the indirect immunofluorescence method, and the virus titers detected by the two methods have better correlation (figure 5).
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
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