WO2021186003A1 - Formats d'écoulement latéral améliorés pour écoulement optimisé et sensibilité accrue - Google Patents

Formats d'écoulement latéral améliorés pour écoulement optimisé et sensibilité accrue Download PDF

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Publication number
WO2021186003A1
WO2021186003A1 PCT/EP2021/057027 EP2021057027W WO2021186003A1 WO 2021186003 A1 WO2021186003 A1 WO 2021186003A1 EP 2021057027 W EP2021057027 W EP 2021057027W WO 2021186003 A1 WO2021186003 A1 WO 2021186003A1
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Prior art keywords
sample
lateral flow
flow device
zone
conjugate
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PCT/EP2021/057027
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English (en)
Inventor
Robert Porter
Mohamad TAKWA
Vasiliki FRAGKOU
Milo Thomas WRIGHT
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Aegirbio Ab
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Publication of WO2021186003A1 publication Critical patent/WO2021186003A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—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

Definitions

  • the present invention relates to lateral flow devices and methods for controlling the interactions between reagents and components of a sample.
  • Lateral flow devices or simply strip tests, are usually self-contained, portable devices that are easy to use, fast, and inexpensive. Lateral flow devices can be stored at ambient temperature, have a long shelf life, and provide diagnostic results without complex sample processing or additional equipment, making them ideal for both point- of-care and field-based diagnostic uses.
  • the purpose of these devices are to detect a target compound in a liquid sample, where the detection can be done both semi-quantitatively or quantitatively. For some lateral flow devices it is not necessary to quantitate the target compound, it is enough to detect or not detect the presence of a target substance, such as pregnancy tests or screening for microbial infections.
  • a typical lateral flow device is composed of several porous materials, onto which assay reagents are striped, sprayed, or spotted, then dried for storage in spatially distinct locations, and through which analyte, in a clinical or environmental sample, and assay reagents are transported by capillary action.
  • a sample pad is used for receiving a sample, localized at one end of the strip. The sample will then flow by capillary action through a conjugate pad, where conjugates, comprising indicator particles (such as colloidal gold) together with detection/capture molecules specific for the target compound, have been deposited and dried. When the sample gets in contact with the dried conjugates, they dissolve and bind to the target compound in the sample, thereby forming a complex.
  • test lines contain target-specific molecules, e.g. antibodies.
  • a visually present test line indicates that the target compound has bound to the test line, and that it therefore is present in the sample.
  • the lateral flow device typically comprises a control line, which binds and immobilizes unbound conjugates. A visually present control line indicates that the test has operated correctly.
  • lateral flow devices While lateral flow devices have gained widespread use, other methods, for example other antibody-based tests such as ELISA, are typically associated with a superior sensitivity compared to conventional lateral flow devices.
  • the sensitivity of the lateral flow device is still inferior to comparable analytical instruments, limiting the use of lateral flow devices.
  • the present inventors have realized that although the interactions between the sample and the reagents, such as the conjugates, are an essential component in the generation of a visible signal, they can also have a detrimental effect to the sensitivity and specificity of lateral flow devices. Therefore, by controlling the interactions the negative effects can be mitigated.
  • conjugates are deposited on a conjugate pad and allowed to interact with the sample upon dissolution, may not result in optimal interactions between the sample and the conjugates.
  • a target compound is present in the sample, a substantial amount of conjugates may be dissolved and arrive at the test line without having been given the opportunity to interact with the target compound, potentially leading to inconclusive test results.
  • the agglutinate may further act to restrict fluid flow of the device and alter the displacement of the sample and/or conjugates.
  • a network of conjugates and target compounds may be formed for example if the target compound comprises multiple copies of the epitope towards which the conjugate is directed. Additionally, for a competitive assay, if the conjugate reaches the target line before the sample, the sensitivity and specificity of the test decreases.
  • the invention relates to a lateral flow device for detecting the presence of at least one target compound in a sample, the lateral flow device comprising:
  • a membrane layer comprising at least one test line, the at least one test line including an immobilized capturing agent for capturing the at least one compound in the sample;
  • a reagent zone comprising:
  • a sample loading zone for receiving the sample; wherein the sample loading zone is located, along the axial length of the lateral flow device, between the conjugate zone and the at least one test line.
  • While the analysis of a sample, comprising a target compound having multiple copies of an epitope, by conventional lateral flow devices may lead to formation of large complexes.
  • the present disclosure may prevent this from occurring. Thereby, the fluid flow through the device may not be restricted by large complexes which may act to exclude at least a part of the sample and/or the conjugates from reaching the test lines and/or control lines.
  • An important aspect of the present disclosure is therefore mitigation of the drawbacks associated with complex formation, at undesirable locations of the lateral flow device, which could result in non-optimal flow behaviour and, as a result, a decreased sensitivity and specificity.
  • the location of the sample loading zone, with respect to the conjugate zone and the test lines, has been revealed to be an important factor for achieving lateral flow devices with an optimal flow behaviour.
  • the lateral flow device may comprise conjugates, typically provided in a dry state as part of the conjugate zone.
  • the conjugates may be provided as part of a buffer solution and/or a running buffer. In such a case, the running buffer and/or buffer solution may also be referred to as a conjugate solution.
  • the conjugates comprise a label and at least one conjugate protein binders. The conjugates may thereby be configured to bind to the one or more target compounds and, thereby provide an indication of the presence of the target compound in the sample.
  • the indicator for the presence of the target compound may be a visually observable line at the test line, or the absence of a visually observable line at the test line, preferably in combination with a visually observable line at the control line.
  • a sandwich assay generates a visually observable line, if the target compound is present in the sample, while a competitive assay would result in no visually observable line at the test line.
  • the lateral flow device may be configured with a combination of a number of sandwich assay test lines and a number of competitive test lines. In general, it may be a preference that the control line is visually present for indicating that the test has operated correctly
  • the conjugate zone is typically located near an end of the lateral flow device, such as a proximate end.
  • the proximate end may be put in contact with a buffer solution, potentially comprising the conjugates.
  • the solution will thereafter, by capillary action, flow through the device, and may act to drive the sample towards the test line, while maintaining a control of the interactions between the sample and the conjugates.
  • the sample reaches the test line before the conjugates, thereby the capturing agent may bind to the target compound, if provided in a sandwich assay format, and the conjugates may thereafter bind and form a visually observable indication of the presence of the target compound.
  • the lateral flow device comprises a filter for filtering a sample before contacting the membrane layer.
  • the filter may be provided to cover at least a part of the sample loading zone.
  • the filter may be a porous structure with pores in the size range between 0.1 and 1 pm.
  • the filter may for example be configured to filter out mucus structures of the sample.
  • the kit for detecting the presence of a target compound in a sample, the kit comprising:
  • a lateral flow device comprising: o a membrane layer, comprising at least one test line, the at least one test line comprising an immobilized capturing agent for capturing the at least one target compound in the sample; and o a reagent zone comprising:
  • sample loading zone for receiving the sample; wherein the sample loading zone is located, along the axial length of the lateral flow device, between the conjugate zone and the at least one test line.
  • the kit further comprises a buffer solution for mixing with the sample.
  • the buffer solution may be selected to decrease non-specific binding when performing the test, dissolve components, such as mucus, of the sample, stabilizing the sample, and/or to adjust the viscosity of the sample.
  • the kit may be provided with a receptacle containing the buffer solution.
  • the sample is to be mixed with the buffer solution at a ratio of between 1:5 and 1:15, such as at a ratio between 1:7 and 1:12.
  • the sample is a saliva sample.
  • Saliva samples have been shown to comprise numerous relevant disease markers, and the process of obtaining saliva samples is associated with limited inconvenience for the patient, as compared to collection of other sample types, such as a nasopharyngeal sample. For example 0.1-1 ml of saliva may rapidly be collected from the mouth of a patient and has been shown to allow for sensitive quantitative and/or qualitative measurements of a target analyte in a sample.
  • the kit may advantageously be provided, in the dry unused state, with a conjugate zone comprising conjugates.
  • the conjugates may for example have been striped, sprayed, or spotted, then dried on the conjugate zone before use of the lateral flow device.
  • a fluid such as the running buffer, may act to displace the conjugates towards the one or more test lines, a control line and/or an absorption pad.
  • the conjugates may be provided as part of the running buffer.
  • the kit may for example comprise a receptacle retaining the running buffer, and may further be configured such that the running buffer/conjugate solution is applied to the lateral flow device by exposing the proximate end of the lateral flow device to the running buffer/conjugate solution.
  • the fluid may act to transport the sample to the test line without the formation of a significant amount of complexes comprising the conjugate and the target compound at undesirable locations.
  • the invention relates to a method for detecting the presence of at least one target compound in a sample, the method comprising the following steps:
  • a lateral flow device comprising:
  • reagent zone comprising:
  • a sample loading zone for receiving the sample; wherein the sample loading zone is located, along the axial length of the lateral flow device, between the conjugate zone and the at least one test line.
  • indicators may either be the presence of a visible line or the absence of a visible line.
  • the test line is a sandwich assay or a competitive assay.
  • the lateral flow device may further be configured with a combination of one or more sandwich assays and one or more competitive assays. Due to the high sensitivity, a wide range of target compounds can be detected, even at low concentrations.
  • the target compound may be a biomarker for a viral disease, such as coronavirus infection.
  • the capturing agent, at the test line may in such a case comprise a synthetic fragment of the virus, such as an epitope. In this arrangement, the epitope of the test line, may be substantially identical, such as functionally identical, to the binding counterpart of the conjugate.
  • the running buffer may be added to substantially the same location of the lateral flow device as the sample.
  • Fig. 1 shows a schematic illustration and the use of a lateral flow device, according to a specific embodiment of the present disclosure.
  • Fig. 2 shows a schematic illustration and the use of a lateral flow device comprising a conjugate zone that, before use, had been preloaded with conjugates, according to a specific embodiment of the present disclosure.
  • Fig. 3 shows schematic illustrations of a lateral flow device comprising three test lines and a control line, and its use, according to a specific embodiment of the present disclosure..
  • Fig. 4 shows schematic illustrations of possible outcome of measurements of patient blood by a lateral flow device for viral infections, according to a specific embodiment of the present disclosure.
  • Fig. 5 shows lateral flow devices following measurements of IgG, IgM and virus particles, according to a specific embodiment of the present disclosure..
  • Fig. 6 shows a schematic illustration of a lateral flow device for serological assay IgG/lgM, according to a specific embodiment of the present disclosure.
  • Fig. 7 shows a schematic illustration of a lateral flow device for detection of virus particles, according to a specific embodiment of the present disclosure..
  • Fig. 8 shows lateral flow devices following measurements of virus particles, according to a specific embodiment of the present disclosure.
  • Fig. 9 shows a schematic illustration of a lateral flow device according to a specific embodiment of the present disclosure.
  • Fig. 10 shows a schematic illustration of a lateral flow device according to a specific embodiment of the present disclosure.
  • the present invention relates to a lateral flow device for detecting the presence of a target compound in a sample.
  • the device may comprise a membrane layer and a loading zone. Said loading zone may be provided as a part of the membrane layer or may be a separate component, but preferably in fluidic contact with the membrane layer.
  • the membrane layer may comprise one or more test lines comprising immobilized capturing agent(s) specific for one or more target compounds. Typically, each test line comprises a specific type of capturing agent. Thereby, each test line is directed towards a specific target compound.
  • the lateral flow device may comprise a loading zone.
  • Said loading zone may comprise a conjugate zone and a sample loading zone, wherein the conjugate zone may comprise the conjugates and/or wherein the conjugate zone is configured to receive the conjugates, typically after the sample/sample solution has been provided to the sample loading zone.
  • the sample loading zone and the conjugate zone may be provided as part of a single unit, for example a reagent zone or a reagent pad. However, the sample loading zone and the conjugate zone may further be provided as separate units, preferably still in fluidic contact. It is a preference that the sample loading zone is located, along the axial length of the lateral flow device, between the conjugate zone and the at least one test line.
  • the lateral flow device comprises an absorption pad at a distal end of the lateral flow device.
  • the proximate end of the device comprises the conjugate zone.
  • the buffer solution is applied to the device by dipping the proximate end of the device into said buffer solution. Consequently, it may be a preference that the conjugate zone is located at, or near, the proximate end of the device. In general, it is a preference that the conjugate zone is located towards the proximate end of the device with respect to the sample loading zone.
  • the sample loading zone is located between the conjugate zone and the test line, thereby allowing the sample to flow before the conjugate, which will decrease the risk of the sample and conjugate mixing and potentially aggregating, thereby increasing the risk of them not reaching the test lines.
  • conjugate refers to two molecules that are bound to each other.
  • it may refer to a particle, such as gold, latex or any other colloidal material, bound to the target compound or a protein containing the epitope or epitopes of the target compound.
  • the particle can also be bound to other detection/capture molecules specific for the target compound.
  • aptamer refers to a single-stranded oligonucleotide (single- stranded DNA or RNA molecule) that can bind specifically to its target with high affinity.
  • the aptamer can be used as a biosensor element capable of binding to a molecule in a detection/analysis system, and thus has been recognized as a substitutive for antibody.
  • the aptamers can be used as molecules targeting various organic and inorganic materials, including toxins, unlike antibodies, and once an aptamer binding specifically to a certain material is isolated, it can be consistently reproduced at low costs using automated oligomer synthesis methods.
  • affimer refers to small, engineered non-antibody binding proteins that can bind target molecules and are designed to mimic the molecular recognition characteristics of monoclonal antibodies.
  • Affimer reagents are suitable for use in for example biosensors and point-of-care diagnostics.
  • polymer refers to macromolecular materials having at least five repeating monomeric units, which may or may not be the same.
  • a molecularly imprinted polymer is a polymer that has been processed using the molecular imprinting technique which leaves cavities in the polymer matrix with an affinity for a chosen template molecule.
  • Molecularly imprinted polymers possess the most important features of biological receptors - recognition.
  • Molecularly imprinted polymers can comprise cross linked polymers. They can also comprise amorphous metal oxides or zeolites. Metal oxides and zeolites can be imprinted using a variety of known techniques. In some cases, the cavities or pores produced are an induced fit for polymers of the imprinting molecules.
  • the conjugate zone of said lateral flow device comprises conjugates.
  • the conjugate zone in the dry unused state, may comprise at least one type of conjugate.
  • the conjugates may thereby have been preloaded on the device, such as on a conjugate pad.
  • Conjugates may for example be applied to the conjugate zone with the use of an air jet dispensing platform or by immersion.
  • Several machines with hollow fibre dispensers used to stripe nitrocellulose membranes can also be configured with an air jet spray apparatus to dispense the conjugates onto the conjugate zone.
  • the conjugate zone does not comprise conjugates.
  • a conjugate comprises a label and at least one conjugate protein binder, such as multiple conjugate protein binders.
  • the conjugate protein binders may be configured for binding to an immobilized capturing agent, at any of the test lines. In such cases, the presence of a target compound at a test line, may result in the conjugate not being able to bind to the immobilized capturing agent.
  • the conjugate protein binder may further be configured to bind to a target compound. In such cases the absence of a target compound at a test line may result in the conjugate not being immobilized at the test line.
  • the conjugate protein binder, in combination with the capturing agent and the target compound may be configured for binding according to a sandwich assay or a competitive assay. It should be appreciated that a lateral flow device comprising multiple lines, such as test lines and control lines, may comprise lines configured for sandwich assay format and lines configured for competitive assay format.
  • the label is a metal nanoparticle, such as a gold nanoparticle, and/or a coloured latex particle.
  • the capturing agent may further be any other type of sol-based material.
  • a sol is a colloid made out of very small solid particles in a continuous liquid medium. Colloidal gold is the most widely used label in lateral flow devices, it has an intense colour and no development process is needed for visualization. Moreover, it has high stability in both liquid and dried forms.
  • Another popular label is latex, which can be tagged with a variety of detector reagents such as coloured or fluorescent dyes, and magnetic or paramagnetic components.
  • latex As latex can be produced in multiple colours, it has an application in multiplex assays, which require discrimination between numerous lines.
  • Carbon such as carbon nanotube, fluorescent labels, or enzymatic modification of the labels, may also be used to improve the sensitivity of the assay.
  • the label may further be a fluorescent nanoparticle, such as a quantum dot.
  • the conjugate protein binder is selected from the list including an antibody, an Ig unit, an aptamer, an affimer, an RNA molecule, a DNA molecule, an organic polymer, or a fragment thereof.
  • the conjugate may comprise a single or multiple conjugate protein binders.
  • the organic polymer is a molecularly imprinted polymer.
  • the membrane layer of said lateral flow device comprises multiple test lines for capturing and/or detecting the presence of multiple target compounds in the sample.
  • a multiplexed lateral flow device preferably comprises multiple test lines, wherein each test line comprises a binding agent for a specific target compound.
  • the lateral flow device may thereby for example detect the presence of pathogens, such as virus or bacteria, while simultaneously having test lines comprising binding agents directed at capturing and/or detecting the presence of IgG and IgM in the sample.
  • pathogens such as virus or bacteria
  • test lines comprising binding agents directed at capturing and/or detecting the presence of IgG and IgM in the sample.
  • the presence of IgG and IgM should here be understood as an elevated concentration of IgG and IgM, as to normal reference values, such as reference values for the patient group of the individual which is diagnosed.
  • the pathogen such as a virus
  • the immune system produces IgM antibodies.
  • the immune system commences producing a more effective and long-term IgG response in order to eradicate the infection and prevent a future re-infection.
  • a positive IgM test indicates that the subject has been infected and that the subject’s immune system has started responding to the pathogen.
  • the subject may still be infected, or the subject may have recently recovered from the pathogen infection.
  • IgG antibodies In most subjects IgG is developed within seven to ten days after manifestation of symptoms of the pathogen infection has begun, and the IgG antibodies remain in the blood after an infection has passed. Consequently, IgG antibodies indicate that the subject has had a viral infection in the recent past and has developed antibodies that may protect the subject from future infection.
  • the presence of IgG, the presence of IgM and the presence of pathogens, such as bacteria and/or virus may be used.
  • the clinical staging may result in six different scenarios as shown in the table below.
  • test strip that is capable of measuring all three parameters, according to table 1, will aid in the triage and allows for a rapid decision of a need for medical intervention.
  • Clinical symptoms of an infection such as COVID-19, may be taken into account during triage. For example, if a measurement by the lateral flow test strip reveals that the subject has developed IgG antibodies but no IgM or pathogens are detected, the clinical symptoms may be used to classify whether the subject is coming to an end of the infection, or whether the immune system is not capable of eliminating the pathogen, and thereby requires urgent medical intervention.
  • each test line comprises a different immobilized capturing agent, such as a first capturing agent, a second capturing agent, a third capturing agent and/or a fourth capturing agent.
  • the lateral flow device has a test line comprising a first capturing agent, another test line comprising a second capturing agent and yet another test line comprising a third capturing agent.
  • the lateral flow device may further comprise even yet a further test line comprising a fourth capturing agent.
  • the first capturing agent may be selected to capture IgM antibodies
  • the second capturing agent may be configured for capturing IgG antibodies
  • the third capturing agent may be configured for capturing pathogen particles, complexes thereof or fragments thereof.
  • the fourth line may be a control line configured to provide a response, typically a visual response, of an accurate measurement.
  • each test line is configured such that it can detect the presence of a different target compound.
  • the lateral flow device is configured for operation as a sandwich assay.
  • sandwich assays generally are used for larger compounds since they tend to have multiple binding sites.
  • the sample will come in contact with the conjugate and migrate together towards the test line of the lateral flow device, where a visually present test line indicates the presence of the target compound.
  • the sandwich assay format is typically used for detecting larger analytes that have at least two binding sites, or epitopes.
  • an antibody to one binding site is conjugated to the nanoparticle, and an antibody to another binding site is used for the assay’s test line.
  • the analyte will bind to both the antibody-nanoparticle conjugate and to the antibody on the test line, yielding a positive signal.
  • the sandwich format results in a signal intensity at the test line that is directly proportional to the amount of analyte present in the sample. Regardless of the quantity of analyte in the sample, an anti-species antibody at the control line will bind the nanoparticle, yielding a strong control line signal that demonstrates that the assay is functioning correctly.
  • the competitive format is typically used for detecting analytes when antibody pairs are unavailable or if the analyte is too small for multiple antibody binding events, such as steroids and drugs.
  • the test line typically contains the analyte molecule, usually a protein-analyte complex, and the conjugate pad contains the detection antibody-nanoparticle conjugate. If the target analyte is present, the analyte will bind to the conjugate and prevent it from binding to the analyte at the test line. If the analyte is not present, the conjugates will bind to the analyte at the test line, yielding a signal.
  • the signal intensity is inversely proportional to the amount of analyte present in the sample.
  • the control line will bind the nanoparticle conjugate with or without the analyte providing confidence that the assay is working correctly.
  • the conjugate can be said to be a detection particle, as it may, in certain embodiments of the present disclosure, provide an observable signal at a test line regarding the presence of a specific particle, such as a pathogen, in the sample.
  • the lateral flow device is configured for operation as a competitive assay.
  • competitive assays generally are used for smaller compounds, but is also preferable for compounds with multiple epitopes.
  • the lateral flow device simultaneously functions as both a sandwich assay and a competitive assay.
  • the membrane layer of said test directly contacts the test line.
  • the immobilized binding agent may thereby be immobilized to a part of the membrane layer.
  • the conjugate zone of said test is located at a proximate end of the lateral flow device, such as wherein the test line is towards the distal end of the lateral flow device with respect to said conjugate zone.
  • the lateral flow device further comprises an absorbent pad, located at a distal end of the lateral flow device.
  • the absorbent pad may for example be provided as part of the reagent zone.
  • An absorbent pad allows for controlled release of sample and conjugate onto the lateral flow device and to filter undesired components.
  • the absorbent pad may be in any type of material that absorbs water, such as cellulose fibre or woven meshes.
  • the application pad comprises the conjugate zone and/or the sample loading zone, such as a conjugate pad and/or a sample pad.
  • the membrane layer comprises the reagent zone.
  • the absorbent pad may be configured to increase the total volume of sample that can enter the test strip.
  • the bed volume of any membrane is finite, and having an absorbent pad at the distal end of the test strip can increase the volume of sample that can be run across the membrane as it acts as a sponge for the additional volume.
  • the presence of an absorbent pad can contribute to the reduction of non-specific binding and sensitivity. This may be accomplished due to the additional volume that can run across the test line washing non-specifically bound material off the test line, and allowing for an increase in totally analyte concentration to reach the test line.
  • the lateral flow device further comprises an application pad, and wherein the application pad comprises the conjugate zone and/or the sample loading zone.
  • the lateral flow device comprises a backing layer in an inert material.
  • Inert materials are materials which have minimal or almost no chemical or biological activity when present in the environment. It is a strong preference that the membrane layer comprises or consists of a porous layer for wicking of a liquid sample.
  • the membrane layer of said test comprises or consists of nitrocellulose, cellulose acetate or paper.
  • the lateral flow device comprises a cassette, and/or wherein the lateral flow device is configured such that is can be operated as a dipstick
  • said capture protein binder is selected from the list including an antibody, an aptamer, an affimer, an RNA molecule, a DNA molecule, an organic polymer, the target compound, a derivative of the target compound, or a fragment thereof.
  • the capture protein binder may for example comprise a virus particle, or a fragment thereof, for example attached to a colloidal particle at one or more of the test lines.
  • said organic polymer is a molecularly imprinted polymer.
  • the lateral flow test typically comprises multiple test lines, where each test line is configured to capture and/or detect a different compound.
  • the lateral flow device comprises multiple test lines, it is a preference that the device comprises one test line configured for capturing IgM, one test line configured for capturing IgG, one test line configured for capturing a pathogen particle, such as a virus particle.
  • the lateral flow device typically also comprises a test line for providing an indication of an accurate measurement.
  • any of the capturing agents comprises a capture protein binder, typically wherein each capturing agent has a unique capture protein binder.
  • the capture protein binder may be selected from the list including an antibody, an aptamer, an affimer, an RNA molecule, a DNA molecule, an organic polymer, the target compound, a derivative of the target compound or a fragment thereof.
  • the capture protein binder may for example comprise a virus particle, or a fragment thereof, for example attached to a colloidal particle at one or more of the test lines.
  • said organic polymer is a molecularly imprinted polymer.
  • the capturing agent and/or conjugate of the lateral flow device disclosed herein is specific for an epitope of the target compound.
  • said target compound comprises multiple copies of the epitope.
  • the membrane layer of the lateral flow device disclosed herein comprises a control line.
  • said control line comprises an immobilized capturing agent specific for binding to the conjugate.
  • said control line comprises the target compound, or a fragment thereof.
  • the lateral flow device comprising a reagent zone (2) and a membrane layer (3).
  • the test lines are here shown as visually observable and comprises a first test line (11), a second test line (12), and a third test line (13).
  • the device furthermore comprises a control line (14).
  • the lateral flow device is targeting triaging of viral infections.
  • the first test line is configured to detect the presence of virus in the sample, thereby comprising a capturing agent specific for an epitope of the surface of the virus particle (19)
  • the second test line is configured to detect the presence of IgM
  • the capturing agent may consequently be an anti-human IgM antibody (20)
  • the third test line is configured to detect the presence of IgG
  • the capturing agent may consequently be anti-human IgG antibody (21).
  • the control line is configured to target a conjugate, such as a non related control line conjugate, and may consequently be an antibody targeting this conjugate (22).
  • the invention in another aspect, relates to a kit for detecting the presence of a target compound in a sample.
  • the kit may comprise a lateral flow device and reagents.
  • the kit may comprise a running buffer, for example that acts as a chase buffer, the kit may alternatively or additionally comprise a buffer solution to be mixed with the sample thereby forming a sample solution that is to be added to the lateral flow device.
  • the buffer solution may for example act to stabilize and/or modify the viscosity of the sample.
  • the kit may comprise a lateral flow device comprising a membrane layer.
  • the membrane layer may comprise at least one test line comprising an immobilized capturing agent.
  • the lateral flow device may comprise a reagent zone, such as for receiving sample, conjugates, and/or buffer solution.
  • the reagent zone may comprise a conjugate zone for receiving the running buffer, and/or a sample loading zone for receiving the sample. It is a preference that the sample loading zone is located, along the axial length of the lateral flow device, between the conjugate zone and the at least one test line.
  • the lateral flow device comprises an absorption pad at a distal end of the lateral flow device. Near the opposite end, the proximate end, of the strip, it may be advantageous to have the conjugate zone located.
  • the running buffer may be applied to the conjugate zone by dipping the proximate end of the lateral flow device in the buffer solution. It is a preference that the conjugate zone is nearer the proximate end than the sample loading zone.
  • the kit comprises a buffer solution, the buffer solution comprising sodium chloride, bovine serum albumin, and a borate buffer. In one embodiment of the present disclosure, the kit comprises a buffer solution, the buffer solution consisting of sodium chloride, bovine serum albumin, and a borate buffer.
  • the kit comprises a buffer solution, the buffer solution comprising 1 M sodium chloride, 1% w/v bovine serum albumin, and 0.1M borate buffer.
  • the kit comprises a buffer solution, the buffer solution consisting of 1 M sodium chloride, 1% w/v bovine serum albumin, and 0.1M borate buffer.
  • the kit comprises a lateral flow device configured as disclosed elsewhere herein.
  • the conjugate zone of said kit comprises, in the dry unused state, the conjugates.
  • said conjugates are provided in a dry state, and upon application of a mobile phase, said conjugates will flow substantially with the mobile phase.
  • said kit comprises a receptacle comprising the conjugates, as part of a conjugate solution.
  • the lateral flow device is lowered into the conjugate solution in a conjugation well without lowering the sample loading zone in the buffer.
  • the conjugates in the conjugate solution will flow into the membrane and continue along the lateral flow device.
  • the conjugates are configured such that upon application of the running buffer, said conjugates will flow substantially with the running buffer, such as from a proximate end of the lateral flow device to a distal end of the lateral flow device.
  • the conjugates are provided as part of the running buffer, or such that the conjugates can be mixed with the running buffer before application to the lateral flow device.
  • the kit comprises a receptacle for retaining the running buffer.
  • the kit is configured such that the running buffer may be applied to an end of the lateral flow device, such as the proximate end, by pipetting, and/or such that the running buffer may be applied to the lateral flow device by dipping an end of said lateral flow device, such as the proximate end, in the receptacle.
  • the invention relates to a method for detecting the presence of at least one target compound in a sample.
  • the method may comprise the provision of a kit as disclosed herein.
  • the method may comprise a lateral flow device and reagents.
  • the kit may comprise a running buffer and/or a buffer solution.
  • the method may comprise the provision of a lateral flow device comprising a membrane layer.
  • the membrane layer may comprise at least one test line comprising an immobilized capturing agent.
  • the lateral flow device may comprise a reagent zone, such as for receiving sample, conjugates, and/or buffer solution.
  • the reagent zone may comprise a conjugate zone for receiving the running buffer, and/or a sample loading zone for receiving the sample.
  • the sample loading zone is located, along the axial length of the lateral flow device, between the conjugate zone and the at least one test line.
  • the lateral flow device comprises an absorption pad at a distal end of the lateral flow device.
  • the proximate end, of the strip it may be advantageous to have the conjugate zone located.
  • the running buffer may be applied to the conjugate zone by dipping the proximate end of the lateral flow device in the buffer solution. It is a preference that the conjugate zone is nearer the proximate end than the sample loading zone.
  • the method may further comprise the step of applying the sample to the sample loading zone.
  • the lateral flow device, and the sample volume is configured and/or selected such that the majority of the sample progress towards the absorption pad, and does not interact with any present conjugates.
  • the method may comprise the step of applying the running buffer to the conjugate zone. Thereby, the running buffer may reconstitute the conjugates, if provided preloaded on the lateral flow device. Further the method may comprise the step of waiting a sufficient amount of time for the sample and the conjugates to flow over the test lines. The sufficient time may for example be until the control line is visually observable.
  • the method may comprise the step of detecting, for each test line, the presence of at least one indicator. The method may thereby be used for detecting the presence of at least one target compound in the sample.
  • the method may also comprise the step of mixing the sample with the buffer solution, wherein the volumetric ratio between the buffer solution and the sample is between 5 parts buffer solution and 1 part sample and 15 part buffer solution and 1 part sample, thereby obtaining a sample solution.
  • This step may be important for reducing the viscosity of the sample and allowing a sensitive and specific detection of the at least one target compound.
  • the method may comprise the step of applying the sample solution to the sample loading zone. Further the method may comprise the step of waiting a sufficient amount of time for the sample and the conjugates to flow over the test lines. The sufficient time may for example be until the control line is visually observable.
  • the method may comprise the step of measuring, such as detecting, for each test line, the presence of at least one indicator. The method may thereby be used for detecting the presence of at least one target compound in the sample.
  • the kit is configured as disclosed elsewhere herein, and/or wherein the lateral flow device is configured as disclosed elsewhere herein.
  • the application of the sample and/or the application of the running buffer is configured such that a substantial fraction of the sample is allowed to flow over the test line before the conjugates.
  • the sample is first allowed to flow over the test lines before loading the conjugate.
  • this prevents the conjugate and sample from mixing prior to reaching the test line, thereby avoiding aggregation on the test lines.
  • the lateral flow device of said method is provided with the conjugates present at the conjugate zone, such as in the dry unused state.
  • the running buffer of said method does not comprise conjugates.
  • the lateral flow device of said method is provided without the conjugates present at the conjugate zone.
  • the running buffer of said method comprises conjugates
  • the running buffer of said method is added to the loading zone, such as the conjugate zone, and/or a proximate end of the lateral flow device, at or near the conjugate zone.
  • the running buffer is first applied to an area between the sample loading zone and the conjugate zone, followed by a second application of running buffer to the conjugate zone and/or the proximate end of the lateral flow device, wherein the first application is carried out for a predetermined time and/or consists of application of a predetermined volume.
  • said running buffer is applied to the loading zone by dipping an end of the lateral flow device in the running buffer.
  • the sufficient time is the time lapsed until the control line is expected to be detectable, such as visually detectable.
  • the indicators of the method disclosed herein are a number of immobilized conjugates comprising the label, such as forming a visually detectable line.
  • a major fraction of the sample is allowed to migrate from the sample loading zone to the test lines and contact the capturing agents without the presence of conjugates, such as before the conjugates.
  • said conjugates are allowed to migrate from the loading zone to the test lines and, if the target compound is bound to the capturing agent and the test line, are present, said conjugates bind to their respective test lines, thereby forming a detectable signal.
  • said detectable signal indicates the presence of the target compound in the test sample.
  • the target compound of the method disclosed herein is a biomarker for an infectious disease.
  • said infectious disease is a viral disease.
  • said viral disease is a coronavirus infection.
  • the coronavirus infection may be of the type SARS-CoV-2, also referred to as COVID-19.
  • the membrane layer of the method disclosed herein comprises a test line that comprises or consists of an immobilized capturing agent specific for IgM antibodies, such as human IgM antibodies, such as a first test line.
  • said membrane layer comprises a test line that comprises or consists of an immobilized capturing agent specific for IgG antibodies, such as human IgG antibodies, such as a second test line.
  • said membrane layer comprises a test line that comprises or consists of an immobilized capturing agent specific for a biomarker of a disease, such as disease-specific antibodies, such as a third test line.
  • IgG antibodies is the most common type of antibody found in the blood circulation. A person skilled in the art will know that IgM antibodies are the first antibodies to be produced in the response to an initial exposure to an antigen. Therefore, IgM levels in the blood are indicative of an active infection. IgG antibodies, on the other hand, are generated following class switching and maturation of the antibody response, thus they participate predominantly in the secondary immune response, thereby indicating a long-term response to an infection.
  • the sample comprises or consists of whole blood, serum, plasma, nasal secretions, sputum, urine, saliva and/or stool.
  • the sample comprises or consists of saliva.
  • the sample has been pre-treated, such as by mixing with a buffer solution, that may comprise anticoagulants.
  • Anticoagulants are additives that inhibit for example blood and/or plasma from clotting, ensuring that the constituent that is to be measured, is non-significantly changed prior to the analytical process.
  • lateral flow devices typically are limited to analysis of urine, blood, or water samples, other kinds of samples may in many cases be more readily accessible or may comprise a higher amount of the target analyte.
  • lateral flow devices typically are limited to analysis of urine, blood, or water samples
  • other kinds of samples may in many cases be more readily accessible or may comprise a higher amount of the target analyte.
  • the incompatibility between conventional lateral flow devices and specific sample types results in that these samples cannot be measured using conventional lateral flow devices, or at best, that the sensitivity is limited.
  • the present invention relates to a method for detecting the presence of at least one target compound in a sample, the method comprising:
  • a lateral flow device comprising i. a membrane layer, comprising at least one test line, the at least one test line including an immobilized capturing agent for capturing the at least one target compound in the sample; ii. a sample loading zone for receiving the sample, the sample loading zone in fluidic connection with the test line;
  • the sample is a saliva sample.
  • Obtaining saliva samples is associated with limited inconvenience for the patient, as compared to collection of other sample types, such as a nasopharyngeal sample.
  • 0.1-1 ml of saliva may be rapidly collected from the mouth of the patient and has been shown to allow for sensitive quantitative and/or qualitative measurements of a target analyte in a sample.
  • an obtained saliva sample is between 0.1 and 0.2 ml, consequently it is a preference that the volume of the buffer solution is between 0.5 ml and 3 ml.
  • the obtained sample solution may be poured or dripped, e.g. pipetted, onto the sample loading zone.
  • the sample preferably a saliva sample
  • the buffer solution may be selected to decrease non-specific binding when performing the test, dissolve components, such as mucous, of the sample, stabilizing the sample, and/or to adjust the viscosity of the sample.
  • the method of the present disclosure provides several advantages over previously known methods.
  • One advantage is that sensitive and highly specific measurements are possible without a need for a running buffer.
  • a running buffer may be used, but the method and kits of the present disclosure can be used successfully without the need of adding a running buffer.
  • Another advantage of the method and kits of the present disclosure is that aggregation in the sample is avoided by pre-treating the sample with a suitable buffer solution to obtain a sample solution.
  • a further advantage of the method and kits of the present disclosure is that non-specific binding (NSB) to the immobilized capturing agent is minimized or even completely avoided.
  • the method and kits of the present disclosure allow a more efficient detection of the presence of a target compound in a sample by minimizing, such as reducing, the waiting time required for the sample solution to migrate to the membrane layer comprising the immobilized capturing agent.
  • said method does not need the use of a running buffer.
  • the presently disclosed method is capable of achieving highly sensitive and specific measurement without the need for a running buffer.
  • the buffer solution is an aqueous solution and/or wherein the sample is mixed with the buffer solution at a volumetric ratio of 1 part sample to between 2 and 15 parts buffer solution, more preferably between 5 and 10 parts buffer solution, yet more preferably between 9 and 10 parts buffer solution.
  • the buffer solution is advantageously mixed with the sample , as a buffer solution is mixed with the sample to obtain a sample solution, and the buffer solution present in the sample solution is sufficient to mobilize the analytes of the sample and/or the conjugates present at the conjugate zone, such as in the dry unused state.
  • the sample has been pre-treated, such as by mixing with a buffer solution that comprises sodium chloride, bovine serum albumin, and a borate buffer.
  • the sample has been pre-treated, such as by mixing with a buffer solution, that consists of sodium chloride, bovine serum albumin, and a borate buffer.
  • a buffer solution that consists of sodium chloride, bovine serum albumin, and a borate buffer.
  • the sample has been pre-treated, such as by mixing with a buffer solution that comprises 1 M sodium chloride, 1% w/v bovine serum albumin, and 0.1 M borate buffer. It has been found that a buffer comprising at least one, preferably all, these components effectively reduces viscosity of the sample, in particular wherein the sample is saliva, and thereby helps in preventing formation of aggregates within the sample, such as within the sample solution.
  • the sample has been pre-treated, such as by mixing with a buffer solution that consists of 1 M sodium chloride, 1% w/v bovine serum albumin, and 0.1 M borate buffer.
  • the sample is diluted in the buffer solution by mixing between 5 parts buffer solution and 1 part sample and 15 part buffer solution and 1 part sample, thereby obtaining a sample solution.
  • the sample is diluted in the buffer solution by mixing 1 part sample and at least 5 parts buffer solution, more preferably at least 6 parts buffer solution, more preferably at least 7 parts buffer solution, more preferably at least 8 parts buffer solution, even more preferably at least 9 parts buffer solution, most preferably at least 10 parts buffer solution, such as 11 parts buffer solution, or 12 parts buffer solution, or even 13 parts buffer solution, or 14 parts buffer solution, such as 15 parts buffer solution.
  • the sample is diluted in the buffer solution by mixing 1 part sample and 10 parts buffer solution.
  • the sample is saliva, and it is diluted in the buffer solution by mixing between 5 parts buffer solution and 1 part sample and 15 part buffer solution and 1 part sample, preferably about 10 parts buffer solution and 1 part sample, thereby obtaining a sample solution.
  • diluting the sample in buffer solution as described herein is important for reducing the viscosity of the sample, for example wherein the sample is saliva, and thus preventing formation of aggregates within the sample, such as within the sample solution.
  • the membrane layer has not been provided with a blocking agent, such as after plotting the test and/or control line. This helps to minimize the time required for measurement, such to minimize and/or reduce the time sufficient for the sample solution to arrive at the test lines, and for the control line to be detectable, such as visually detectable.
  • a common blocking agent is a solution comprising bovine serum albumin (BSA), which is typically applied to the membrane layer and allowed to dry. Other types of blocking agent are known to the person skilled in the art.
  • the capturing agents are configured for capturing a coronavirus, such as a human coronavirus, or fragments thereof.
  • the capturing agents are configured for binding to a spike protein, such as SARS-CoV S protein and/or SARS-CoV-2 S protein.
  • the method and/or the kits disclosed herein are suitable for diagnosis of an infectious disease, such as for diagnosis of COVID-19.
  • the present invention relates to a kit for detecting the presence of a target compound in a sample, the kit comprising:
  • a buffer solution for diluting the sample wherein said buffer solution comprises sodium chloride, bovine serum albumin, and a borate buffer;
  • a lateral flow device comprising:
  • a membrane layer comprising at least one test line, the at least one test line comprising an immobilized capturing agent for capturing the at least one target compound in the sample;
  • the kit may advantageously be used for detecting the presence of a target compound in a saliva sample.
  • the kit is suitable for running the method as disclosed elsewhere herein.
  • Fig. 1 shows schematic representations of a configuration of a lateral flow device according to a specific embodiment of the present disclosure.
  • Fig. 1A shows a schematic representation of a lateral flow device (1) of the present disclosure.
  • the device comprises a reagent zone (2), a membrane layer (3) and an absorption pad (4).
  • the reagent zone comprising a sample loading zone (5) and a conjugate zone (6).
  • the conjugate zone does not comprise conjugates.
  • Fig. 1 B shows the device during use, wherein a buffer solution (8) is applied to the lateral flow device, exemplified here by dipping said device into a receptacle (7) retaining the buffer solution comprising the conjugates.
  • the buffer solution is thereby allowed to travel from the proximate end of the lateral flow device, to the distal end of the device.
  • Fig. 2 shows schematic representations of a configuration of a lateral flow device according to a specific embodiments of the present disclosure.
  • Fig. 2A shows a schematic representation of a lateral flow device (1) of the present disclosure.
  • the device comprises a reagent zone (2), a membrane layer (3) and an absorption pad (4).
  • the reagent zone comprising a sample loading zone (5) and a conjugate zone (6).
  • the conjugate zone comprises conjugates.
  • Fig 2B shows the lateral flow device during use, wherein a buffer solution (8) is applied to the lateral flow device, exemplified here by dipping said device into a receptacle (7) retaining the buffer solution.
  • the buffer solution initially does not comprise conjugates, however upon contact with the conjugate zone, the conjugates are displaced by the buffer solution towards the absorption pad.
  • Fig. 3A shows schematic representation of a lateral flow device (1) comprising three test lines.
  • the lateral flow device comprising a reagent zone (2) and a membrane layer (3).
  • the test lines are here shown as visually observable and comprises a first test line (11), a second test line (12), and a third test line (13).
  • the device furthermore comprises a control line (14).
  • the lateral flow device is targeting triaging of viral infections.
  • the first test line is configured to detect the presence of virus in the sample, thereby comprising a capturing agent specific for an epitope of the surface of the virus particle (19)
  • the second test line is configured to detect the presence of IgM
  • the capturing agent may consequently be an anti-human IgM antibody (20)
  • the third test line is configured to detect the presence of IgG
  • the capturing agent may consequently be anti-human IgG antibody (21).
  • the control line is configured to target a conjugate, such as a non related control line conjugate, and may consequently be an antibody targeting this conjugate (22).
  • conjugates (15) may have been preloaded onto the lateral flow device.
  • the conjugates comprising one or more conjugate protein binders (16) and a label (17), such as gold sol.
  • a blood sample (18)
  • the sample flows mainly towards the test line and the distal end of the lateral flow device, thereby avoiding mixing with the conjugates.
  • the running buffer is provided to the conjugate zone, thereby acting to displace the conjugates.
  • the conjugates may directly bind to an immobilized capturing agent, or bind to a target compound that has bound to an immobilized capturing agent, additionally test lines may be configured such that the conjugates does not bind, if a target compound has already bound to the test line.
  • Fig. 4 shows various outcomes of a viral infection test comprising three test lines, comprising a first test line (11) comprising anti-virus antibodies (19), such as anti COVID-19 antibodies, a second test line (12) comprising anti-human IgM antibodies (20), a third test line (13) comprising anti-human IgG antibodies (21) directed at capturing IgM, and a control line (14) comprising antibodies directed at capturing non- related control line conjugates (22).
  • a first test line (11) comprising anti-virus antibodies (19), such as anti COVID-19 antibodies
  • a second test line (12) comprising anti-human IgM antibodies (20)
  • a third test line (13) comprising anti-human IgG antibodies (21) directed at capturing IgM
  • a control line (14) comprising antibodies directed at capturing non- related control line conjugates (22).
  • the presence of a visually present test line may either indicate the presence of the target compound or the absence of the target compound in the sample, depending on the configuration of the specific test line.
  • binding of a virus particle makes conjugates comprising synthetic fragments of virus as the conjugate protein binder not able to bind to the first test line, thereby producing an absence of a visual line.
  • the conjugates may comprise conjugate protein binders directed at the respective target compounds, thereby producing visual lines if the target compound is present.
  • test lines for human IgG and IgM antibodies function as a sandwich assay, where a visible line indicates the presence of these antibodies in the blood
  • the test line for the target compound functions as a competitive assay, where a visible line indicates the absence of antibodies against the virus.
  • the test line for the virus (11) is not visible, indicating the virus is present.
  • the control line (14) is also visible, as well as the lines for IgM (12) and IgG (13) antibodies.
  • Fig. 4B the control line and test line for human IgM antibodies are visible, suggesting an active infection, and that the test subject is infectious.
  • Fig. 4A the test line for the virus (11) is not visible, indicating the virus is present.
  • the control line (14) is also visible, as well as the lines for IgM (12) and IgG (13) antibodies.
  • the control line and test line for human IgM antibodies are visible, suggesting an active infection, and that the test subject is infectious.
  • control line the control line, test lines for the target compound, and human IgG antibodies are present, suggesting that the test subject has had an infection (and no longer has antibodies against the target compound), and is now immune and non-infectious.
  • control line, test lines for the target compound, and test lines for human IgG and IgM antibodies are present on the lateral flow device, suggesting a previous infection, and that the test subject could be contagious.
  • control line and test line for the target compound are visible, suggesting that there is no active infection and that the test subject should be immunized.
  • Fig. 4F only the control line is visible, suggesting an active infection, but no immune reaction, indicating that the test subject is in urgent need of hospital care.
  • a three line lateral flow device was fabricated comprising three test lines and one control line.
  • the device comprised a membrane layer, wherein said three test lines and one control line were located.
  • Said test lines and control lines comprising immobilized capturing agents for capturing COVID-19 virus particles, IgM, and IgG, Fig. 3A-B.
  • the reagent zone was located at a proximate end of the device, and was in fluidic connection with the membrane layer.
  • the sample was added to the sample loading zone, located between the conjugate zone and the test zone. Four whole blood samples were used, said samples comprising:
  • sample 1 For sample 1 all four lines were visually present, for sample 2 only the virus test line and the control line were present, Fig 5A. Sample 3 shows only the control line while sample 4 shows a positive IgG.
  • Example 2 Sensitive serum assay
  • test zone comprises immobilized S-protein, spike protein, (23) while the conjugates comprised anti-lgG gold sol (24) and anti-lgM gold sol (25), Fig. 6.
  • Sample was added to the reagent zone prior to addition of conjugates.
  • Example 3 Virus test format
  • the coronavirus has multiple copies of the same epitope making it challenging to produce a virus assay in a conventional format. Mixing of the conjugate and the sample too early (before reaching the test zone) typically reduces the sensitivity and leads to aggregations of the gold label and may prevent further flow in the device.
  • a device comprising immobilized Anti-COVID-19 antibodies at the test line was used, Fig. 7. After the sample had been allowed to contact the test line, the conjugate solution was added comprising anti-covid-19 gold sol.
  • the sample comprised trimeric S-Protein to mimic the Virus.
  • Sample 1 was a negative serum sample
  • sample 2 was virus positive serum.
  • the present sandwich assay format allows for the sample to interact with the test line prior to the conjugate particle interacting with said test line.
  • the conjugate particle is capable of binding to any present virus homologue.
  • a lateral flow test device was fabricated by assembling components according to Fig.
  • a lateral flow test device (29) comprised of a nitrocellulose membrane (30) provided on an inert backing card (31).
  • the membrane comprises a test line (32) and may advantageously further comprise a control line (33).
  • the loading zone comprises two sample/conjugate pads (34), wherein at least one comprises conjugates, such as gold conjugates, for enabling a visual line at the test line or control line.
  • the exemplified device comprises a Saliva filter pad (36) for filtering of the saliva sample and/or a sample solution comprising a saliva sample.
  • the saliva filter is a porous structure with pores in the size range between 0.1 and 1 pm.
  • the device further comprises an end pad (37) for preventing leakage.
  • the end pad may be provided, at the proximal end of the device, in a hydrophobic material.
  • an absorbent pad (38) At the distal end of the device, across the test lines.
  • the sample/conjugate pads were pre-treated with a buffer solution.
  • Said solution was a tris buffered saline solution comprising BSA, PVP and Tween 20.
  • the saliva filter pad was pre-treated with a PBS solution comprising BSA, NaCI and Tween 20.
  • the nitrocellulose membrane was pre-treated (optional step) with a tris buffered saline solution comprising BSA, NaCI, BSA, sucrose and PVP.
  • Plotting and blocking of the nitrocellulose membrane was performed by a standard plotter by plotting a solution of anti-SARS-CoV2 nucleocapsid protein mAb mixed with anti-2019-nCoV-2 antibody to give a total antibody concentration of 0.5 mg/ml.
  • the nitrocellulose membrane was blocked by a TBS solution with NaCI, BSA, Sucrose and PVP.
  • a lateral flow device for diagnosing COVID-19 by detection of protein antigen from SARS-CoV-2 in saliva samples was fabricated.
  • Example 5 Detection of protein antigen from SARS-CoV-2 in saliva samples from individuals suspected of COVID-19
  • the lateral flow test used was an immuno-chromatographic membrane assay that uses highly sensitive antibodies to detect SARS-CoV-2 Spike protein from saliva specimens.
  • a SARS-CoV-2 specific antibody and a control antibody are immobilized onto a membrane support as two distinct lines and combined with other reagents/pads to construct a test strip, as shown in Fig. 10.
  • the figure illustrates a lateral flow test device (29) comprising a nitrocellulose membrane (30), on an inert backing card (31).
  • the membrane comprises a test line (32) and a control line (33).
  • the device has a sample/conjugate pad (34) comprising gold conjugates (35), an (optional) saliva filter pad (36), and a sink pad/absorbent pad (38).
  • the lateral flow test strip consists of: 1. A backing card structure to support the different layers
  • a nitrocellulose membrane comprising the solid phase of the assay, which comprises two distinct areas, one where the test line is plotted and one where the control line is plotted.
  • the mobile phase consisted of two parts:
  • Both conjugates are diluted in a gold spraying buffer, containing high amounts of sucrose and protein, in order to enrich the particles’ surface and improve the stability of the conjugates. Both gold conjugates are sprayed at a concentration of 10 OD each.
  • the nitrocellulose is plotted with anti-2019 nCoV (S1) to form the test line and rabbit anti mouse IgG to form the control line. After the plotting the NC is being blocked in order to remove any non-specific binding and aggregation of the gold conjugate.
  • a saliva sample is added to the lateral flow device and the test line is visually inspected after 30 minutes.
  • Subjects that are both positive and negative for COVID-19 infection are tested using the lateral flow test strip.
  • two visual lines appear shortly after adding the saliva sample to the test line, the first visual line indicating that the virus particles are correctly detected and the second visual line (control line) indicating the validity of the test.
  • a single visual line appears shortly after adding the saliva sample to the test line, the single visual line (control line) indicating the validity of the test.
  • the lateral flow test is capable of correctly diagnosing COVID-19 by detection of protein antigen from SARS-CoV-2 in saliva samples.
  • the SARS-CoV-2 isolate REMRQ0001/Human/2020/Liverpool is used for the serial dilutions and Vero E6 cells (C1008; African green monkey kidney cells) are used to propagate the virus.
  • Vero E6 cells C1008; African green monkey kidney cells
  • Cells are maintained in Dulbecco’s minimal essential medium (DM EM) containing 10% foetal bovine serum (FBS) and 0.05 mg/ml gentamicin at 37°C with 5% C02 and FBS concentration was reduced to 4% for viral propagation.
  • DM EM minimal essential medium
  • FBS foetal bovine serum
  • gentamicin 0.05 mg/ml gentamicin
  • Sensitivity (Observed Positive Results/Actual Positive Results) x100
  • the specificity of the COVID-19 saliva test is evaluated by testing potentially cross reactive microorganisms.
  • the microorganisms are tested in triplicate in the absence (cross-reactivity) or presence of SARS-CoV-2 virus (interference) in saliva samples.
  • the microorganisms include Adenovirus, Human Metapneumovirus (hMPV), Parainfluenza virus 1-4, Influenza A & B, Enterovirus, Respiratory syncytial virus, Rhinovirus, Haemophilus influenzae, Streptococcus pneumoniae, Streptococcus pyogenes, Candida albicans, Pooled human nasal wash, Bordetella pertussis, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, Staphylococcus aureus, Staphylococcus epidermidis
  • Lateral flow tests were fabricated comprising a porous phase of nitrocellulose, a sample pad and an absorbent pad.
  • the test line was either sprayed with Anti-2019-n-CoV(S1) targeting the more prominent S protein on the surface of the virus, and Hu mAb to SAR- CoV-2 nucleocapsid protein targeting a less prominent N protein inside the virus.
  • test lines were sprayed by a solution comprising either of the two antibodies at various concentrations between 0.1 and 5 mg/ml.
  • Saliva samples positive for Covid-19 were tested, and it was concluded that devices having a test line directed at the S protein of the virus had better sensitivity than those that were directed at the N protein.
  • Lateral flow tests were fabricated comprising a porous phase of nitrocellulose, a sample pad and an absorbent pad.
  • the test line was sprayed with Anti-2019-n-CoV(S1) targeting the more prominent S protein on the surface of the virus.
  • the control line was Ms mAb to human IgG.
  • the saliva mix buffer formula was further optimized by testing buffers with different amounts of protein, salt and detergents. The optimal results were obtained by 0.1 M borate buffer with 1M NaCI and 1% BSA. Multiple different formulas were assessed and this one resulting in no measurable non-specific binding. The ratio of the saliva mix buffer was also tested. Despite the high dilution of the saliva, a dissolution ratio between the saliva and buffer of 1:10 worked the best (one part saliva). The results are provided in the table below.
  • a method for detecting the presence of at least one target compound in a sample comprising: a. providing a buffer solution; b. providing a lateral flow device comprising i. a membrane layer, comprising at least one test line, the at least one test line including an immobilized capturing agent for capturing the at least one target compound in the sample; ii.a sample loading zone for receiving the sample, the sample loading zone in fluidic connection with the test line; c. mixing the sample with the buffer solution, thereby obtaining a sample solution; d. applying the sample solution to the sample loading zone; e. measuring the presence of the at least one target compound in the sample.
  • the sample comprises or consists of whole blood, serum, plasma, nasal secretions, sputum, urine, saliva and/or stool, preferably wherein the sample is a saliva sample.
  • volumetric ratio between the buffer solution and the sample is between 5 parts buffer solution and 1 part sample and 15 part buffer solution and 1 part sample.
  • the volumetric ratio between the buffer solution and the sample is 1 part sample and at least 5 parts buffer solution, more preferably at least 6 parts buffer solution, more preferably at least 7 parts buffer solution, more preferably at least 8 parts buffer solution, even more preferably at least 9 parts buffer solution, most preferably at least 10 parts buffer solution, such as 11 parts buffer solution, or 12 parts buffer solution, or even 13 parts buffer solution, or 14 parts buffer solution, such as 15 parts buffer solution.
  • the buffer solution comprises sodium chloride, bovine serum albumin, and a borate buffer.
  • the buffer solution comprises 1 M sodium chloride, 1% w/v bovine serum albumin, and 0.1M borate buffer.
  • the capturing agents are configured for capturing a coronavirus, such as a human coronavirus, or fragments thereof.
  • capturing agents are configured for binding to a spike protein, such as SARS-CoV S protein and/or SARS-CoV- 2 S protein.
  • the lateral flow device comprises a conjugate zone
  • said conjugate zone comprises in a dry unused state conjugates for binding to the target compound and/or the immobilized capturing agent.
  • control line comprises a further immobilized capturing agent specific for binding to the conjugate.
  • measuring the presence of the at least one target compound in the sample comprises detecting for each test line, the presence of at least one indicator, such as a visible line.
  • the indicators are a number of immobilized conjugates comprising the label, such as forming a visually detectable line.
  • the membrane layer comprises a test line that comprises or consists of an immobilized capturing agent specific for IgM antibodies, such as human IgM antibodies, such as a first test line.
  • the membrane layer comprises a test line that comprises or consists of an immobilized capturing agent specific for IgG antibodies, such as human IgG antibodies, such as a second test line.
  • the membrane layer comprises a test line that comprises or consists of an immobilized capturing agent specific for a biomarker of a disease, such as disease-specific antibodies, such as a third test line. 22. The method according to any one of the preceding items, wherein the lateral flow device is configured for operation as a sandwich assay and/or as a competitive assay.
  • a kit for detecting the presence of a target compound in a sample comprising: a. a buffer solution for diluting the sample, wherein said buffer solution comprises sodium chloride, bovine serum albumin, and a borate buffer; and b. a lateral flow device comprising: i. a membrane layer, comprising at least one test line, the at least one test line comprising an immobilized capturing agent for capturing the at least one target compound in the sample; ii. a sample loading zone for receiving the sample, the sample loading zone in fluidic connection with the test line.

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  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un dispositif à écoulement latéral pour détecter la présence d'au moins un composé cible dans un échantillon, le dispositif à écoulement latéral comprenant : une couche de membrane, comprenant au moins une ligne de test, l'au moins une ligne de test comprenant un agent de capture immobilisé pour détecter la présence de l'au moins un composé dans l'échantillon ; une zone de réactif, comprenant : i. une zone de conjugué pour recevoir un tampon de migration, et ii. une zone de chargement d'échantillon pour recevoir l'échantillon ; la zone de chargement d'échantillon étant située, le long de la longueur axiale du dispositif d'écoulement latéral, entre la zone de conjugué et l'au moins une ligne de test.
PCT/EP2021/057027 2020-03-18 2021-03-18 Formats d'écoulement latéral améliorés pour écoulement optimisé et sensibilité accrue WO2021186003A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120031206A1 (en) * 2009-04-09 2012-02-09 Arkray, Inc. Analysis method, sample analysis tool, method for preventing back-flow of sample solution, and method for preventing increase in background
CN106290880A (zh) * 2015-05-27 2017-01-04 徐州雷森生物科技有限公司 一种用于犬冠状病毒的免疫层析用试剂组合物及检测方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120031206A1 (en) * 2009-04-09 2012-02-09 Arkray, Inc. Analysis method, sample analysis tool, method for preventing back-flow of sample solution, and method for preventing increase in background
CN106290880A (zh) * 2015-05-27 2017-01-04 徐州雷森生物科技有限公司 一种用于犬冠状病毒的免疫层析用试剂组合物及检测方法

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