EP4147050A1 - Procédé pour la détection de corona virus - Google Patents
Procédé pour la détection de corona virusInfo
- Publication number
- EP4147050A1 EP4147050A1 EP21724288.2A EP21724288A EP4147050A1 EP 4147050 A1 EP4147050 A1 EP 4147050A1 EP 21724288 A EP21724288 A EP 21724288A EP 4147050 A1 EP4147050 A1 EP 4147050A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- coronavirus
- binding agent
- cov
- antigen
- sample
- 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
Links
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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
Definitions
- the disclosure provides tests, for example immunoassays, for use in detecting pathogens and/or antigens in samples, in particular, the disclosure provides point or care/point of need type tests.
- SARS-CoV-2 severe acute respiratory syndrome coronavirus
- COVID-19 coronavirus disease
- the SARS-CoV-2 virus is considered to be easily transmissible, although researchers are still working to understand how it spreads. Data so far has shown that it spreads from person to person among those in close contact (within about 6 feet, or 2 meters).
- the virus spreads by respiratory droplets released when someone with the virus coughs, sneezes or talks. These droplets can be inhaled or land in the mouth or nose of a person nearby. Therefore, efforts to date to contain the virus have focused on aggressive testing of individuals to find and isolate infected people.
- test kits described herein combine the benefits of the PCR or ELISA tests by directly detecting SARS-CoV-2, (without the need for specialised equipment) with the benefits of the traditional antibody LFAs which are POC but cannot detect the virus elements directly.
- the present disclosure provides new and improved methods for the detection of pathogens, for example viral pathogens, in samples.
- the methods, tests and assays described herein not only permit the identification of an infection (for example a Coronavirus infection) in a sample, but can also be used as a means to identify the infectious status or “infectivity level” of a subject to an extent that PCR and any other prior art assays cannot.
- an infection for example a Coronavirus infection
- infected subjects can report as negative by DNA/RNA analysis, but positive for antigens.
- the patient is therefore infectious, but perhaps asymptomatic with insufficient pathogen present to cause infection.
- a sample for example a biological fluid (including but not limited to blood, serum, plasma or saliva) may be rapidly and reliably tested for the presence of one or more pathogens and/or any component (for example an antigen) thereof.
- a biological fluid including but not limited to blood, serum, plasma or saliva
- One particular application of the methods described herein is in the detection of SARS-CoV- 2 in a sample.
- a method or assay of this disclosure is capable of detecting as few as about 1 x10 2 plaque forming units (PFU)/mL, 2x10 2 PFU/mL, about 5 x10 2 PFU/mL, about 1 x10 3 PFU/mL, about 1.5 x10 3 PFU/mL, about 2 x10 3 PFU/mL, about 5 x10 3 PFU/mL or about 1 x10 4 PFU/mL.
- PFU plaque forming units
- a method for detecting Coronavirus or a Coronavirus antigen in a sample comprising: contacting the sample with a Coronavirus I Coronavirus antigen binding agent under conditions which permit binding between the binding agent and any Coronavirus or Coronavirus antigen present in the sample and detecting binding agent/ Coronavirus complexes and/or binding agent/ Coronavirus antigen complexes, wherein the Coronavirus / Coronavirus antigen binding agent is not an antibody.
- a method for detecting Coronavirus or a Coronavirus antigen in a sample comprising: contacting the sample with a Coronavirus I Coronavirus antigen binding agent under conditions which permit binding between the binding agent and any Coronavirus or Coronavirus antigen present in the sample and detecting complexes comprising the binding agent and Coronavirus/ Coronavirus antigen, wherein the Coronavirus /Coronavirus antigen binding agent is not an antibody.
- Coronavirus / Coronavirus antigen binding agent may be one of a number of binding agents used in a method of this disclosure. Accordingly, the methods may use additional (for example second) Coronavirus / Coronavirus antigen binding agents. As noted, below, nothing about the terms first or second (as used herein) is intended to dictate the order in which these agents are used in the assays. Rather the terms are intended to distinguish one binding agent from another and it is the case that one (first) binding agent may be added before, concurrently with or after another (or second binding agent). Possible second bind agents are described elsewhere.
- binding agent/ Corona virus complexes i.e. complexes in which the binding agent has become bound to Coronavirus present in the sample
- binding agent/ Corona virus antigen complexes i.e. complexes in which the binding agent has become bound to Coronavirus antigen present in the sample
- Coronavirus as used herein should be understood as relating to those viruses which belong to the Corona viridae family; these are a group of enveloped, positive-sense, single-stranded RNA viruses. Coronavirus causes respiratory tract infections in humans including for example, common cold type diseases and more severe acute respiratory syndromes. SARS-CoV, SARS-CoV-2 and MERS-CoV are examples of Coronaviruses that have emerged as pathogenic in humans. In humans, SARS-CoV is known to cause the disease known as severe acute respiratory syndrome (SARS) and SARS-CoV-2 causes the disease referred to as COVID-19.
- SARS severe acute respiratory syndrome
- COVID-19 severe acute respiratory syndrome
- Coreaviridae or “Coronavirus” is derived from the distinctive shape of the virus which contains a number of crown-like projections ("peplomers” or “spikes”).
- peplomers or “spikes”
- S spike protein
- N nucleocapsid protein.
- the S-protein mediates host receptor binding, fusion and entry.
- the S-protein contains a number of domains or regions referred to as S1 , S2 and or S-receptor binding domain (RBD).
- S-protein For convenience and throughout this specification, reference is made to the “S-protein”, this term should be understood as embracing not only the whole or complete S-protein, but also any host receptor binding fragment thereof and/or any S1 protein/domain, any S2 protein/domain and or the receptor binding domain of the S-protein (S-RBD).
- S-protein may also embrace pre-fusion forms of the S-protein, including trimeric forms. Without wishing to be bound by theory, the S-protein exists in a trimeric form on the virus with multiple s1 , s2, and RBD subunits.
- the S-protein from some Coronavirus binds to the angiotensin converting enzyme 2 (ACE2) expressed on the surface of host cells.
- ACE2 angiotensin converting enzyme 2
- the S-protein of MERS-CoV may bind dipeptidyl peptidase 4 (DPP4).
- Coronavirus structural proteins may be referred to as Coronavirus antigens.
- S-protein may be referred to as a “Coronavirus antigen”.
- the method of this disclosure may be a method for detecting the presence of the Coronavirus S-protein in a sample.
- any Coronavirus antigen for example Coronavirus S-protein
- any Coronavirus antigen may be present in a sample as free antigen or as antigen which is part of a whole or fragmented virus particle.
- any Coronavirus antigen for example Coronavirus S-protein
- a method of this disclosure may provide a method for detecting, in a sample, one or more of:
- a method for detecting SARS-CoV-2 and/or a SARS-CoV-2 antigen in a sample may comprise: contacting the sample with a first SARS-CoV-2/SARS-CoV-2 antigen binding agent under conditions which permit binding between the binding agent and any SARS-CoV-2 or SARS-CoV-2 antigen present in the sample; and detecting binding agent/SARS-CoV-2 complexes and/or binding agent/SARS-CoV-2 antigen complexes, wherein the first SARS-CoV-2/SARS-CoV-2 antigen binding agent is not an antibody.
- binding agent/SARS-CoV-2 complexes i.e. a complex in which the binding agent has become bound to SARS-CoV-2 present in the sample
- binding agent/SARS-CoV-2 antigen complexes i.e. complexes in which the binding agent has become bound to SARS-CoV-2 antigen present in the sample
- the methods of this disclosure may be used to diagnose Coronavirus infections and/or diseases in subjects.
- the methods described herein may find application in the diagnosis of COVID-19, SARS and/or MERS - and a range of other Coronavirus related diseases.
- the methods described herein may be used to detect the presence of Coronavirus and/or Coronavirus antigens in environmental samples including, for example samples of soil, dust, water, air, and/or on, within or from environmental swabs.
- samples found to contain Coronavirus or Coronavirus antigens may have been provided by or obtained from subjects that have or have had a Coronavirus infection or a disease and/or condition associated therewith.
- the methods of this disclosure may be adapted for use as point of care or point of need tests.
- the sample may comprise blood (whole blood or a fragment thereof such as serum or plasma), a biopsy, cells, a tissue scraping, a mucosal or tissue secretion, mucus, mucus, saliva, pulmonary surfactant and fluid from a washing procedure (for example the fluid obtained from a lung or bronchoalveolar lavage washing procedure).
- blood whole blood or a fragment thereof such as serum or plasma
- tissue scraping a mucosal or tissue secretion
- mucus mucus
- mucus saliva
- pulmonary surfactant and fluid from a washing procedure for example the fluid obtained from a lung or bronchoalveolar lavage washing procedure.
- the sample may be provided by or obtained from any subject to be tested.
- the subject may be any human or animal subject.
- the subject may be suspected of having a Coronavirus infection and/or a disease or condition associated with or caused, or contributed to, by a Coronavirus.
- the subject may have had or may be convalescing from a Coronavirus infection and/or a disease or condition associated with or caused, or contributed to, by a Coronavirus.
- the subject may be asymptomatic and/or known to have been in contact with another (human or animal) subject that has or has had a Coronavirus infection and/or a disease or condition associated with or caused, or contributed to, by a Coronavirus.
- the subject may have tested negative (by (RT)-PCR, ELISA or some other type of test) for a Coronavirus infection and/or Coronavirus associated disease or condition.
- a first Coronavirus or Coronavirus antigen binding agent may comprise a moiety which binds to and/or has affinity and/or specificity for a Coronavirus antigen.
- agents which have affinity and/or specificity for a particular antigen will be referred to as “binding to” that antigen.
- binding includes, but is not limited to, covalent binding, electrostatic binding, hydrogen bonding, hydrophobic binding and the like.
- the first Coronavirus or Coronavirus antigen binding agent may comprise a moiety which binds to the Coronavirus S-protein.
- the agent which binds a Coronavirus S- protein may bind to a fragment, domain or portion of the Coronavirus (S)-protein (for example the S1 domain, the S2 domain, S-RBD, or combinations thereof).
- a method which is for the detection of SARS-CoV-2 in a sample may exploit an agent which binds a SARS-CoV-2 antigen, for example the SARS-CoV-2 S-protein.
- the agent which binds the SARS-CoV-2 antigen may bind to a fragment, domain or portion of the SARS-CoV- 2 S-protein.
- a method which is for the detection of SARS-CoV in a sample may exploit an agent which binds a SARS-CoV antigen, for example the SARS-CoV S-protein.
- the agent which binds the SARS-CoV antigen may bind to a fragment, domain or portion of the SARS-CoV S- protein.
- a method which is for the detection of MERS-CoV in a sample may exploit an agent which binds a SARS-CoV antigen, for example the MERS-CoV S-protein.
- the agent which binds the MERS-CoV antigen may bind to a fragment, domain or portion of the MERS-CoV S- protein.
- An agent for use in a method of this disclosure may comprise, for example, angiotensin converting enzyme 2 (ACE2) or a Coronavirus or Coronavirus antigen binding fragment thereof.
- ACE2 may be used where the method is for the detection of SARS-CoV-2, a SARS- CoV-2 antigen, the SARS-CoV-2 S-protein, SARS-CoV, a SARS-CoV antigen or the SARS- CoV S-protein.
- An agent for use in a method of this disclosure may comprise, for example, dipeptidyl peptidase 4 (DPP4) or a Coronavirus or Coronavirus antigen binding fragment thereof.
- DPP4 dipeptidyl peptidase 4
- Coronavirus or Coronavirus antigen binding fragment thereof may comprise, for example, dipeptidyl peptidase 4 (DPP4) or a Coronavirus or Coronavirus antigen binding fragment thereof.
- DPP4 may be of particular use where the method is for the detection of MERS-CoV, a MERS-CoV antigen or the MERS-CoV S-protein.
- ACE2 will embrace all ACE2 fragments which retain an ability to bind to Coronavirus and/or a Coronavirus antigen (in particular SARS-CoV-2, the SARS- CoV-2 S-protein, SARS-CoV or the SARS-CoV S-protein).
- ACE2 will embrace fragments (for example truncated forms of ACE2) and/or dimeric/multimeric forms, which retain an ability to bind any Coronavirus S-protein and/or the S1 domain, S2 domain or S-RBD domain thereof.
- DPP4 will embrace all DPP4 fragments which retain an ability to bind to Coronavirus and/or a Coronavirus antigen (in particular MERS-CoV of the MERS-CoV S-protein).
- Coronavirus bind ACE2/DPP4, these proteins (ACE2 or DPP4) will, when brought into contact with a sample containing ACE2- bindng or DPP4-binding Coronavirus, bind thereto and form a complex therewith.
- ACE2 or DPP41 Coronavirus and/or ACE2 or DPP4/Coronavirus antigen complexes can then be detected as used as means to confirm the presence of Coronavirus and/or Coronavirus antigen in the sample.
- ACE2 may be used as an agent to bind SARS-CoV-2, SARS-CoV-2 S protein, SARS-CoV and/or SARS-CoV S protein, present in a sample.
- An advantage associated with the use of ACE2 as an agent to bind any SARS-CoV-2, SARS-CoV-2 S protein, SARS-CoV and/or SARS-CoV S protein, present in a sample is that the assay is not so vulnerable to a loss of sensitivity owing to a viral mutation.
- DPP4 may be used as an agent to bind MERS-CoV or MERS-CoV S protein present in a sample.
- the disclosure provides a method for detecting Coronavirus and/or a Coronavirus antigen in a sample may comprise: contacting the sample with ACE2/DPP4 under conditions which permit binding between the ACE2/DPP4 and any Coronavirus or Coronavirus antigen present in the sample and detecting ACE2/DPP4 -.Coronavirus complexes and/or ACE2/DPP4 -.-.Coronavirus antigen complexes.
- ACE2/DPP4 wCoronavirus complexes that is a complex comprising ACE2/DPP4 bound to a Coronavirus
- ACE2/DPP4 -/.Coronavirus antigen complexes a complex in which ACE2/DPP4 is bound to a Coronavirus antigen
- the test sample was provided by and/or obtained from a subject that has or has had a Coronavirus infection or a disease or condition caused or contributed to by a Coronavirus.
- ACE2/SARS-CoV-2 complexes that is a complex comprising ACE2 bound to a SARS-CoV-2
- ACE2/ SARS-CoV-2 antigen complexes a complex in which ACE2 is bound to a SARS-CoV-2 antigen
- the test sample was provided by and/or obtained from a subject that has or has had a SARS-CoV-2 infection or a disease or condition caused or contributed to by SARS-CoV-2 (for example COVID-19).
- An exemplary (human) ACE2 sequence for use as a first SARS-CoV-2 binding agent in a method of this disclosure is deposited as Q9BYF1 (UniProtKB); that sequence is reproduced below as SEQ ID NO: 1
- SEQ ID NO: 1 human ACE2 sequence
- residues marked in grey highlight are thought to be important in binding to SARS-CoV-2 - in particular to the SARS-CoV-2 S-protein. These residues form a domain, all or part of which interacts with, binds to and/or has affinity for, the SARS-CoV-2 S-protein and/or the S-RBD part thereof (see below for a description of the S-RBD).
- the region spanning residues 24-416 of the ACE2 protein may referred to as defining or containing the SARS-CoV-2 binding interface.
- a method of this disclosure may use, as a first Coronavirus (antigen) SARS-CoV-2 (antigen) binding agent, a peptide having or comprising a sequence of:
- SARS-CoV-2 binding fragment embraces any fragment of the ACE2 protein which functions to bind SARS-CoV-2 and/or the S-protein expressed thereby.
- DPP4 Dipeptidyl peptidase 4
- SEQ ID NO: 3 human DPP4 sequence
- region spanning residues 267-392 of the DPP4 protein may referred to as defining or containing a Coronavirus or Coronavirus S-protein SARS-CoV-2 binding interface.
- a method of this disclosure may use, as a first Coronavirus or Coronavirus antigen binding agent, a peptide having or comprising a sequence of:
- Coronavirus or MERS-CoV binding fragment embraces any fragment of the DPP4 protein which functions to bind Coronavirus or MERS- CoV and/or an antigen or S-protein expressed thereby.
- a method for detecting a Coronavirus, a Coronavirus antigen, MERS-CoV or a MERS-CoV in a sample may comprise: contacting the sample with an DPP4 protein or a Coronavirus or MERS-CoV binding fragment thereof, under conditions which permit binding between the DPP4 protein the Coronavirus or MERS-CoV binding fragment thereof and any Coronavirus, a Coronavirus antigen, MERS-CoV or MERS-CoV antigen present in the sample and detecting DPP4 protein or Coronavirus or MERS-CoV binding fragment thereof bound to Coronavirus, a Coronavirus antigen, MERS-CoV antigen or MERS-CoV.
- the invention further embraces methods which use sequences which exhibit some degree of sequence identity and/or homology to any of the sequences described herein.
- the first SARS-CoV-2 binding agent may comprise a protein sequence which is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or homologous to the sequences represented by any of SEQ ID NOS: 1-4 described herein. Note, all useful homologous or identical sequences will be able to bind SARS-CoV-2 or at least the S-protein thereof.
- a method for detecting SARS-CoV-2 in a sample may comprise: contacting the sample with an ACE2 protein or a SARS-CoV-2 binding fragment thereof, under conditions which permit binding between the ACE2 protein or SARS-CoV-2 binding fragment thereof and any SARS-CoV-2 present in the sample and detecting ACE2 protein or SARS-CoV-2 binding fragment thereof bound to SARS-CoV-2.
- the first Coronavirus/ Coronavirus antigen binding agent of any of the methods described herein may be conjugated to (or bound to/associated with) some form of detectable particle, label or tag.
- the first Coronavirus/ Coronavirus antigen binding agent for use in any of the methods described herein may be labelled or tagged for detection.
- the first binding agent may comprise an optically detectable label or tag.
- the first binding agent may comprise a fluorescent or chemiluminescent label or tag.
- the first binding agent may be conjugated to biotin (i.e. it may be biotinylated).
- the first binding agent may be fused or conjugated to a peptide tag.
- Suitable peptide tags may include, for example peptide tags which comprise one or more amino acids.
- a suitable peptide tag may comprise multiple histidine amino acids - a ‘His-tag’.
- the first binding agent may comprise a nanoparticle.
- Suitable nanoparticles may include any to which a first binding agent of this disclosure can be covalently or passively bound and/or immobilised, for example, to the surface.
- a nanoparticle for use may comprise, consist of or consist essentially of a gold nanoparticle and/or a gold-coated nanoparticle (for example a gold coated silica nanoparticle).
- the nanoparticle may not comprise a gold nanoparticle.
- the nanoparticle may comprise a non-gold nanoparticle.
- any nanoparticle/visualisation agent which has been used in a lateral flow assay can be applied to the methods described herein.
- the nanoparticle may comprise any (inorganic) element which has found application as a nanoparticle/visualisation agent in assays of the type described herein
- the first binding agent may comprise a coloured latex beads.
- the size of the nanoparticle may vary depending on a number of factors. Again, without wishing to be bound to any theory, larger particle may allow more target binding and may be easier to visualise.
- Nanoparticles of between 50 nm and 500 nm in diameter may be useful. Nanoparticles with a diameter of about 100 nm, about 200 nm, about 300 nm, or about 400 nm may be used. Nanoparticles with a diameter of >100 nm, >200 nm and/or >300 nm may be used.
- the nanoparticles may each be the same or of different sizes. For example, larger particles may be capable of being bound to more of the first binding agent (there is more surface area to which the first binding agent may be bound and/or immobilised). Without wishing to be bound by theory, it is suggested that by increasing the amount of first biding agent available to bind Coronavirus and/or Coronavirus antigen in a sample, it is possible to improve the sensitivity of an assay of this disclosure.
- a method of this disclosure may use:
- any of binding agents (i)-(iii) above may bind a SARS-CoV-2 antigen, a SARS- CoV antigen or MERS-CoV antigen and may be either covalently or passively conjugated to a gold nanoparticle or some other detectable (for example optically detectable) tag.
- the first binding agent may be immobilised to or impregnated into/held within a substrate.
- the first binding agent may be held within or impregnated into a nitrocellulose membrane. This may be impregnated through the use of a tag (for example, biotin).
- a tag for example, biotin
- the first agent may mobilise through the substrate (for example the nitrocellulose membrane). Solubilisation may occur upon the addition of a sample.
- a method of this disclosure may comprise an optional wash step.
- the method may be subject to a wash so as to remove any sample from the system.
- the wash may also remove any material which has not bound to the first binding agent.
- a wash may use a wash buffer.
- a suitable drying buffer may comprise (5%) Sucrose, (3%) BSA, (0.5%) Tween 20, (0.1 %) sodium azide and (10 mM) Borate buffer.
- the pH of this buffer may be above about 6.9. Without wishing to be bound by theory, the presence of sucrose in this buffer may slow down the travel sample through the membrane in the washing step.
- Other wash buffers may comprise, for example, phosphate buffered saline (PBS).
- the methods described herein may use a second agent, which second agent also binds the Coronavirus or Coronavirus antigen.
- first and second as used herein do not necessarily refer to the order in which the binding agents are added to, or contacted with, the sample. Rather, the terms “first” and “second” primarily refer to the fact that the methods and assays of this disclosure may use two agents capable of binding a Coronavirus antigen (in other words, each binding agent is one of at least two used in any of methods or assays described herein) - either one or those agents may be added to or contacted with the sample, before, concurrently with, or after, the other.
- any of the disclosed methods or assays may involve contacting a sample with what is described herein as the “first coronavirus binding agent” to form (if the sample contains Coronavirus/ Coronavirus antigen) first coronavirus binding agent wCoronavirusl Coronavirus antigen complexes and then contacting the sample (or any first coronavirus binding agent wCoronavirusl Coronavirus antigen complexes) with what is described herein as the second (or additional) coronavirus binding agent.
- the disclosed methods or assays may involve contacting a sample with what is described herein as the “second coronavirus binding agent” to form (if the sample contains Coronavirus/ Coronavirus antigen) second coronavirus binding agent:: Coronavirus/ Coronavirus antigen complexes and then contacting the sample (or any second coronavirus binding agent:: Coronavirus/ Coronavirus antigen complexes) with what is described herein as the first (or additional) coronavirus binding agent.
- any of the disclosed methods or assays may involve contacting a sample with both the “first Coronavirus binding agent” and the “second Coronavirus binding agent.
- a method for detecting Coronavirus or a Coronavirus antigen in a sample comprising: contacting the sample with a first Coronavirus/ Coronavirus antigen binding agent under conditions which permit binding between the binding agent and any Coronavirus or Coronavirus antigen present in the sample to form a first agent /Coronavirus or a first agent/ Coronavirus antigen, complex; contacting (the sample with) a second Coronavirus/ Coronavirus antigen binding agent under conditions which permit binding between any complexes and the second binding agent; detecting first agent/Coronavirus/second agent complexes (that is a complex in which both the first and second agents are bound to a Coronavirus) and/or first agent I Coronavirus antigen/second agent complexes (that is a complex in which both the first and second agents are bound to a
- the first binding agent may be contacted with the sample, before, concurrently with, or after the second binding agent.
- a method which includes the use of first and second binding agents may include an optional washing step.
- the method may include at least two washing steps.
- a first wash step may be executed after addition of the sample to the first binding agent and before addition of the second binding agent.
- the second optional washing step may be executed after addition of the second binding agent and before the detecting step.
- the second binding agent may comprise any molecule which binds to a Coronavirus and/or a Coronavirus antigen - including any or all of the Coronavirus types described herein (e.g. SARS-CoV-2, SARS-CoV and MERS-CoV) or any or all of the Coronavirus antigens described herein, for example the Coronavirus S-protein, the SARS-CoV-2 S-protein, the SARS-CoV S-protein or the MERS-CoV S-protein).
- a Coronavirus antigen - including any or all of the Coronavirus types described herein (e.g. SARS-CoV-2, SARS-CoV and MERS-CoV) or any or all of the Coronavirus antigens described herein, for example the Coronavirus S-protein, the SARS-CoV-2 S-protein, the SARS-CoV S-protein or the MERS-CoV S-protein).
- the second agent may be the same as the first binding agent.
- the second binding agent may also comprise ACE2 and/or DPP4 (where the terms “ACE2” and “DPP4” embrace all Coronavirus or Coronavirus antigen binding fragments of ACE2 and/or DPP4 as defined herein).
- the second agent may comprise an antibody.
- the second agent may comprise an antibody which binds to (i.e. has affinity and/or specificity for) a Coronavirus and/or a Coronavirus antigen.
- the second agent may bind the same target as the first binding agent.
- the second agent may bind a different site on the same target as the first binding agent.
- the second agent may bind the S2 domain of the Coronavirus S-protein.
- the second agent may comprise an anti-S2 antibody (that is an antibody with specificity for the S2 domain of the SARS-CoV-2 Spike protein S2 domain.
- the second agent may be conjugated.
- the second agent may be conjugated to a detectable moiety.
- the second agent may be conjugated or bound to a detectable moiety, for example an optically detectable moiety (a gold coated nanoparticle or the like).
- the second agent may be conjugated to, for example, europium nanoparticles and/or carbon nanoparticles.
- the second agent may be biotinylated (a biotinylated agent may bind to the PSA that is immobilised on the surface of a substrate, for example a nitrocellulose membrane).
- the second agent may comprise an antibody.
- the second agent may comprise a biotinylated antibody.
- the second agent may comprise a biotinylated antibody which binds to Coronavirus and/or a Coronavirus antigen.
- the second binding agent may be immobilised to or impregnated into/held within a substrate.
- the second binding agent may be held within or impregnated into a nitrocellulose membrane.
- the second agent may mobilise through the substrate (for example the nitrocellulose membrane). Solubilisation may occur upon the addition of a sample.
- the second agent may comprise an antibody which binds SARS-CoV-2 and/or a SARS-CoV-2 antigen.
- the second agent may comprise an antibody which binds SARS-CoV and/or a SARS-CoV antigen.
- the second agent may comprise an antibody which binds MERS-CoV-2 and/or a MERS-CoV antigen.
- a method of this disclosure may involve a control test or assay in which the results obtained from a test sample are compared with the results obtained from positive and/or negative control samples.
- a negative control sample may comprise a sample known to lack Coronavirus and/or Coronavirus antigens.
- the negative control may exploit a sample which does not Coronavirus, SARS, SARS-CoV-2 or MERS-CoV or any antigens therefrom.
- a positive control sample may comprise a sample which contains (or has been spiked with) a Coronavirus, SARS, SARS-CoV-2, MERS-CoV or any antigen therefrom.
- a positive control may function simply to show that a test has run and functioned correctly.
- a positive control may comprise an antibody that binds to a component expected to be present in the test sample.
- the positive control may comprise an antibody which binds to salivary amylase or the like.
- test sample and the control sample may be the same.
- test sample is, or comprises blood (or a fragment thereof such as serum or plasma), so too is, or does, the control sample.
- the disclosure provides a method wherein the first and second binding agents are added to a sample to be tested.
- the sample (with the added first and second binding agents) may then be incubated for a period of time.
- the period of incubation may last seconds, minutes or hours.
- the period of time may be 5s, 10s, 15s, 20s, 30s, 60s, 2 min, 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50, min, 1 hr, 2 hr or 3 hr.
- a period of 10 s may be useful in the case of a sample comprising S-RBD and S1+S2 domain and a time period of about 15 min may be useful in the case of a sample that comprises the S1 protein domain).
- the sample may then be added to a system which comprises a capture agent, wherein the capture agent binds to the biotinylated second agent.
- the capture agent may comprise polystrepavidin (PSA).
- PSA polystrepavidin
- the capture agent may be immobilised at or on a test line in substrate.
- the sample (with added first and second binding agents which may have bound to any Coronavirus or Coronavirus antigen present in the sample), may then be added to the substrate and allowed or caused to flow there through.
- the substrate may comprise an absorbent pad that draws the sample through the substrate.
- the first and/or second binding agents may comprise either ACE2 or DPP4.
- the disclosure further provides an immunoassay for the detection of Coronavirus in a sample, wherein the immunoassay comprises a labelled agent which binds a Coronavirus and/or a Coronavirus, wherein the labelled agent does not comprise an antibody.
- the immunoassay may comprise a lateral flow assay.
- a lateral flow assay may comprise a sample pad (for receiving the sample), a test line, a control line and an absorbent pad (to draw the sample through the assay).
- a lateral flow assay of this disclosure may be for the detection of Coronavirus, a Coronavirus antigen, SARS-CoV-2, SARS-CoV, MERS-CoV, the S-protein of SARS-CoV-2, the S-protein of SARS-CoV or the S-protein of MERS-CoV.
- a lateral flow assay of this disclosure may comprise the first and second binding agents described herein.
- the lateral flow assay may contain an ACE2 protein (again for the avoidance of doubt, the term “ACE2” includes proteins having the sequences defined herein and also any functional fragments thereof).
- the lateral flow assay may comprise a nanoparticle coated with or bound to multiple copies of the ACE2 protein.
- useful nanoparticles may comprise inorganic elements and/or particles containing organic materials, including, but not limited to, gold.
- the lateral flow assay may contain a DPP4 protein (again for the avoidance of doubt, the term “DPP4” includes proteins having the sequences defined herein and also any functional fragments thereof).
- the lateral flow assay may comprise a nanoparticle passively conjugated with or covalently bound to multiple copies of the DPP4 protein.
- useful nanoparticles may comprise inorganic elements, including, but not limited to, gold.
- the lateral flow assay may further comprise an antibody which binds the same target as the ACE2 protein.
- the antibody may also bind the Coronavirus S-protein.
- An antibody for use in a lateral flow assay of this innovation may comprise an anti-S2 antibody - that is an antibody with specificity/affinity for the S2 domain of the SARS-Cov-2 spike protein.
- a lateral flow assay of this disclosure may comprise a nanoparticle comprising multiple copies of an ACE2 protein (as defined herein) and an anti-S2 antibody.
- a sample is brought into contact with the ACE2 conjugated nanoparticle under conditions which permit binding between the ACE2 component of the nanoparticle and any Coronavirus/ Coronavirus S-protein present in the sample.
- a nanoparticle/S-protein complex will form via binding between the ACE2 component of the nanoparticle and the S-protein present in the sample (present in any pre-fusion, trimeric or post-fusion form or as S-protein expressed on the surface of a Coronavirus particle).
- the nanoparticle/S-protein complex may then mobilise through the assay whereupon it will contact the second binding agent.
- the second binding agent will also bind the Coronavirus/ Coronavirus S-protein component of the nanoparticle/S-protein complex.
- the second binding agent may bind a different site on the S-protein and may comprise a label or tag (referred to hereinafter as a ‘capture moiety’) - for example, the second binding agent may bind the S2 domain of the S-protein.
- this stage of the assay will yield a complex comprising the first binding agent (immobilised to a nanoparticle) and the second binding agent.
- the lateral flow assay may further comprise a test line.
- the test line may comprise an immobilised agent which binds the capture moiety of the second binding agent.
- the immobilised agent may be described as a capture agent - where the capture agent is formulated to bind to the capture moiety of the second binding agent. Accordingly the test line (of the lateral flow assay) may comprise a capture agent.
- the capture agent may comprise a moiety which binds to (or which has specificity and/or affinity for) the capture moiety of the second binding agent.
- the capture agent may comprise an agent which binds to biotin.
- the capture agent may comprise polystreptavidin (PSA) or streptavidin.
- the control may comprise a moiety which binds ACE2. This moiety may be immobilised to a control line. Any Ace2 which has not been bound by spike protein will bind to the binding moiety. If the Ace2 is bound to a detectable nanoparticle (for example a coloured nanoparticle) the control line will become visible as the detectable/coloured nanoparticles accumulate at the control line.
- a detectable nanoparticle for example a coloured nanoparticle
- the disclosure provides a lateral flow assay for detecting Coronavirus or a Coronavirus antigen in a sample, said lateral flow assay comprising: a first Coronavirus / Coronavirus antigen binding agent; and a second Coronavirus / Coronavirus antigen binding agent; wherein the first binding agent is not an antibody.
- the lateral flow assay may further comprise a capture agent which comprises a moiety which binds to the second Coronavirus / Coronavirus antigen binding agent,
- the disclosure provides a lateral flow assay for detecting Coronavirus or a Coronavirus antigen in a sample, said lateral flow assay comprising: a nanoparticle conjugated ACE2; and a biotin conjugated antibody which binds the S2 domain of the Coronavirus S-protein.
- the lateral flow assay may further comprise a capture agent which comprises a moiety which binds biotin.
- a lateral flow assay of this disclosure may receive a sample, for example a sample of blood, saliva, nasal fluid, mucus, including nasal mucus, sweat, or faeces, or combinations thereof.
- the sample may be brought into contact with the first binding agent - which binding agent (as described herein) binds to Coronavirus or a Coronavirus antigen.
- the first binding agent may comprise ACE2 which binds to Coronavirus S-protein.
- the first binding agent may be conjugated to some form of detectable label - e.g. a gold nanoparticle.
- Coronavirus or Coronavirus antigen present in the sample will bind to the first agent. This forms Coronavirus/first binding agent complexes or Coronavirus antigen/first binding agent complexes.
- the absorbent pad part of the lateral flow assay will draw any complexes through the substrate of the lateral flow assay.
- the substrate may comprise nitrocellulose.
- any complexes being drawn through the sample are then contacted with the second binding agent of the lateral flow assay.
- the second binding agent comprises an agent which also binds the Coronavirus or Coronavirus antigen (perhaps at a different site from the first binding agent)
- the second binding agent will bind any complexes (which complexes comprise Coronavirus or Coronavirus antigen) as they move through the lateral flow assay.
- This forms a further, or secondary complex comprising (i) first binding agent; (ii) Coronavirus or Coronavirus antigen and (iii) second binding agent.
- the second binding agent may comprise a (capture) moiety which is bound by the capture agent present in the test line. Therefore as the secondary complexes are drawn through the sample and come into contact with the capture agent immobilised at the test line, the secondary complexes are also immobilised.
- Immobilisation of the secondary complexes at the test line causes the appearance of a line - which line can be seen by virtue of the optically detectable label present on the first binding agent.
- the presence or appearance of a line may mean that the sample has been obtained from a subject that has or has had a Coronavirus infection of a disease caused or contributed to by a Coronavirus.
- a sample Prior to use, a sample may be subject to some preparation or activation protocol.
- the sample may, for example be treated by the addition of reagents and buffers. These buffers may neutralise certain enzymes and/or may increase or facilitate the flow of a sample into and through and assay.
- a sample may be subject to a viral lysis protocol before use.
- the sample may be treated with a composition designed to break up and/or lyse any coronavirus present in the sample.
- a protocol which breaks up or lyses the virus in a sample before use, may improve the sensitivity of the assay.
- a sample to be tested may be contacted with an activating agent.
- the activating agent may increase binding between any Coronavirus or Coronavirus antigen present in the sample and the first binding agent.
- the activating agent may increase binding between ACE2 (when used as the first binding agent) and the S-protein (or components thereof).
- the activating agent may comprise a serine protease to prime the Coronavirus or any antigen thereof, for binding to the first agent.
- the activating agent may comprise TMPRSS2 and/or trypsin.
- the activating agent may comprise a serine protease inhibitor.
- the serine protease inhibitor may comprise, for example, TMPRSS2 or Furin.
- Inhibitors of this type may be added to the sample prior to use in a method or assay of this disclosure.
- this disclosure provides a method for treating a sample for use in a method of detecting a Coronavirus or Coronavirus antigen in the sample, said method comprising contacting the sample with a serine protease, trypsin or TMPRSS2.
- the protease (for example) may be used at a concentration of about 32-0.25 nm.
- a sample may be contacted with a Mcllvaine buffer supplemented with 0.3 % DDAO at a neutral pH (for example, pH 7.4).
- a neutral pH for example, pH 7.4
- a method of detecting Coronavirus or a Coronavirus antigen in a sample may comprise
- Any of the methods of this disclosure may be further used to diagnose or triage Coronavirus infections and/or disease, including for example COVID-19 in a point of case or point of need setting.
- the methods may be (as stated above), applied to samples obtained form or provided by symptomatic and/or asymptomatic subjects.
- a sample may be further treated before being subjected to a method of this disclosure.
- the pH of a sample may be adjusted.
- the pH of the sample may be adjusted so that it is above about pH8.
- All of the methods of this disclosure may be combined or performed simultaneously or concurrently with other methods for the detection or diagnosis of other infections, antigens and/or diseases.
- the methods may be combined and/or performed simultaneously or concurrently with other immunoassays.
- the methods of this disclosure may find application in the research setting where they may be used to determine the binding affinity/kinetics/activity of a virus - for example a Coronavirus.
- the methods of this disclosure may be used to determine the efficacy of particular drug candidates.
- a test agent may be added prior to addition of the first binding agent (or concurrently therewith) prior to addition of the first binding agent (or concurrently therewith) a test agent may be added. That test agent may compete the first agent for binding to the Coronavirus and/or Coronavirus antigen. Where a test agent is found to compete with the first agent, that test agent may be useful in the treatment and/or prevention of Coronavirus infections and/or diseases or conditions associated with a Coronavirus infection. Potentially useful test agents may be identified as a negative result in an assay of this disclosure where the sample used is known to contain Coronavirus ora Coronavirus antigen.
- Figure 1 A) positive line indicating the presence of biotinylated S1 antibody, B) Biotinylated Rabbit IgG was used for control.
- S-RBD (0.25 mg/mL) was diluted 1 :10000.
- 20-2.5uL (test 1-5) of 1 :10000 S-RBD was mixed with 2 uL of 1 :50 dilution of (1 mg/mL) biotinylated S1 mab. 10 or 20 uL was added to the sample and the mixture was incubated for 15 min before running the strip. Sample was run through the strip and washed with 10 uL of drying buffer.
- Figure 4 Schematic diagram demonstrating the sensitivity of the lateral flow assay.
- Figure 5 Schematic illustration of A) Wet assay of Viral protein, B) Lateral flow assay format demonstrating viral antigen-NP and S-RBD specific antibody sandwiched around the virus, C) Lateral flow assay with viral antigen-NP and antibody sandwiched around the viral protein.
- Figure 6 Comparison of A) Wet assay with B) dry assay.
- Figure 7 Comparison of asymmetric assay with S1 protein present versus S-RBD protein.
- Figure 9 Comparison of spiked saliva sample application: 15 ⁇ g/ml OD20 gold 4 ⁇ l dried.
- Figure 10 Comparison of gold volumes, 10 and 20 minute read times, RBD saliva spike 250 ng/ml (50:50)
- Figure 11 spiked saliva with RBD (1;1), OD20 gold 15 ⁇ g/ml using 8 ⁇ l deposition, 1% T20 PBS-B running buffer, 20 minute run time.
- Figure 12 cartoon showing the arrangement at the test line of an example lateral flow assay.
- nitrocellulose strip 16 contains a test line having immobilised thereof a quantity PSA (14) immobilised thereon.
- a nanoparticle having immobilised thereof a quantity of ACE2 (2: or a Coronavirus/S-protein binding fragment thereof) is contacted with a sample thought to contain either Coronavirus and/or the Coronavirus S- protein under conditions which permit binding between any Coronavirus and/or the Coronavirus S-protein present in the sample and the ACE2 bound to the nanoparticle.
- the antigen (the Coronavirus S-protein) is represented by components 4 (the RBD domain), 6 (the S1 domain) and 8 (the S2 domain).
- the result of this step of the assay is the formation of a complex between the S-protein (specifically the S1 domain thereof) and the ACE-2 component bound to the nanoparticle.
- the assay system further comprises an antibody (10) with specificity for the S2 domain of the S-protein (8). That antibody (10) is biotinylated (12).
- the assay permits the formation of a further complex with antibody 10. This complex is then captured at the test line by binding between the biotin moiety (12) of the antibody (10) and the PSA (14) at the test line (18). Because a test line contains multiple PSA (14) moieties, multiple nanoparticles become immobilised and can be detected.
- the method of detection may vary depending on the type of nanoparticle, but it may be optically detectable r the nanoparticle being a coloured bead or the like.
- Figure 13 Cartoon showing an exemplar assay format.
- the Coronavirus spike protein is shown as comprising three domains, the RBD domain (42), the S1 domain (44) and the S2-domain (46).
- a sample (20) comprising spike protein is applied to a device which comprises a nitrocellulose strip (21).
- the strip (21) comprises a sample pad (22) for receiving the sample, a conjugation pad (24) comprising a first spike protein binding agent (26) and a second conjugation pad (28) comprising a molecule which itself comprises a nanoparticle (32) with ACE2 (30) (or a spike binding fragment thereof) immobilised thereto.
- the sample (20) moves through the nitrocellulose strip (21) by capillary action/wicking.
- the sample maybe ‘pulled’ or drawn through by an absorption pad located at a distal point of the strip (21 : not shown).
- the sample 20 and any spike protein therein (42, 44, 46) arrives at conjugation pad (24) which comprises an antibody (26) with specificity for the S2 domain (46) of the spike protein.
- the antibody is biotin labelled.
- the result of this interaction is the formation of a spike protein (42, 44, 46)::antibody complex (26).
- This complex will then move through to conjugation pad 2 (28) which comprises the Ace2 (30) bound nanoparticle (32).
- conjugation pad 2 (28) there will form a further complex comprising not only the spike protein (42, 44, 46) and antibody (26), but also the Ace2 (30) bound nanoparticle (32).
- This larger complex will then continue to move through the strip (210 towards the text line (34) and control line (36).
- Test line (34) may have immobilised thereon s quantity of PSA - this will bind the biotin moiety of the antibody (26).
- the control line (36) may comprise an Ace2 binding moiety (38).
- a test line forms because of complex binding to the PSA of the test line (via biotin on the anti-S2 antibody (26)) free ACE2/nanoparticle conjugates (30/32) bind to moiety (38) at the control line to form a red line.
- Biotinylation of antibodies specific to S-RBD is prepared by using Lightning-Link® Rapid Biotin Conjugation Kit (Type B) following advised protocol:
- ACE2 Angiotensin-Converting Enzyme 2
- conjugation buffers Tris, Borate, MES, TAPS and BES
- OD1 -OD10 gold nanoparticles in size ranges 10-150 nm (100-500uL). The mixture was left on a shaker for 45 min at room temperature (21 C). 1 uL of (300 mg/ml_) of Probumin was added to the mixture and vortexed. Then left for 30 min at room temperature (21 C).
- drying buffer pH range > 6, 1 mM Borate, 2-5 % sucrose, 1-3 % BSA, 0.1 -2 % Tween 20 or Tween 80, 0.1 % sodium azide in filtered deionised water
- 900 uL drying buffer pH range > 6, 1 mM Borate, 2-5 % sucrose, 1-3 % BSA, 0.1 -2 % Tween 20 or Tween 80, 0.1 % sodium azide in filtered deionised water
- Plotting of PSA on nitrocellulose strips was performed by preparing a solution of 4.7 uL of 4.36 mg/ml of PSA, 4.1 uL of 10 % sucrose, 3.6 uL of PBS buffer and 29 uL filtered and deionised water. The mixture was used to plot a line on 30 cm nitrocellulose membrane CN140 with a plotting rate of 0.1 mm/s. The membrane was dried at 40 C and attached to a card together with an absorbent pad. The membrane was cut in 3 mm to yield 100 strips. See Figure 3.
- Example 2 - parts of a useful assay may include:
- Procedure for the formation of asymmetric sandwich Mix together 20-2.5 uL of saliva, serum or throat swab containing S-RBD, S1+S2 and S1 protein solution in 0-20 uL drying buffer with 2 uL of conjugated S-RBD specific antibodies and 5-20 uL of ACE-2 conjugated to gold nanoparticles 40 nm. 6. Add to the nitrocellulose strips and if the saliva, serum or throat swab contained viral proteins then a line appears on the strip.
- the amount and type of gold nanoparticle could be modified such that the intensity of the band generated in the presence of SARS-COV-2 proteins increased more progressively based on the viral protein concentration. This would allow the LFA to be used to measure how well another compound binds to ACE2 in competition with viral proteins. This could be used to test potential drug candidates for treatment of SARS- COV-2 rapidly and cheaply compared to other lab based techniques (mostly ELISA). Note current formulation designed to give as much signal as possible with as little viral protein as possible.
- This LFA setup described is likely to be applicable to other viruses (other than SARS- COV-2). As viruses all need to bind to some protein to gain entry into cells such a protein might substitute for ACE2 and along with an antibody specific to alternative virus the same type of sandwich LFA could be constructed.
- Both the second binding agent for example the specific antibody
- first binding agent e.g. angiotensin-Converting Enzyme 2 (ACE2)
- ACE2 angiotensin-Converting Enzyme 2
- Gold nanoparticles (an option for tagging or labelling the first binding agent) could be changed to a wide range of other materials. Other metals could potentially be used. Also, other fluorescent or coloured compounds are potential substitutes. Any nanoparticles could be used. 3.
- the test could be arranged in a variety of formats and conjugations. For example, the methods of this invention could be adapted to take place in ELISA format, ELIspot format, as Dot blots, radioimmunoassays and the like.
- the wet assay format has been optimised into a dry assay format. Moreover, sensitivity has been optimised by titrating RBD, attenuated SARS-CoV-2 or SARS-CoV-2 viruses spiked into negative saliva samples on the dry assay format to evaluate the assay performance.
- the assay is evaluated using clinical samples to confirm those results obtained from the abovementioned mentioned titrations.
- Plotting procedure 1 mg/ml of human monoclonal anti SARS-CoV-2 Spike antibody is plotted on a CN140 membrane with a rate of 0.1 uL/mL to form a test line.
- Spray procedure 40 nm OD20 gold colloid conjugated with 15 ⁇ g/ml ACE2 with 20 mM MES pH 5.3 was prepared and sprayed onto the conjugation pad. Strips were closed using 1-well Kanani housing tops and bottoms and passed through a cassette sealer. The devices were placed in a labelled aluminium pouch with sufficient desiccant before sealing.
- the concentration of gold is at OD2015 ⁇ g/ml ACE2 and 8 ⁇ l of the material was deposited centrally onto the conjugate pad.
- RBD spikes are diluted to the required concentration, or if testing with saliva, the initial concentration is doubled as RBD and saliva are combined 50:50.
- Gold is at OD20 (15 ⁇ g/ml ACE2 from R&D); 8 ⁇ l deposited centrally onto the conjugate pad.
- SARS-CoV2 virus drooled saliva, universal buffer, 12 SARS-CoV-2 Ag tests (as disclosed herein), eppendorf tubes, pipettes, tips and a timer.
- the sample was vortexed and inverted to ensure appropriate mixture to create a homogeneous sample. Dilutions were performed as follows to make up 5x10 3 , 2x10 3 and 5x10 2 and 0 pfu/mL from the provided 3x10 4 PFU/mL viral stock solution as follows.
- Dilutions were performed as follows to make up 5x10 3 , 2x10 3 and 5x10 2 and 0 PFU/mL from provided 3x10 4 PFU/mL viral stock solution as follows:
- Titration was performed in singles and a limit of detection of 5x10 2 PFU/mL was visible. Second titration on 20 healthy saliva samples were performed.
- SARS-CoV-2 isolate Culture materials, General consumables: filter tips, micropipette, vortex, PPE, reagent reservoirs, towels etc., Category 3 facilities and PPE, Human Coronavirus 229E, OC43, NL63, Influenza A and B, Rhinovirus and Adenovirus, 4 SARS-CoV-2 Rapid Antigen Tests devices (as described herein).
- This validation protocol will evaluate the specificity of the test by testing potentially cross-reactive microorganisms.
- the following microorganisms were tested in triplicate. The results were interpreted by two operators, each blinded to the result of the other. If a discrepant result was obtained, a third operator was called for a result. Invalid results are to be repeated once.
- Trimeric spike protein 1 mg/mL, Mcllvaine buffer with 0.3% DDAO pH 7.4.
- 1 mg/ml_ of Trimeric spike was diluted in saliva to make a 5 ng/mL solution.
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Abstract
La présente invention concerne des procédés nouveaux et améliorés pour la détection d'agents pathogènes, par exemple des pathogènes viraux, dans des échantillons. Les procédés, les tests et les dosages permettant l'identification d'une infection, par exemple une infection par le Coronavirus, dans un échantillon, mais peuvent également être utilisés en tant que moyen pour identifier l'état infectieux ou le "degré d'infectivité" d'un sujet. L'invention concerne un procédé de détection d'un antigène dans un échantillon, ledit procédé comprenant: la mise en contact de l'échantillon avec un agent de liaison à l'antigène, l'agent de liaison à l'antigène n'étant pas un anticorps.
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EP4220166A1 (fr) * | 2022-02-01 | 2023-08-02 | Universitat Rovira I Virgili (URV) | Procédé de détection in vitro d'anticorps dans un échantillon |
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CN111024954A (zh) * | 2020-03-09 | 2020-04-17 | 深圳市易瑞生物技术股份有限公司 | 联合检测covid-19抗原和抗体的胶体金免疫层析装置及其使用法 |
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