EP4278182A1 - Détection de particules virales par co-précipitation à médiation immunospécifique - Google Patents
Détection de particules virales par co-précipitation à médiation immunospécifiqueInfo
- Publication number
- EP4278182A1 EP4278182A1 EP22701192.1A EP22701192A EP4278182A1 EP 4278182 A1 EP4278182 A1 EP 4278182A1 EP 22701192 A EP22701192 A EP 22701192A EP 4278182 A1 EP4278182 A1 EP 4278182A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- virus
- nanoparticles
- sample
- population
- particles
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Classifications
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- 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
Definitions
- the present invention relates to a method for rapid detection of viral particles in a sample. This method is particularly useful for rapid test of SARS-CoV-2.
- SARS-CoV severe acute respiratory syndrome coronavirus
- MERS-CoV Middle-East respiratory syndrome coronavirus
- SARS-CoV-2 SARS-CoV-2 was discovered in December 2019 in Wuhan, Hubei province of China and was sequenced and isolated by January 2020.
- SARS-CoV- 2 is associated with an ongoing outbreak of atypical pneumonia (COVID-19).
- COVID-19 atypical pneumonia
- the World Health Organization declared the SARS-CoV-2 epidemic a public health emergency of international concern.
- COVID-19 tests can be divided in two categories; PCR-based tests and immuno tests.
- PCR-based tests are designed to detect the virus RNA by amplifying the genetic material thanks to an enzymatic process.
- Immuno tests are, more often, designed to detect the patient antibodies displaying some affinity for the viral particles.
- Immuno-tests although more reliable (they can reach a reported reliability around >95%), are designed to detect the immuno response (the presence of anti-SARS-CoV-2 antibodies), not the viral particles. Thus, such tests cannot detect a COVID-19+ in its first days after the infection, since the production of antibodies by the patient takes about a week, at best. That is a huge drawback, since it has been demonstrated that the average viral load (the average number of viral particles) in saliva reaches its peak (between 10 7 to 10 8 ) before or in the first days after the apparition of the symptoms. So, the immuno tests cannot detect patients that are highly infectious until several days. There are some immuno tests designed to detect directly the viral particles. Those usually are highly reliable (about >95%) but, until now, are very expensive (Ravi et al. Biosens Bioelectron. 2020 Oct 1; 165: 112454).
- the present invention relates to very fast and low cost test protocol, which could be applied in the first days after the infection to detect the viral particles.
- This test takes only a few minutes (about 10 min) and it is almost costless in terms of reagents and analyzing equipment (about one American dollar). It can allow any laboratory to create a SARS-CoV-2 tests. All those characteristics make this test an excellent candidate to a massive program of COVID-2019 screening.
- the present invention relates to an in vitro method for detecting and/or quantifying particles of a virus in a sample, said method comprising a) incubating a sample suspected of containing virus particles with two populations of nanoparticles coated with one or several ligands that bind to said virus particles, under conditions allowing the formation of aggregates comprising at least one nanoparticle of the first population, at least one nanoparticle of the second population and at least one virus particle, wherein the nanoparticles of the first population have a settling velocity lower than the nanoparticles of the second population and are capable of directly or indirectly generating a detectable signal, b) concentrating said aggregates in order to form a precipitate, said concentration being carried out under conditions allowing settling of said aggregates while nanoparticles of the first population which are not comprised in said aggregates remain in suspension, and c) detecting and/or quantifying the signal emitted by the nanoparticles of the first population in the precipitate, thereby detecting and/or quantifying said aggregates, where
- an in vitro method for detecting and/or quantifying particles of a virus in a sample comprising a') contacting a sample suspected of containing virus particles with one or several ligands that bind to said virus particles, a”) coating said one or several ligands on two populations of nanoparticles, wherein the nanoparticles of the first population have a settling velocity lower than the nanoparticles of the second population and are capable of directly or indirectly generating a detectable signal, a'”) incubating the mix comprising the sample and the nanoparticles coated with one or several ligands under conditions allowing the formation of aggregates comprising at least one nanoparticle of the first population, at least one nanoparticle of the second population and at least one virus particle, b) concentrating said aggregates in order to form a precipitate, said concentration being carried out under conditions allowing settling of said aggregates while nanoparticles of the first population which are not comprised in said aggregates remain in suspension, and c)
- the method of the invention may further comprise after step b) and before step c) the separation of the precipitate from the supernatant, and optionally the addition of a reagent that generates a signal in the presence of the nanoparticles of the first population.
- step a) or step a' is carried out in a reaction mix without any distinct layer of different density.
- the nanoparticles may be metal nanoparticles such as gold, silver, platinum, iron, zinc, cerium or thallium nanoparticles, silica nanoparticles, polymeric nanoparticles or quantum dots, preferably metal nanoparticles, more preferably gold nanoparticles.
- the nanoparticles are spherical nanoparticles, spheroidal nanoparticles, rod-shaped particles or star-shaped nanoparticles.
- the nanoparticles may be spherical or spheroidal.
- the nanoparticles of the first population and of the second population have a different shape.
- the nanoparticles of the two populations have similar apparent mass density and the two populations have different mean particle sizes, the first population having a mean particle size smaller than the mean particle size of the second population.
- the nanoparticles of the first population may have a mean particle size of 10 nm to 80 nm, preferably 20 nm to 50 nm and/or the nanoparticles of the second population may have a mean particle size of 100 nm to 200 nm, preferably 120 nm to 180 nm.
- Said one or several ligands may be selected from antibodies and aptamers, preferably are monoclonal and/or polyclonal antibodies directed against said virus particles.
- concentration in step b) may be carried out by centrifugal settling or gravitational settling.
- the nanoparticles of the first population may be capable of directly generating a signal that can be detected colorimetrically (e.g., visually) or spectrophotometrically.
- the sample is preferably a biological fluid sample, preferably saliva, nasopharyngeal, urine, fecal, blood, plasma, milk or mucus sample, more preferably saliva, nasopharyngeal, urine, fecal, blood, plasma, milk or mucus sample, even more preferably a nasopharyngeal sample or a saliva sample.
- a biological fluid sample preferably saliva, nasopharyngeal, urine, fecal, blood, plasma, milk or mucus sample, more preferably saliva, nasopharyngeal, urine, fecal, blood, plasma, milk or mucus sample, even more preferably a nasopharyngeal sample or a saliva sample.
- the virus may be a virus pathogenic for humans, plants or animals.
- the virus may be selected from the group consisting of coronavirus, Ebola virus, hepatitis virus, in particular hepatitis A, B, and C viruses, retrovirus, in particular HIV, influenza virus, herpes virus, in particular varicella-zoster virus and pseudorabies virus, adenovirus, polyomavirus, in particular human polyomavirus, papilloma virus, in particular human papilloma virus, parvovirus, in particular human parvovirus, Mumps virus, rotavirus, in particular human rotavirus, enteroviruses, dengue virus, respiratory syncytial virus (RSV), rubella virus, classical swine fever virus, circovirus (including porcine circovirus PCV-1, PCV-2 and PCV-3), porcine reproductive and respiratory syndrome virus, flavivirus, in particular bovine viral diarrhea virus, porcine epidemic diarrhea virus, Sindbis virus, baculovirus,
- the virus may be selected from the group consisting of coronavirus, Ebola virus, hepatitis virus, HIV, influenza virus, herpes virus, adenovirus, human polyomavirus, human papilloma virus, human parvovirus, Mumps virus, human rotavirus, enterovirus, dengue virus, respiratory syncytial virus and rubella virus.
- the virus is selected from the group consisting of coronavirus, flavivirus and orthopneumovirus, in particular from the group consisting of SARS coronavirus, bovine viral diarrhea virus (BVDV) and respiratory syncytial virus (RSV).
- the virus is a coronavirus, preferably SARS-CoV-2.
- the present invention also relates to the use of the method of the invention in determining whether a subject is affected with a viral infection, wherein the sample is a biological sample from the subject, preferably a nasopharyngeal sample, a milk sample or a saliva sample, more preferably a nasopharyngeal sample or a saliva sample.
- the sample is a biological sample from the subject, preferably a nasopharyngeal sample, a milk sample or a saliva sample, more preferably a nasopharyngeal sample or a saliva sample.
- the viral infection may be a coronavirus infection, preferably SARS-CoV-2 infection, and the sample may be a nasopharyngeal sample or a saliva sample;
- the viral infection may be an orthopneumovirus infection, preferably respiratory syncytial virus infection, and the sample may be a nasopharyngeal sample or a saliva sample;
- the viral infection may be a flavivirus infection, preferably bovine viral diarrhea virus infection, and the sample may be a milk sample or a saliva sample.
- kit for detecting and/or quantifying particles of a virus in a sample comprising
- nanoparticles preferably gold nanoparticles
- the nanoparticles of the first population having a settling velocity lower than the nanoparticles of the second population and being capable of directly or indirectly generating a detectable signal
- said nanoparticles being coated with one or several ligands that bind to said virus particles, preferably one or several monoclonal and/or polyclonal antibodies directed against said virus particles, and/or
- nanoparticles preferably gold nanoparticles
- the nanoparticles of the first population having a settling velocity lower than the nanoparticles of the second population and being capable of directly or indirectly generating a detectable signal
- one or several ligands that bind to said virus particles preferably one or several monoclonal and/or polyclonal antibodies directed against said virus particles, and - optionally a reaction buffer, a negative control sample, a positive control sample, and/or one or several single-use microtubes suitable for centrifugation, and/or a leaflet providing guidelines to use the kit.
- Figure 1 Cell supernatant solutions with a viral load around 10 7 particles by ml (A and D), MOCK supernatant without virus (B and E) and PBS (C and F) incubated with the 30 nm GNP only (A, B and C) or with both GNPs, 30 nm and 150 nm, (D, E and F) conjugated with a polyclonal antibody.
- the arrow shows the red pellet.
- FIG. 2 Cell supernatant solutions with a viral load around 10 7 particles by ml (A) and MOCK supernatant without virus (B) incubated with both GNPs, 30 nm and 150 nm, conjugated with the monoclonal antibody. The arrow shows the red pellet.
- FIG. 3 Cell supernatant solutions with a viral load around 10 7 particles by ml of inactivated virus incubated with both GNPs, 30 nm and 150 nm, conjugated with the monoclonal antibody.
- the arrow shows the red pellet.
- FIG. 4 Cell supernatant solutions with a viral load around 2.10 6 viral particles by ml (E, F and G) and MOCK supernatant without virus (A, B and C) incubated with both GNPs, 30 nm and 150 nm, conjugated with the monoclonal antibody.
- FIG. 1 Cell supernatant solutions with a viral load around 10 6 and 5.10 5 particles by ml (A and C, respectively) and MOCK supernatant without virus (B and D) incubated with both GNPs, 30 nm and 150 nm, conjugated with the monoclonal antibody.
- the inventors herein provided a very fast and low cost test to detect virus particles in a sample with high reliability.
- This test can be applied in the first days after the infection to detect the viral particles without any help of sophisticated analyzing equipment.
- the principle of this test was exemplified in the experimental section to detect SARS-CoV-2.
- two sizes of gold nanoparticles, a larger and a smaller one, covalently conjugated with antibodies anti-SARS-COV-2 were mixed with an infected supernatant.
- the largest nanoparticles which were visually colorless, precipitated after a few minutes under mild centrifugation speed and have the function of precipitating the viral particles into a pellet.
- the present invention relates to an in vitro method for detecting and/or quantifying particles of a virus in a sample, said method comprising a) incubating a sample suspected of containing virus particles with two populations of nanoparticles coated with one or several ligands that bind to said virus particles, under conditions allowing the formation of aggregates comprising at least one nanoparticle of the first population, at least one nanoparticle of the second population and at least one virus particle, wherein the nanoparticles of the first population have a settling velocity lower than the nanoparticles of the second population and are capable of directly or indirectly generating a detectable signal, b) concentrating said aggregates in order to form a precipitate, said concentration being carried out under conditions allowing settling of said aggregates while nanoparticles of the first population which are not comprised in said aggregates remain in suspension, and c) detecting and/or quantifying the signal emitted by the nanoparticles of the first population in the precipitate, thereby detecting and/or quantifying
- step a) of the method may be replaced by the following steps a'), a”) and a'”): a') contacting a sample suspected of containing virus particles with one or several ligands that bind to said virus particles, a”) coating said one or several ligands on two populations of nanoparticles, wherein the nanoparticles of the first population have a settling velocity lower than the nanoparticles of the second population and are capable of directly or indirectly generating a detectable signal, and a'”) incubating the mix comprising the sample and the nanoparticles coated with one or several ligands under conditions allowing the formation of aggregates comprising at least one nanoparticle of the first population, at least one nanoparticle of the second population and at least one virus particle.
- Steps a'), a”) and a'”) can be performed simultaneously or sequentially.
- Viral particles detected and/or quantified by the method of the invention may be particles of any virus, in particular any virus that is pathogenic for humans, plants or animals, preferably for humans or animals, more preferably for humans.
- viruses include, but are not limited to, coronavirus, ebola virus, hepatitis virus, in particular hepatitis A, B, and C viruses, retrovirus, in particular HIV, influenza virus, herpes virus, in particular varicella-zoster virus and pseudorabies virus, adenovirus, polyomavirus, in particular human polyomavirus, papilloma virus, in particular human papilloma virus, parvovirus, in particular human parvovirus, Mumps virus, rotavirus, in particular human rotavirus, enteroviruses, dengue virus, respiratory syncytial virus (RSV), rubella virus, classical swine fever virus, circovirus (including porcine circovirus PCV-1, PCV-2 and PCV-3), porcine reproductive and respiratory syndrome virus, flavivirus, in particular bovine viral diarrhea virus, porcine epidemic diarrhea virus, Sindbis virus, baculovirus, cytomegalovirus, vesicular stomatitis
- the particles to be detected and/or quantified may belong to a virus selected from the group consisting of coronavirus, ebola virus, hepatitis virus, HIV, influenza virus, herpes virus, adenovirus, human polyomavirus, human papilloma virus, human parvovirus, Mumps virus, human rotavirus, enteroviruses, dengue virus, respiratory syncytial virus (RSV) and rubella virus.
- a virus selected from the group consisting of coronavirus, ebola virus, hepatitis virus, HIV, influenza virus, herpes virus, adenovirus, human polyomavirus, human papilloma virus, human parvovirus, Mumps virus, human rotavirus, enteroviruses, dengue virus, respiratory syncytial virus (RSV) and rubella virus.
- the virus may be of any shape and any size.
- the virus may be enveloped or non-enveloped, may have helical, icosahedral, prolate or complex structure.
- viral particles to be detected and/or quantified have an average size ranging from 10 nm to 400 nm, preferably from 18 nm to 400 nm, more preferably from 20 nm to 300 nm, even more preferably an average size ranging from 50 nm to 200 nm.
- the virus is selected from the group consisting of coronavirus, flavivirus and orthopneumovirus, in particular from the group consisting of SARS coronavirus, bovine viral diarrhea virus (BVDV) and respiratory syncytial virus (RSV).
- coronavirus flavivirus and orthopneumovirus
- SARS coronavirus bovine viral diarrhea virus (BVDV)
- BVDV bovine viral diarrhea virus
- RSV respiratory syncytial virus
- the virus is a coronavirus, preferably a SARS coronavirus. In a preferred embodiment, the virus is SARS-CoV-2.
- the virus is an orthopneumovirus, preferably respiratory syncytial virus (RSV).
- RSV respiratory syncytial virus
- the virus is a flavivirus, preferably bovine viral diarrhea virus (BVDV).
- BVDV bovine viral diarrhea virus
- the sample to be tested may be any sample suspected of containing virus particles.
- the sample may be a biological sample or a sample of any material that may have been contaminated by the virus such as food or water.
- the sample is a biological sample, more preferably a biological sample from a subject.
- the sample may a biological fluid sample or a tissue sample.
- the sample is a biological fluid sample, preferably selected from saliva, nasopharyngeal, urine, seminal fluid, spinal fluid, blood, serum, plasma, fecal sample, milk or mucus sample, more preferably selected from saliva, nasopharyngeal, urine, seminal fluid, spinal fluid, blood, serum, plasma, fecal sample or mucus sample, even more preferably a nasopharyngeal sample or a saliva sample.
- viral particles detected and/or quantified by the method of the invention are particles of a coronavirus, preferably SARS-CoV-2, and the sample is a nasopharyngeal sample or a saliva sample.
- a coronavirus preferably SARS-CoV-2
- the sample is a nasopharyngeal sample or a saliva sample.
- viral particles detected and/or quantified by the method of the invention are particles of an orthopneumovirus, preferably respiratory syncytial virus (RSV), and the sample is a nasopharyngeal sample or a saliva sample.
- RSV respiratory syncytial virus
- viral particles detected and/or quantified by the method of the invention are particles of a flavivirus, preferably bovine viral diarrhea virus (BVDV), and the sample is a milk sample or a saliva sample.
- BVDV bovine viral diarrhea virus
- the sample may be directly used in the method of the invention or may be treated prior to its use. Treatments applied on the sample should be chosen in order to not alter the recognition of the virus particles by the ligands. Such treatments can be easily chosen by the skilled person.
- the sample may be diluted, concentrated, frozen or lyophilized, and/or submitted to solid/liquid separation.
- the cells contained in the sample may be submitted to a cell lysis and optionally, cell debris can be eliminated from the cell lysate.
- the sample may also be treated in order to inactivate virus particles, for example using known chemical treatments (e.g. with p-propiolactone or paraformaldehyde) or physical treatments (e.g. with UV-irradiation).
- the method of the invention may further comprise, before step a), the step of providing a sample from a subject.
- the term "subject" or “patient” refers to an animal, preferably to a mammal. In preferred embodiments, this term refers to a human, including adult, child and human at the prenatal stage. However, this term can also refer to non-human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheeps and non-human primates or non-mammal animals such as fishes and birds, preferably mammal animals.
- the sample may be obtained from a normal/healthy subject, for example for routine screening or testing, or from a subject at risk for or suspected of being infected with the virus to be detected/quantified.
- the subject may also be a subject who have been diagnosed with the viral disease.
- the method of the invention may be used to follow the infection.
- the subject may be asymptomatic (showing no symptoms of the viral infection) or symptomatic (showing symptoms of the viral infection).
- the sample is incubated with two populations of nanoparticles.
- the sample is incubated with more than two populations of nanoparticles, e.g. 3 or 4 populations, are also contemplated.
- the sample is incubated with only two populations of nanoparticles
- nanoparticles of each population having a different settling velocity.
- Sedimentation is the tendency for particles in suspension to settle out and come to rest. Numerous forces can act on a particle to promote settling including gravity, centrifugal acceleration, and the like.
- settling is the falling of suspended particles through liquid and the "settling velocity” or “settling rate” at which suspended particles settle depends on various parameters including the size, the shape and the density of particles as well as the viscosity and density of the medium.
- the settling velocity of nanoparticles can be predicted/measured using any known methods such as methods described by Stokes G. G. in the article “On the effect of internal friction of fluids on the motion of pendulums” (Transactions of the Cambridge Philosophical Society.
- A( ) is the difference between the mass densities of the particle (p p ) and fluid (pf) (kg-m 3 ), and p is the dynamic viscosity of the fluid (Pa s).
- an aspect ratio, AR equal to the long dimension ri/short dimension r2 ratio
- the r value in the above stokes law can be replaced by the Stokes equivalent radius r' (B. R. Jennings and K. Parslow, Particle Size Measurement: The Equivalent Spherical Diameter, Proc. Royal Soc. London. Series A, Mathematical and Physical Sciences, 419, No. 1856. 1988, 137-149):
- the settling rates of the nanoparticles of the first and second populations have to be distinct and defined in order to provide conditions allowing settling of aggregates (comprising at least one nanoparticle of the first population, at least one nanoparticle of the second population and at least one virus particle) while nanoparticles of the first population that are not comprised in said aggregates remain in suspension.
- the settling rate of the nanoparticles of the first population in water is between 1.10 1 m-s 1 and 1.10 -4 m-s 1 at an acceleration of 2500 g and the settling rate of the nanoparticles of the second population in water is between 5.10 -5 m-s 1 and 5. IO -8 m-s 1 at an acceleration of 2500 g.
- the settling rate of nanoparticles mainly depends on their size and density. These two parameters can be adjusted in order to provide a clear gap between the settling rate of nanoparticles of the first population and the settling rate of nanoparticles of the second population.
- the two populations of nanoparticles may have similar or different mean particle sizes and may have similar or different densities.
- similar values refers to substantially the same value. Two values are similar if they do not differ by more than 5%, preferably by more than 2%.
- the term "mean particle size” refers to the statistical mean particle size of a nanoparticle population, prior to any coating with one or several ligands that bind to the virus particles.
- the size of nanoparticles may be assessed using different methods and expressed in different ways. In particular, the size of a nanoparticle may be assessed by measuring the largest dimension of the particle and/or by measuring the hydrodynamic diameter of said nanoparticle.
- the largest dimension of a particle is the largest linear distance between two points on the surface of said particle (e.g., the diameter of a spherical particle, the length of a rod shaped particle, the diagonal of a cube shape particle, the bisector of a triangular shaped particle, etc.).
- the largest dimension of nanoparticles can be measured by electron microscopy such as Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), or cryo-TEM. Electron microscopy measures the projected images of particles deposited onto an electron- transparent substrate. The recording of more than about 50, preferably more than about 100, 150 or 200 nanoparticles per sample should typically be measured for size assessment.
- the hydrodynamic diameter of a nanoparticle is the diameter of an equivalent hard sphere that diffuses at the same rate as the nanoparticle.
- Dynamic light scattering can be used to measure the hydrodynamic diameter of nanoparticles.
- a typical assay protocol may be found in "NIST - NCL Joint Assay Protocol, PCC-I; Measuring the size of nanoparticles in aqueous media using batch-mode dynamic light scattering; version 1.2, revised May 2015. Cumulants analysis yields a mean intensity-weighted size commonly called the mean hydrodynamic diameter (or z-average diameter).
- nanoparticle refers to a particle having a largest dimension or a hydrodynamic diameter of less than 1000 nm, preferably of less than 500 nm, and even more preferably of less than 300 nm.
- mean particle size of a population of nanoparticles refers to the mean largest dimension of the nanoparticles and/or the mean hydrodynamic diameter.
- the mean largest dimension of the nanoparticles may differ from the mean hydrodynamic diameter, in particular when the nanoparticles are not spherical or spheroidal.
- the particles are spherical or spheroidal and the mean particle size may be the mean largest dimension or the mean hydrodynamic diameter. In some other embodiments, the particles are not spherical or spheroidal and the mean particle size is the mean hydrodynamic diameter.
- the populations of nanoparticles can also be defined with their polydispersity indexes.
- polydispersity index is used herein as a measure of the size distribution of an ensemble of particles, e.g., nanoparticles.
- the polydispersity index can be calculated by any method known by the skilled person such as dynamic light scattering.
- each population of nanoparticles is homogeneous in respect of particle size.
- each population of nanoparticles has a low average polydispersity index ("PDI").
- each population has a PDI of less than 0.5, more preferably less than 0.4, more preferably less than 0.3, yet more preferably less than 0.25, and most preferably less than 0.2.
- the term "density” refers to the apparent mass density, i.e. the mass of a particle divided by its apparent volume (i.e., the volume including interior void space).
- the apparent mass density may differ from the true density which refers to the density of a given material, excluding any interior void volume in the material, determined at a given pressure and temperature (e.g., one atmosphere pressure and a temperature of 25°C).
- the density of nanoparticles can be predicted/measured using any method known by the skilled person such as differential centrifugal sedimentation (Minelli et al., Anal. Methods, 2018, 10, 1725-1732). The density of a nanoparticle greatly depends on its material.
- the densities of gold, silver, copper, zinc, platinum and polymer are 19.3 g/cm 3 , 10.49 g/cm 3 , 8.92 g/cm 3 , 7.14 g/cm 3 , 21.45 g/cm 3 , and about 1.1 g/cm 3 respectively.
- nanoparticles of the second population isolated or in the form of an aggregate as defined below, have to settle in a condition wherein isolated nanoparticles of the first population remain in suspension.
- the two populations of nanoparticles have similar mean particle sizes and different apparent mass densities, the nanoparticles of the first population having an apparent mass density lower than the apparent mass density of the nanoparticles of the second population.
- the two populations of nanoparticles have different mean particle sizes and different apparent mass densities.
- the nanoparticles of the first population have an apparent mass density lower than the apparent mass density of the nanoparticles of the second population and/or the first population has a mean particle size smaller than the mean particle size of the second population. More preferably, the nanoparticles of the first population have an apparent mass density lower than the apparent mass density of the nanoparticles of the second population and the first population has a mean particle size smaller than the mean particle size of the second population.
- the two populations of nanoparticles used in the method of the invention have different mean particle sizes and the nanoparticles of the two populations have similar apparent mass densities.
- the apparent mass densities of the two population differ by at least 20%, preferably by at least 50%.
- the nanoparticles of the smaller mean particle size may have a mean particle size between 10 nm to 80 nm, preferably between 20 nm to 50 nm and/or the nanoparticles of the larger mean particle size may have a mean particle size between 100 nm to 200 nm, preferably 120 nm to 180 nm.
- the nanoparticles of the smaller mean particle size have a mean particle size between 10 nm to 80 nm, preferably between 20 nm to 50 nm and the nanoparticles of the larger mean particle size have a mean particle size between 100 nm to 200 nm, preferably 120 nm to 180 nm.
- the nanoparticles used in the method of the invention may have various shapes.
- the shape of the nanoparticles is typically evaluated using electron microscopy such as transmission electron microscopy (TEM).
- TEM transmission electron microscopy
- the nanoparticles may be selected amongst isotropic and anisotropic shaped nanoparticles. They may be symmetrical or unsymmetrical.
- the nanoparticles may be spherical (shaped like a sphere) or spheroidal (roughly spherical). Alternatively, they may be not spherical, such as rod-shaped particles having any aspect ratio (e.g. nanorods and nanowires), star-shaped nanoparticles, cube-shaped nanoparticles, platelet-shaped nanoparticles or particles having any other geometry such as nanoparticles with bipyramidal shapes.
- the nanoparticles are spherical particles, spheroidal particles, rod-shaped particles or star-shaped nanoparticles. More preferably, the nanoparticles are rod-shaped particles or star-shaped nanoparticles.
- the nanoparticles of the first population and of the second population may have different or identical shape.
- the nanoparticles of the two populations have the same shape.
- at least one of the populations of nanoparticles used in the method of the invention have a spherical or spheroidal shape.
- the nanoparticles of the two populations are spherical or spheroidal.
- Nanoparticles used in the present invention may be of any suitable material, in particular any material allowing coating with one or several ligands that bind to the virus particles.
- the nanoparticles may be metal nanoparticles (e.g. nanoparticles of gold, silver, platinum, iron, zinc, cerium, gadolinium or thallium, made of pure metal or their compounds e.g., oxides, hydroxides, sulfides, phosphates, fluorides, or chlorides), silica nanoparticles, polymeric nanoparticles or quantum dots (e.g.
- the nanoparticles are metal nanoparticles or quantum dots. More preferably, the nanoparticles are metal nanoparticles.
- the nanoparticles may also comprise a surface coating affecting their physicochemical behavior such as solubility. In particular, the surface of the nanoparticles may be coated with polymers or inorganic layers such as silicon dioxide.
- the nanoparticles of the first population and the nanoparticles of the second population may be of identical or different material.
- the nanoparticles of the two populations are of identical material.
- at least one of the populations of nanoparticles used in the method of the invention are gold nanoparticles.
- the nanoparticles of the two populations are gold nanoparticles.
- Nanoparticles used in the invention are coated with one or several ligands that bind to the virus particles.
- ligands coated on nanoparticles specifically bind to the virus particles of interest, i.e. the virus particles to be detected and/or quantified.
- the term "specifically binding” is used herein to indicate that the ligands have the capacity to recognize and interact specifically with the virus particles of interest, while having relatively little detectable reactivity with other structures present in the aqueous phase.
- the degree of affinity is much greater than such non-specific binding interactions.
- the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd).
- Kd representing the affinity of a ligand as used herein and the virus particles of interest is from 1.10 7 M or lower, preferably from 1. 10“ s M or lower, and even more preferably from 1. 10“ 9 M or lower.
- the Kd representing the affinity of a ligand as used herein and the virus particles of interest is between 1.10“ 7 M and 1.10 15 M, preferably between 1.10" 8 M and 1.10 15 M, and more preferably between 1.10 9 M and 1.10 15 M.
- ligands as used herein recognize and bind to an external component of the viral particle.
- a target on the viral particles to be recognized by the ligands can be easily chosen by the skilled person, depending on the accessibility of said target and the availability of ligands directed against said target.
- the virus of interest is a nonenveloped virus
- ligands are preferably directed against one or more capsid proteins.
- the virus of interest is an enveloped virus
- ligands are preferably directed against one or more proteins or glycoproteins of the viral envelope.
- the virus to be detected/quantified is a coronavirus and the ligands are directed against spike (S), envelope (E) and/or membrane (M) proteins.
- the ligands are directed against the S protein, more preferably against the SI subunit, the S2 subunit and/or the receptor binding domain (RBD) of the S protein of said coronavirus.
- ligands coated on the nanoparticles may be selected from any peptide, oligonucleotide and/or oligosaccharide ligands that specifically bind to the virus particles of interest.
- peptide oligopeptide
- polypeptide polypeptide
- protein protein
- peptide may comprise residues from any of the naturally occurring amino acids and/or from any non-naturally occurring amino acids.
- oligonucleotide and “polynucleotide” are employed interchangeably and refer to a polymer of nucleotides bonded to one another by phosphodiester bonds, regardless of the number of nucleotides forming said polymer.
- the oligonucleotide may comprise naturally occurring nucleotides and/or non-naturally occurring nucleotides.
- oligosaccharide and “polysaccharide” are employed interchangeably and refer to any linear or branched polymer consisting of monosaccharide residues connected by glycosidic linkages, regardless of the number of residues forming said polymer.
- the oligonucleotide may comprise naturally occurring monosaccharide residues and/or non-naturally occurring monosaccharide residues.
- ligands coated on the nanoparticles may be selected from the group consisting of antibodies and aptamers that specifically bind to the virus particles of interest. More preferably, ligands coated on the nanoparticles may be selected from the group consisting of antibodies that specifically bind to the virus particles of interest.
- ligands coated on the nanoparticles may be selected from the group consisting of antibodies that specifically bind to the virus particles of interest.
- Aptamers are oligonucleotide or peptide molecules that are selected based on specific binding properties to a particular molecule.
- Aptamer ligands may be DNA, RNA, L-RNA or XNA aptamers or peptide aptamers such as affimers and X-aptamers.
- Examples of aptamers that specifically bind to virus particles include, but are not limited to, DNA aptamer selected against COVID-19 Spike Protein (Cambio Cat.# CFA0688); DNA aptamer selected against the Zika envelope protein (Cambio Cat.# ATW0104), and RNA aptamers selected against human influenza B virus hemagglutinin (Gopinath et al., J. Biochem. 2006, 139, 837-846).
- antibody refers to an antibody such as IgG, IgM, IgD and IgA antibodies, or a fragment or derivative thereof such as Fab, Fab', F(ab)2, F(ab')2, F(ab)a, Fv, single-chain Fv (ScFv), bi-specific antibodies, diabodies or VHH, and other fragments capable of binding to target molecule.
- the definition includes polyclonal antibodies and monoclonal antibodies.
- antibodies used as ligands are IgG monoclonal or polyclonal antibodies.
- antibodies that specifically bind to virus particles include, but are not limited to, anti-SARS-CoV-2 spike RBD antibody (Acrobiosystems; Cat.# SAD-S35) and SARS-CoV-2 (COVID-19) spike antibody (GeneTex; Cat.# GTX135356).
- the virus to be detected/quantified is a coronavirus and the ligands are antibodies directed against spike (S), envelope (E) and/or membrane (M) proteins of said virus, preferably against the S protein of said virus, and more preferably against the SI subunit, the S2 subunit and/or the receptor binding domain (RBD) of the S protein of said virus.
- S spike
- E envelope
- M membrane
- the virus to be detected/quantified is a flavivirus, in particular bovine viral diarrhea virus (BVDV) and the ligands are antibodies directed against BVDV surface proteins.
- BVDV bovine viral diarrhea virus
- the virus to be detected/quantified is an orthopneumovirus, in particular respiratory syncytial virus (RSV) and the ligands are antibodies directed against RSV surface proteins.
- RSV respiratory syncytial virus
- Each nanoparticle may be coated with only one ligand (i.e. one or several molecules of the same ligand, e.g. a monoclonal or polyclonal antibody), or with several ligands (i.e. one or several molecules of each ligand, e.g. a monoclonal antibody and a polyclonal antibody or two monoclonal antibodies).
- each nanoparticle is coated with only one ligand.
- Nanoparticles of the same population may be coated with identical ligand(s) or with different ligands.
- nanoparticles of the same population are coated with identical ligand(s), more preferably with only one ligand.
- the nanoparticles of the two populations may be coated with identical or different ligands.
- the nanoparticles of the first population and the nanoparticles of the second population are coated with different ligands (e.g. a monoclonal antibody coated on a nanoparticle population and a polyclonal antibody coated on the other).
- each nanoparticle is coated with only one ligand (i.e. one or several molecules of the same ligand), nanoparticles of the same population are coated with the same ligand, and the two populations of nanoparticles are coated with different ligands.
- the method used to coat the nanoparticles depends on the nature of the ligands and the nature of the nanoparticles and may be easily chosen by the skilled person.
- Ligands may be attached to the nanoparticles through intermolecular attractions between the nanoparticles and ligands such as covalent bonding, chemisorption, and noncovalent interactions.
- the ligands are covalently attached (conjugated) to the nanoparticles.
- the methods for conjugation of ligands on nanoparticles are well known by the skilled person. In particular, numerous physicochemical methods have been described to couple and functionalize several types of nanoparticles with antibodies (see e.g. Oliveira et al.
- step a) of the method of the invention the sample suspected of containing virus particles is incubated with coated nanoparticles as described above, under conditions allowing the formation of aggregates.
- the incubation is carried out in a suitable liquid medium allowing the formation of said aggregates.
- said suitable medium is an aqueous phase compatible with physiological conditions, more preferably a buffer solution such as Phosphate Buffered Saline (PBS) or Tris Buffered Saline (TBS), or a solution of NaCI that mimics physiological conditions (e.g. 150 mM).
- PBS Phosphate Buffered Saline
- TBS Tris Buffered Saline
- NaCI that mimics physiological conditions
- the medium may further comprise additional components such as proteins (e.g. BSA) or preservatives provided that these components do not interfere with the binding of ligands and virus particles.
- the sample is mixed with said suitable liquid medium containing coated nanoparticles in order to form a reaction mix without any distinct layer of different density.
- the time of incubation may be easily chosen by the skilled person and is typically between 10 seconds and 4 hours, preferably between 10 seconds and 1 hour .
- the sample may be incubated with coated nanoparticles for a longer period but without any further advantage.
- the sample is incubated with coated nanoparticles for 1 min to 30 min, more preferably for 2 min to 15 min, and even more preferably for 2 min to 10 min.
- Aggregates are formed as a result of the interactions between virus particles and ligands coated on the nanoparticles.
- Possible aggregates include (i) aggregates comprising at least one nanoparticle of the first population, at least one nanoparticle of the second population and at least one virus particle (tripartite aggregates), (ii) aggregates comprising at least one nanoparticle of the first population and at least one virus particle, and (iii) aggregates comprising at least one nanoparticle of the second population and at least one virus particle.
- aggregates comprising at least one nanoparticle of the first population, at least one nanoparticle of the second population and at least one virus particle are the only form that is detected by the method of the invention.
- aggregates comprising only nanoparticle(s) of the first population and virus pa rticle(s) are not large enough and/or dense enough to alter the solubility of the complex and thus do not settle out under gravity to form a precipitate.
- aggregates comprising only nanoparticle(s) of the second population and virus pa rticle(s) do not emit any signal that can be detected by the method of the invention.
- tripartite aggregates are the only ones of interest in the present invention.
- the concentration of nanoparticles may be also easily chosen by the skilled person.
- the concentration of nanoparticles in the incubation medium is between 1.10 5 nanoparticles per mL and 1.10 13 nanoparticles per mL, preferably between 1.10 8 nanoparticles per mL and 1.10 10 nanoparticles per mL, more preferably between 5.10 9 nanoparticles per mL and 5.10 8 nanoparticles per mL.
- the ratio of nanoparticles of the first population to nanoparticles of the second population may be easily chosen by the skilled person and is typically between 0.02 and 50, preferably between 0.2 and 25, more preferably between 1 and 20.
- step a) of the method of the invention may be replaced by steps a'), a”) and a'”) as defined above. Steps a'), a”) and a'”) can be performed simultaneously or sequentially.
- step a') the sample suspected of containing virus particles is contacted with one or several ligands that bind to said virus particles.
- the ligands are as defined above. They are not coated on nanoparticles and free to interact with the virus particles.
- the ligands are coated on the two populations of nanoparticles as defined above.
- the ligands are conjugated on the nanoparticles. Methods suitable to coat the nanoparticles with the ligands are defined above.
- Step a') can be performed before step a”).
- Steps a') and a”) can be conducted in the same vessel or in distinct vessels.
- additional step(s) such as washing step in order to eliminate unbound ligands can be performed before step a”).
- these two steps, a') and a”) can be performed simultaneously.
- the sample is mixed with nanoparticles and free ligands.
- Ligands are coated on nanoparticles while interacting with the virus particles, if present in the sample.
- step a' the mix comprising the sample and the nanoparticles coated with one or several ligands is incubated under conditions allowing the formation of aggregates comprising at least one nanoparticle of the first population, at least one nanoparticle of the second population and at least one virus particle. Conditions of this incubation are as described above.
- the mix comprising the sample and the coated nanoparticles is mixed with the suitable liquid medium in order to form a reaction mix without any distinct layer of different density.
- Step a' can be performed after step a”).
- Steps a'”) and a”) can be conducted in the same vessel or in distinct vessels.
- additional step(s) such as washing step in order to change the liquid medium can be performed after step a”) and before step a'”).
- steps a'), a”) and a'”) are performed simultaneously.
- step b) of the method of the invention aggregates formed in step a) and comprising at least one nanoparticle of the first population, at least one nanoparticle of the second population and at least one virus particle, are concentrated in order to form a precipitate.
- This concentration step is conducted under conditions allowing settling of said aggregates while nanoparticles of the smaller first population not comprised in said aggregates (i.e. isolated nanoparticles or nanoparticles of aggregates containing only nanoparticles of the first population and virus particles), remain in suspension.
- nanoparticles of the smaller first population not comprised in said aggregates i.e. isolated nanoparticles or nanoparticles of aggregates containing only nanoparticles of the first population and virus particles
- some nanoparticles of the first population i.e. isolated nanoparticles or nanoparticles of aggregates containing only nanoparticles of the first population and virus particles
- aggregates comprising only nanoparticles of the second population and virus particles are not detected by the method of the invention, they can be also settled along with tripartite aggregates without interfering with the result of the analysis.
- Concentration of the tripartite aggregates may be carried out by gravitational settling (i.e. settling based on gravitational acceleration) or by centrifugal settling (settling based on centrifugal acceleration). Settling conditions can be easily chosen and adjusted by the skilled person depending on the settling velocities of the two populations of nanoparticles.
- the incubation medium is preferably allowed to settle during at least 10 min, at least 1 hour, more preferably at least 2 hours.
- the incubation medium is allowed to settle during less than 12 hours, preferably during less than 8 hours.
- concentration step is carried out by centrifugation, preferably mild centrifugation, in order to speed up the settling process.
- the term "mild centrifugation” refers to a centrifugal speed of 300 g to 5000g, preferably 1000 g to 3000 g and more preferably 1500 g to 2500 g.
- the time of centrifugation may be easily adapted by the skilled person depending on the centrifugal speed.
- the incubation medium is centrifuged for 30 sec to 10 min, preferably for 1 min to 10 min and more preferably for 4 min to 6 min.
- concentration step is carried out by centrifugation for 4 min to 6 min at a speed of 1500 g to 2500 g.
- the method may further comprise after step b) and before step c) the separation of the precipitate from the supernatant, preferably by total or partial elimination of the supernatant after centrifugation.
- Tripartite aggregates formed in step a) and settled in step b) are then detected and/or quantified in step c) of the method of the invention.
- This detection/quantification is based on the signal emitted by the nanoparticles of the first population in the precipitate.
- the signal is not detected using dynamic light scattering (DLS).
- DLS dynamic light scattering
- This signal is specific of nanoparticles of the first population which are part of aggregates and are found in the precipitate.
- other components of this precipitate e.g. nanoparticles of the second population, virus particles or other biological components that can be present in the sample, do not interfere with this signal.
- the signal emitted by said nanoparticles of the first population may be directly or indirectly generated by said nanoparticles.
- the nanoparticles of the first population are capable of directly generating a signal that can be detected colorimetrically (e.g., visually) or spectrophotometrically.
- Nanoparticles have optical properties that are sensitive to size, shape, concentration and agglomeration state and that can be used as a signal.
- metal nanoparticles such as gold and silver nanoparticles, strongly interact with specific wavelengths of light.
- the surface plasmon resonance phenomenon causes an absorption of light in the blue-green portion of the spectrum ( ⁇ 450 nm) while red light ( ⁇ 700 nm) is reflected, yielding a rich red color.
- the wavelength of surface plasmon resonance related absorption shifts to longer, redder wavelengths. Red light is then absorbed, and blue light is reflected, yielding solutions with a pale blue or purple color.
- surface plasmon resonance wavelengths move into the infra-red portion of the spectrum and most visible wavelengths are reflected, giving the nanoparticles clear or translucent color.
- the surface plasmon resonance can thus be tuned by varying the size or shape of the nanoparticles, leading to particles with tailored optical properties.
- the surface plasmon resonance of the two populations of nanoparticles has to be tuned in order to clearly distinct the optical properties of each population.
- nanoparticles of the first population are colored nanoparticles, i.e. reflect light in the visible portion of the spectrum, such asgold nanoparticles having a mean particle size of 10 nm to 80 nm.
- Nanoparticles of the second population are preferably chosen in order to have a translucent color, i.e. to reflect light in the infra-red portion of the spectrum, such as gold nanoparticles having a mean particle size of 100 nm to 200 nm.
- nanoparticles of the first population are quantum dots, i.e. nanoparticles that emit light under UV irradiation. Different sized quantum dots emit different colors of light due to quantum confinement. Thus, in this embodiment, nanoparticles of the second population may be unable to emit light under UV irradiation or may be also quantum dots emitted longer wavelengths.
- the nanoparticles of the first population are capable of indirectly generating a signal.
- the signal to be detected is not directly emitted by nanoparticles but by another reagent capable of specific interaction with the nanoparticles of the first population.
- the reagent may be a labeled secondary antibody directed against said nanoparticles, preferably directed against a molecule coated on the nanoparticles, more preferably against a ligand, e.g. an antibody, that bind to the virus particles.
- the secondary antibody may be labelled using any method known by the skilled person.
- the labelling agent may be selected from the group consisting of a fluorescent compound, such as fluorescein or fluorescein derivatives or phycoerythrin, a fluorescent particle such as quantum dot, an enzyme such as horseradish peroxidase or alkaline phosphatase, a chromophore and a radioactive molecule.
- This reagent may be already present in the medium in steps a) and b). Preferably, this reagent is added after step b), more preferably after separation of the precipitate from the supernatant.
- the method may further comprise an additional step of washing in order to eliminate unbound secondary antibodies.
- the signal to be detected and/or quantified can be detected with the naked eye, optionally using magnifying glass, or using any suitable equipment such as a spectrophotometer, depending on the nature of said signal (color, fluorescence, etc.). Detection of a signal reveals the presence of nanoparticles of the first population in the precipitate, i.e. the presence of aggregates and thus the presence of virus particles in the sample.
- Quantification of the signal may be obtained using any method known by the skilled person, in particular by comparing the intensity of the signal with a standard of known concentrations of virus particles, e.g. using a colorimetric chart.
- the present invention also relates to the use of the method of the invention, in determining whether a subject is affected with a viral infection.
- the method is carried out on a sampled obtained from the subject.
- the sample is a biological fluid sample, preferably selected from saliva, nasopharyngeal, urine, seminal fluid, spinal fluid, blood, serum, plasma, milk or mucus sample, more preferably selected from saliva, nasopharyngeal, urine, seminal fluid, spinal fluid, blood, serum, plasma or mucus sample, even more preferably a nasopharyngeal sample or a saliva sample.
- the subject may be a normal/healthy subject, for example for routine screening or testing, or may be a subject at risk for or suspected of being affected with the viral infection.
- the subject may also be a subject who have been diagnosed with the viral disease.
- the method of the invention may be used to follow the infection, in particular to determine if the subject is still affected with said infection or not.
- the subject may be asymptomatic (showing no symptoms of the viral infection) or symptomatic (showing symptoms of the viral infection).
- the viral infection is an infection with a coronavirus, preferably SARS-CoV-2, and the sample is a nasopharyngeal sample or a saliva sample.
- a coronavirus preferably SARS-CoV-2
- the sample is a nasopharyngeal sample or a saliva sample.
- the viral infection is an infection with an orthopneumovirus, preferably respiratory syncytial virus (RSV), and the sample is a nasopharyngeal sample or a saliva sample.
- an orthopneumovirus preferably respiratory syncytial virus (RSV)
- RSV respiratory syncytial virus
- the viral infection is an infection with a flavivirus, preferably bovine viral diarrhea virus (BVDV), and the sample is a milk sample or a saliva sample.
- BVDV bovine viral diarrhea virus
- the present invention relates to a kit comprising
- nanoparticles as defined above, preferably gold nanoparticles, the first population of nanoparticles having a settling velocity lower than the second population and the nanoparticles of the first population being capable of directly or indirectly generating a detectable signal, said nanoparticles being coated with one or several ligands that bind to virus particles of interest, and/or - two populations of nanoparticles as defined above, preferably gold nanoparticles, the first population of nanoparticles having a settling velocity lower than the second population and the nanoparticles of the first population being capable of directly or indirectly generating a detectable signal, and one or several ligands that bind to virus particles of interest.
- kit of the invention for detecting and/or quantifying particles of a virus in a sample according to the method of invention.
- the kit may further comprise a reaction buffer, a negative control sample, a positive control sample, one or several single-use microtubes suitable for centrifugation, colorimetric chart and/or a leaflet providing guidelines to use the kit.
- the nanoparticles are gold nanoparticles, more preferably a first population of gold nanoparticles having a mean particle size of 10 nm to 80 nm and a second population of gold nanoparticles having a mean particle size of 100 nm to 200 nm.
- the ligands are one or several monoclonal and/or polyclonal antibodies directed against said virus particles.
- the reaction buffer is preferably a suitable liquid medium allowing the formation of aggregates comprising nanoparticles and virus particles and as defined above.
- Said reaction buffer is preferable selection from the group consisting of Phosphate Buffered Saline (PBS) or Tris Buffered Saline (TBS).
- PBS Phosphate Buffered Saline
- TBS Tris Buffered Saline
- the reaction buffer may further comprise additional components such as proteins (e.g. BSA) or preservatives.
- the negative control sample may be a non-infected cell culture supernatant, i.e. a supernatant obtained from the culture of cells that are not infected by the virus of interest.
- the positive control sample may be an infected cell culture supernatant, i.e. a supernatant obtained from the culture of cells that are infected by the virus of interest.
- Vero E6 cells A clinical isolate of SARS-CoV-2 was isolated from a respiratory sample from a patient. The virus was isolated and cultured on Vero E6 cells. Vero E6 cells (ATCC) were grown in MEM (Gibco) supplemented with 10% of heat-inactivated FCS (ATCC), 1% of L-Glutamin and 1% of penicillin/streptomycin (Gibco). Briefly, confluent Vero E6 cells were infected by inoculation with 10 pL of virus (10 6 TCID50/mL) in T25 flasks. Once complete cytopathic effect (CPE) was observed, supernatants were collected, clarified by centrifugation at 3000g for 5min and divided into aliquots and stored at -80°C as viral stocks.
- CPE cytopathic effect
- Viral titers were determined in Vero E6 cell monolayers on 96-well plates using a 50% tissue culture infectious dose assay (TCID50) and expressed as loglO TCID50/mL. Serial dilutions of virus samples were incubated at 37°C for 4 days and subsequently examined for cytopathic effect (CPE) in infected cells. SARS-CoV-induced CPE of infected cells was determined by observing rounded, detached cells in close association to each other.
- TCID50 tissue culture infectious dose assay
- CPE cytopathic effect
- Vero E6 cells were inoculated with 10 pl of SARS-CoV 2 (10 6 TCID50/mL) or non-infected supernatant (Mock) in a final volume of 6mL of MEM diluted in PBS at a 1:3 ratio in T25 flasks. Cells were incubated at 37 °C for 3 days. Once complete CPE was observed in infected cultures, supernatants were collected. Mock cultures were collected after three freeze-thaw cycles in order to maintain the same amount of cellular waste products in the supernatant.
- the dry weight of the MOCK supernatant after centrifugation is about 1.5% w/w which is equivalent with the protein content in human body fluids (Shaila et al. J. Indian Soc. Periodontol. 2013, 17 (1), 42-46).
- Viral titer was determined in Vero E6 cell as described previously and estimated at 3.10 7 TCID50/mL. Viral stocks were stored at 4°C for short term use. Inactivated virus
- Virus supernatants were UV inactivated for 30 min at 15cm distance. UV-irradiated supernatants were titrated and stored at 4°C. Inactivation was confirmed when viral titer were below the limit of detection (10 1 - 5 TCID50/mL)
- the suspension was incubated at Room Temperature for 45 minutes, allowing DTSSP to chemisorb onto GNP to form a thiolate monolayer through cleavage of the disulfide bond yielding a terminal succinimidyl ester.
- the suspension was then centrifugated at 9000 rpm for 7 minutes.
- the supernatant containing excess DTSSP was removed and the GNP were resuspended in 1.5 mL of 2mM borate buffer (pH 8.9).
- 40 pg/mL of anti-SARS CoV-2 monoclonal antibody (AcroBiosystem SAD-S35) or anti-SARS COV2 polyclonal antibody (GTX135356) were added and allowed to react for 1.5 hour at Room Temperature.
- GNPs 10 pl of 30 nm GNP (2.10 9 particles by ml) and 5 pl of 150 nm GNP (2.10 s particles by ml) have been incubated by five minutes in a solution with 1 ml supernatant of cell culture and a concentration of about 10 7 viral particles (in an Eppendorf of 1.5 ml). The tube has been centrifuged using the same time and centrifugation speed previous mentioned.
- the test has been done by incubating by five minutes the 10 pl of 30 nm GNP (2.10 9 particles by ml) and 5 pl of 150 nm GNP (2.10 s particles by ml) in an Eppendorf (of 500 pl) with 500 pl of cell culture with different viral loads (as mentioned in the results section) and centrifuged for 5 minutes at 2000g.
- the different virial loads have been reached by diluting the cell culture with the nominal virial load of about 10 7 virus by ml in the MOCK supernatant solutions five, ten and twenty times.
- the MOCK controls and the tests with the virus have been also done with UV-inactivated virus using the same protocol just mentioned (with 500 pl of solution). Tests and controls with the inactivated virus in 500 pl Eppendorf have been repeated at least three times.
- Figures 1A, IB and 1C show the bottom of Eppendorf tubes after incubation, only with the small 30 nm GNP (control), and after centrifugation.
- the solution with the virus around 10 7 virus by ml
- displays a small red pellet (Figure ID). This is in contrast to the two negative controls (MOCK supernatant and PBS) that do not display any red pellet ( Figures IE and IF).
- the control "PBS" shows that without the virus the two types of GNPs do not bind each other (otherwise the 150 nm particles would co-precipitate with the 30 nm particles). Since only the 150 nm GNPs precipitate no clear pellet is observed (since the 150 nm display colorless pellet). Furthermore, the control MOCK shows that no substantial amount of cellular debris forms co-precipitates with the 30 nm and the 150 nm GNPs (in contrast to the virus particles).
- the test has been done in the same conditions but with virus particles inactivated by UV.
- the test worked as well (figure 3) indicating that the target epitope in spike protein do not show a very strong modification change with UV treatment. This indicates that the protocol of inactivation can be used for this antibody to improve the security of the agents in charge of the analysis.
- Figure 4 shows the results of the first dilution (around 2.10 6 viral particles by ml) and Figure 5 shows the results of the second (about 1.10 6 particles by ml; Fig. 5A) and the third (5.10 5 particles by ml, fig. 5C) dilution.
- E, F and G shows the results of the first dilution (around 2.10 6 viral particles by ml)
- Figure 5 shows the results of the second (about 1.10 6 particles by ml; Fig. 5A) and the third (5.10 5 particles by ml, fig. 5C) dilution.
- a clear pellet was observed for all samples and all dilutions.
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Abstract
La présente invention concerne un procédé de détection ou de quantification rapide de particules virales dans un échantillon. Ce procédé est particulièrement utile pour effectuer un test rapide du SARS-CoV-2.
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