CN111888470A - Flushing fluid with rabies virus neutralizing effect and activity detection method thereof - Google Patents

Flushing fluid with rabies virus neutralizing effect and activity detection method thereof Download PDF

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CN111888470A
CN111888470A CN202010808614.8A CN202010808614A CN111888470A CN 111888470 A CN111888470 A CN 111888470A CN 202010808614 A CN202010808614 A CN 202010808614A CN 111888470 A CN111888470 A CN 111888470A
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rabies
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赵静
刁萍萍
周东
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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    • 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
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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Abstract

The invention discloses a flushing fluid with the effect of neutralizing rabies virus and an activity detection method thereof, and the method comprises the steps of preparing the flushing fluid; detecting the antiviral activity of the flushing fluid; and (4) determining the use place of the flushing liquid. The invention has the beneficial effects that: can clean the wound and neutralize the virus in the wound, and has certain therapeutic effect.

Description

Flushing fluid with rabies virus neutralizing effect and activity detection method thereof
Technical Field
The invention relates to a flushing fluid, in particular to a flushing fluid with the effect of neutralizing rabies viruses and an activity detection method thereof, belonging to the technical field of rabies prevention and treatment measures.
Background
Rabies, one of the most common acute infectious diseases to date in humans, has the etiology of Rabies virus (Rabies virus), which is susceptible to both humans and all warm-blooded animals. According to statistics of a disease prevention and control center in China, 969 rabies cases and 873 death cases occur in 2014 in China. The fatality rate of rabies is close to 100%, although humanized anti-rabies virus antibody drugs have been greatly developed in recent years, no very effective therapeutic drug is developed at present, and therefore, rabies vaccines are still the most effective means for preventing and treating rabies at present. The rabies virus genome is nonsegmented single-strand negative-strand RNA, has a total length of about 12kb, and respectively encodes five main structural proteins, namely an enzyme protein (L), a glycoprotein (G), a matrix protein (M), a phosphorylated protein (P) and a nucleoprotein (N). Wherein, the G protein and the N protein contain a plurality of antigenic sites and can induce and generate anti-rabies virus antibodies.
The World Health Organization (WHO) recommends post-rabies-exposure prophylaxis, which is mainly undertaken by three main measures, namely (1) effective wound irrigation; (2) timely inoculating rabies vaccine for human and (3) injecting anti-rabies virus immunoglobulin. The ultimate goal of the three measures is to clean the rabies virus in the bite site effectively in time and eliminate it before it enters the nervous tissue. Three links are absent and impossible. At present, rabies vaccine and anti-rabies immunoglobulin for human use are mature commercial products. However, in the effective wound irrigation process, generally, running water irrigation is adopted, and in order to improve the irrigation effect, some outpatients of hospitals adopt a pressurized irrigation water gun and properly add soap liquid, so that the aim of finally irrigating viruses as much as possible is fulfilled. This step of wound irrigation is critical and a more effective wound irrigation solution is needed to more effectively remove rabies virus from the wound.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a washing solution having a neutralizing activity against rabies viruses and a method for detecting the activity of the washing solution.
The invention realizes the purpose through the following technical scheme: an irrigation solution having efficacy in neutralizing rabies virus, comprising: 0.9% physiological saline, tween-20, humanized anti-rabies monoclonal antibody with titer not less than 100IU/ml, EDTANa2And chitosan.
A preparation method of a washing liquid with the effect of neutralizing rabies viruses comprises the following steps: adding 10mL tween-20 into 500mL of 0.9% physiological saline to obtain humanized anti-rabies antibody with titer not less than 0.5IU/mL, and adding EDTANa2And adding 0.9% physiological saline to 1000mL after chitosan is added, filtering and sterilizing, and keeping at 2-8 deg.C for later use.
A method for detecting the activity of washing liquid with the effect of neutralizing rabies viruses comprises the following steps:
preparing a washing liquid, namely preparing an experimental material, and preparing the washing liquid according to a formula;
step two, detecting antiviral activity of flushing liquid, performing cell level activity detection by referring to a 'Rapid Fluorescence Focus Inhibition Test (RFFIT)' method for detecting rabies antibody activity in three parts of 'Chinese pharmacopoeia' 2015 edition, and testing the horizontal use effect of animals by taking SPF-level Kunming mice as test objects;
and step three, determining the use place of the washing liquid, wherein the components of the washing liquid are pharmaceutical grade raw materials, and the washing liquid does not contain any preservative or irritant component, is safe, nontoxic and high in stability, can be directly used for replacing the conventional running water wound washing in a rabies treatment clinic, and can also be prepared into a concentrated solution to be matched with a washing device in the treatment clinic for dilution.
As a further scheme of the invention: in the first step, the required experimental materials comprise 0.9% of normal saline, tween-20, high titer human anti-rabies antibody (the titer is not less than 100IU/mL), EDTANa2And chitosan.
As a further scheme of the invention: in the first step, the preparation formula of the flushing liquid is as follows: 500mL of 0.9% physiological saline is taken, 10mL of Tween-20 and a proper amount of humanized anti-rabies antibody (the final titer is not less than 0.5IU/mL) are added,trace EDTANA2And adding 0.9% physiological saline to 1000mL after chitosan is added, filtering and sterilizing, and keeping at 2-8 deg.C for later use.
As a further scheme of the invention: in the second step, the activity detection at the cellular level comprises the following steps:
(1) sampling: taking a sterile 96-microporous plate for cell culture, and adding 100 mu l of DMEM medium containing 10% fetal calf serum into each hole;
(2) sample dilution: firstly, carrying out 3-time serial dilution on a standard substance and a novel flushing liquid, wherein the standard substance is diluted to 9 times, and the flushing liquid is diluted to 3 times;
(3) sample adding: adding 50 mul of the 9 times diluted standard substance into a 96-pore plate in the 2.1.1 step, namely, the 1 st pore is diluted by 27 times; taking 50 mul of the novel washing liquid diluted by 3 times to be put on a plate, namely diluting the 1 st hole by 9 times;
(4) and (3) secondary dilution: adding 50 μ l of the mixture from each well of the 1 st row to the second row by using a row gun, continuously taking 50 μ l of the mixture to the 3 rd row after mixing, and performing 3-fold serial dilution until the 12 th row;
(5) adding poison: adding 50 μ l rabies virus CVS strain with fixed titer into the above micropores, and neutralizing in a constant temperature incubator at 37 deg.C for 1 hr;
(6) standing culture: adding 50 μ l BSR cells into each well after neutralization, and culturing in a constant temperature incubator at 37 deg.C for 24 hr;
(7) and (4) detecting a result: after the culture, the culture supernatant is discarded, 80% cold acetone is added for fixation for 20 minutes, 50 mu.l of FITC-labeled anti-nucleoprotein monoclonal antibody is added for incubation for 1hr, then the supernatant is discarded, the plate is washed by PBS solution for 1 time, and then the fluorescence infection condition of each hole is observed under an inverted fluorescence microscope.
As a further scheme of the invention: in the second step, when the horizontal use effect test of the experimental animals is carried out, the three-level exposure condition of the bite wound is simulated, different combinations are adopted for intervention, and each group is repeated for three times.
The invention has the beneficial effects that: the preparation method of the flushing liquid with the effect of neutralizing rabies viruses is reasonable in design, can play a role in cleaning wounds, can neutralize viruses in the wounds, and has a certain treatment effect.
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FIG. 1 is a schematic view of a process for preparing a structure according to the present invention;
FIG. 2 is a schematic diagram of the process for detecting the activity at the cellular level according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
An irrigation solution having efficacy in neutralizing rabies virus, comprising: 0.9% physiological saline, tween-20, human anti-rabies antibody with titer not less than 100IU/ml, EDTANa2And chitosan.
A preparation method of a washing liquid with the effect of neutralizing rabies viruses comprises the following steps: adding 10mL tween-20 into 500mL of 0.9% physiological saline to obtain humanized anti-rabies antibody with titer not less than 0.5IU/mL, and adding EDTANa2And adding 0.9% physiological saline to 1000mL after chitosan is added, filtering and sterilizing, and keeping at 2-8 deg.C for later use.
As shown in FIG. 1 and FIG. 2, a method for detecting the activity of an irrigation solution having a neutralizing effect on rabies viruses comprises the following steps:
preparing a washing liquid, namely preparing an experimental material, and preparing the washing liquid according to a formula;
step two, detecting antiviral activity of flushing liquid, performing cell level activity detection by referring to a 'Rapid Fluorescence Focus Inhibition Test (RFFIT)' method for detecting rabies antibody activity in three parts of 'Chinese pharmacopoeia' 2015 edition, and testing the horizontal use effect of animals by taking SPF-level Kunming mice as test objects;
and step three, determining the use place of the washing liquid, wherein the components of the washing liquid are pharmaceutical grade raw materials, and the washing liquid does not contain any preservative or irritant component, is safe, nontoxic and high in stability, can be directly used for replacing the conventional running water wound washing in a rabies treatment clinic, and can also be prepared into a concentrated solution to be matched with a washing device in the treatment clinic for dilution.
Further, in the embodiment of the present invention, in the first step, the required experimental materials include 0.9% physiological saline, tween-20, high titer recombinant human anti-rabies monoclonal antibody (1000IU/mL), EDTANA2And chitosan.
Further, in the embodiment of the present invention, in the first step, the preparation formula of the rinsing liquid is: taking 500mL of 0.9% physiological saline, adding 10mL of Tween-20, a proper amount of humanized anti-rabies monoclonal antibody (the final concentration is not lower than 0.5IU/mL), and trace amount of EDTANa2And adding 0.9% physiological saline to 1000mL after chitosan is added, filtering and sterilizing, and keeping at 2-8 deg.C for later use.
Further, in the embodiment of the present invention, in the second step, the activity detection at the cellular level includes the following steps:
(1) sampling: taking a sterile 96-microporous plate for cell culture, and adding 100 mu l of DMEM medium containing 10% fetal calf serum into each hole;
(2) sample dilution: firstly, carrying out 3-time serial dilution on a standard substance and a novel flushing liquid, wherein the standard substance is diluted to 9 times, and the flushing liquid is diluted to 3 times;
(3) sample adding: adding 50 mul of the 9 times diluted standard substance into a 96-pore plate in the 2.1.1 step, namely, the 1 st pore is diluted by 27 times; taking 50 mul of the novel washing liquid diluted by 3 times to be put on a plate, namely diluting the 1 st hole by 9 times;
(4) and (3) secondary dilution: adding 50 μ l of the mixture from each well of the 1 st row to the second row by using a row gun, continuously taking 50 μ l of the mixture to the 3 rd row after mixing, and performing 3-fold serial dilution until the 12 th row;
(5) adding poison: adding 50 μ l rabies virus CVS strain with fixed titer into the above micropores, and neutralizing in a constant temperature incubator at 37 deg.C for 1 hr;
(6) standing culture: adding 50 μ l BSR cells into each well after neutralization, and culturing in a constant temperature incubator at 37 deg.C for 24 hr;
(7) and (4) detecting a result: after the culture, the culture supernatant is discarded, 80% cold acetone is added for fixation for 20 minutes, 50 mu.l of FITC-labeled anti-nucleoprotein monoclonal antibody is added for incubation for 1hr, then the supernatant is discarded, the plate is washed by PBS solution for 1 time, and then the fluorescence infection condition of each hole is observed under an inverted fluorescence microscope.
The results show that the virus fluorescence of each well of the normal saline group is more than 90% of infection, namely no neutralization activity, and the virus fluorescence of the first 1-2 wells of the standard and the novel washing solution is not present, which indicates that the virus is neutralized, and the fluorescence is gradually enhanced and the neutralization activity of the virus is gradually reduced along with the increase of the dilution ratio, and the detailed chart is shown in figure 1. The in vitro cytology level test result shows that the washing liquid has obvious inhibition effect on rabies virus, the effective antibody titer calculated by a Reed-Much method can reach 1.8IU/mL, and according to WHO technical documents, the effective protection on the rabies virus can be generated when the neutralizing antibody titer is more than 0.5IU/mL
Further, in the embodiment of the present invention, in the second step, when the use effect test of the test animal level is performed, the bite three-level exposure condition is simulated, the intervention is performed by adopting different combinations, and each group is repeated three times, and the experimental result shows that the protection rate is only 3.3% for the running water washing, and the protection rate is 36.7% for the common running water washing and vaccine-adding group. The novel flushing liquid can achieve the protection rate of 76.7 percent and is far higher than that of a common running water flushing and vaccinating group, so that the novel flushing liquid not only has the running water flushing effect, but also has a better rabies virus neutralizing effect. The flushing effect is obviously better than that of the common flushing liquid.
The results of the rinse solution test animals are shown in the following table:
Figure BDA0002630084510000061
the components contained in the product are pharmaceutical grade raw materials, no preservative or irritant component is contained, the product is safe and nontoxic, and the product has high stability and is recommended to be stored at low temperature in the shade. The washing liquid can directly replace the conventional running water wound washing in rabies treatment clinics, and can also be prepared into concentrated liquid to be matched with a washing device in the treatment clinics for dilution.
In conclusion, the experimental results of the cell level and the experimental animal level show that the novel flushing liquid has certain rabies virus neutralization activity, the protection rate can reach more than 70% under the condition that the flushing liquid is only used for treatment after the test mouse level simulation three-level exposure, and the effect is far greater than the traditional running water flushing effect. The washing liquid can directly replace the conventional running water wound washing in rabies treatment clinics, and can also be prepared into concentrated liquid to be matched with a washing device in the treatment clinics for dilution.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (7)

1. A washing solution having a neutralizing effect against rabies viruses, comprising: 0.9% physiological saline, tween-20, human anti-rabies antibody with titer not less than 100IU/ml, EDTANa2And chitosan.
2. The preparation method of the washing solution with the effect of neutralizing rabies viruses according to claim 1 is characterized by comprising the following steps: adding 10mL Tween-20 into 500mL of 0.9% physiological saline to obtain final titer of not less than 0.5IU/mLHuman anti-rabies antibody, EDTANA2And adding 0.9% physiological saline to 1000mL after chitosan is added, filtering and sterilizing, and keeping at 2-8 deg.C for later use.
3. The method for detecting the activity of the washing solution having the effect of neutralizing rabies viruses according to claim 1, wherein the method for detecting the activity comprises:
preparing a washing liquid, namely preparing an experimental material, and preparing the washing liquid according to a formula;
step two, detecting antiviral activity of flushing liquid, performing cell level activity detection by referring to a 'Rapid Fluorescence Focus Inhibition Test (RFFIT)' method for detecting rabies antibody activity in three parts of 'Chinese pharmacopoeia' 2015 edition, and testing the horizontal use effect of animals by taking SPF-level Kunming mice as test objects;
and step three, determining the use place of the washing liquid, wherein the components of the washing liquid are pharmaceutical grade raw materials, and the washing liquid does not contain any preservative or irritant component, is safe, nontoxic and high in stability, can be directly used for replacing the conventional running water wound washing in a rabies treatment clinic, and can also be prepared into a concentrated solution to be matched with a washing device in the treatment clinic for dilution.
4. The method for detecting the activity of an irrigating solution having the effect of neutralizing rabies viruses as claimed in claim 3, wherein: in the first step, the required experimental materials comprise 0.9% of normal saline, tween-20, high titer humanized anti-rabies antibody and EDTANa2And chitosan.
5. The method for detecting the activity of an irrigating solution having the effect of neutralizing rabies viruses as claimed in claim 3, wherein: in the first step, the preparation formula of the flushing liquid is as follows: adding 10mL tween-20 and humanized anti-rabies antibody into 500mL of 0.9% physiological saline, adding EDTANA2And adding 0.9% physiological saline to 1000mL after chitosan is added, filtering and sterilizing, and keeping at 2-8 deg.C for later use.
6. The method for detecting the activity of an irrigating solution having the effect of neutralizing rabies viruses as claimed in claim 3, wherein: in the second step, the activity detection at the cellular level comprises the following steps:
(1) sampling: taking a sterile 96-microporous plate for cell culture, and adding 100 mu l of DMEM medium containing 10% fetal calf serum into each hole;
(2) sample dilution: firstly, carrying out 3-time serial dilution on a standard substance and a novel flushing liquid, wherein the standard substance is diluted to 9 times, and the flushing liquid is diluted to 3 times;
(3) sample adding: adding 50 mul of the 9 times diluted standard substance into a 96-pore plate in the 2.1.1 step, namely, the 1 st pore is diluted by 27 times; taking 50 mul of the novel washing liquid diluted by 3 times to be put on a plate, namely diluting the 1 st hole by 9 times;
(4) and (3) secondary dilution: adding 50 μ l of the mixture from each well of the 1 st row to the second row by using a row gun, continuously taking 50 μ l of the mixture to the 3 rd row after mixing, and performing 3-fold serial dilution until the 12 th row;
(5) adding poison: adding 50 μ l rabies virus CVS strain with fixed titer into the above micropores, and neutralizing in a constant temperature incubator at 37 deg.C for 1 hr;
(6) standing culture: adding 50 μ l BSR cells into each well after neutralization, and culturing in a constant temperature incubator at 37 deg.C for 24 hr;
(7) and (4) detecting a result: after the culture, the culture supernatant is discarded, 80% cold acetone is added for fixation for 20 minutes, 50 mu.l of FITC-labeled anti-nucleoprotein monoclonal antibody is added for incubation for 1hr, then the supernatant is discarded, the plate is washed by PBS solution for 1 time, and then the fluorescence infection condition of each hole is observed under an inverted fluorescence microscope.
7. The method for detecting the activity of an irrigating solution having the effect of neutralizing rabies viruses as claimed in claim 3, wherein: in the second step, when the horizontal use effect test of the experimental animals is carried out, the three-level exposure condition of the bite wound is simulated, different combinations are adopted for intervention, and each group is repeated for three times.
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