Patent Application
20230264200
The present invention generally relates to the field of assay devices and method for performing assays, such as immunoassays. In particular, the present invention relates to an automated quantitative assay device and a method of performing heterogenous assay suitable for point of care/use applications.
An assay is an analytical procedure for qualitatively or quantitatively assessing the presence, amount or the functional activity of a target entity (known as an analyte) in a sample, such as a biological sample. Assays are common laboratory procedures in the medical, pharmacological, environmental/molecular biological fields, used to detect analytes such as drug compounds, biochemical substances or particular cell types.
Many assays, particularly those used to make quantitative measurement of an analyte are complex procedures, which must be performed by skilled personnel. As such, they can be time consuming and expensive to conduct, and samples to be assayed are often sent away to dedicated laboratory facilities with results available days or weeks later. There is always an associated risk of mishandling, like non maintenance of temperature during transit, agglutination of samples, mixing of samples and sometimes breach of threshold time between collection of sample and actual testing.
“Point of care” diagnostic tests (or, for non-clinical applications, “point of use” testing) can convey certain benefits over laboratory tests. Point-of-care (POC) is one of the largest growing trend for the diagnosis and cure of a disease. The terms “point of care” and “point of use” are generally understood to include diagnostic tests/assays in which samples, such as patient specimens, are assayed at or near the sampling location such that completion of the assay and any follow-up action based on the results can be completed within the same patient/problem encounter. The Point of Care (POC) generally includes the assay which are performed on a sample or specimen extracted from patient's body and tested near the sampling location so that any follow up action, i.e. any treatment if required after diagnosing the patient's sample or specimen could be done on the spot without any such delay. POC devices are widely used to rapidly and easily diagnose a disease as well as provide cure or prevention to fight against a disease or infection.
Medical/veterinary applications of point of care testing include intensive/emergency care encounters, tests conducted in hospital wards, general practice, outpatient clinics, veterinary surgeries, and the like. Non-medical applications may be found in workplaces and homes. Point of use (i.e. nonclinical) applications include quality control testing, for example in the manufacture and packaging of food or pharmaceuticals, domestic, chemicals, or testing of water quality.
All these applications share the common requirement of a rapid turnaround and communication of results to guide decisions. In addition, point of use/care assays must be comparatively simple to perform, yet yield robust and reliable results. This combination of features is difficult to achieve and, as a consequence, such assays may provide only qualitative or semi-quantitative results. For example, many simple lateral flow type tests for pregnancy or stigmatized diseases such as HIV may provide a coarse “yes” or “no” result, to provide an indication as to whether further laboratory testing is required. However, the results cannot in themselves inform clinical decisions.
The capability of conducting such competitive or sandwich assays in a point of care/use setting is in principle very desirable. Diagnosis without the need for samples to be referred to centralised testing laboratories can be particularly important in resource-limited settings, such as medical encounters in remote locations. The elimination of lengthy delays also has the potential to improve clinical outcomes more generally, by preventing patients from leaving a treatment conduit during the delay before results are available.
The requirement for an assay to be robust and reliable, and sufficiently simple to be performed in a point of use/care setting presents a significant barrier to implementation, and many types of assays are considered to be unsuitable for any type of point of care/use application. Efforts have been made to design assays which can be performed in a single step, by mixing reagents in a “one pot reaction” (also known as a “homogeneous” assay). However, even when this has been possible, the quality of data obtainable is comparatively poor. For example, enzyme-linked immunoassays (ELISAs) are sensitive to “noise” caused by components of the sample, or matrix. In order to obtain quantitative or semi-quantitative results, such assays must therefore include a complex series of reagent and rinse solution washing steps in order to remove the unwanted components. So called “matrix effects”, which are a barrier to point-of-care applications are described for example by Chiu et al., Journal of Laboratory Automation, June 2010, 233-242. The influence of matrix effects on the quality of data and assay efficiency is also described for example by Saab et al., International Journal of High throughput Screening, 2010:1, 81-98, and Imbert, P. E. et.al., Assay Drug Dev Technol., 2007, June; 5 (3):363-72.
Nevertheless, U.S. Pat. No. 11/908,071 discloses a dual path immunoassay device, wherein the invention include test cells with a first sorbent material defining a first flow path for a solution, a second sorbent material defining a second flow path distinct from the first flow path for a sample, and a test site with immobilized antigens or antibodies or other ligand binding molecules such as aptamers, nucleic acids, etc. located at the junction of the first and second sorbent materials for identifying one or more ligands. The first and second sorbent strips touch each other at the test site location. The test has a disadvantage of being qualitative chromatographic assay.
US20160195524A1 discloses an automated system for performing a heterogeneous assay comprising of an assay cassette for use in performing a heterogeneous assay, the assay cassette comprising of a fluid conduit and one or more chambers in the fluid conduit, from which a measurement may be acquired from a sample using a cassette reader device. Also disclosed is a tablet or bead for use with the assay system, which may be incorporated in the cassette. The tablet or bead may comprise one or more reagents to be used in the assay, in a soluble matrix. Use of an acridan or acridinium ester label may enable a sensitive measurement to be rapidly acquired. The assay may be configured to be performed by a clinician at the point of use or care.
US2013/0143328A1 discloses an automated assay fluid dispensing system includes a database that associates assay protocols with assay procedures, the procedures including a first assay procedure specifying dissimilar first and second channel procedures for driving first and second channels of a fluid-dispenser cassette.
U.S. Pat. No. 14/427,880 discloses a point-of-care lateral flow immunochromatographic assay for direct detection of enteroviruses. In particular, the present disclosure relates to a point-of-care lateral flow immunochromatographic assay for detection of the etiologic agents of Hand, Foot and Mouth Disease (HFMD), using antibodies specific for enteroviruses.
Simplifying such assays sufficiently to enable them to be performed at the point of care/use without the need for specialist training has therefore remained a challenge. Accordingly, there remains a need for improved methods and apparatus for performing heterogeneous assays. In view of the cited prior art, the present invention aims to overcome the drawbacks of the prior art as well as to provide a quick, heterogeneous, simple and quantitative immunoassay device and method for performing immunoassay at point of care/use. This particular patent application is non-limiting to measurements of analytes associated with the COVID 19 outbreak.
Accordingly, the primary object of the invention is to provide a quick, heterogeneous, simple and quantitative immunoassay device.
Another object of the invention is to provide a method for performing heterogeneous and quantitative immunoassay.
Another object of the invention is to provide automated quantitative assay device suitable for point of care/use applications.
Another object is to provide an automated immunoassay device and method for performing immunoassay at point of care/use.
Yet another object of the invention is to provide an automated immunoassay device and method for performing immunoassay for measurement of analytes associated but not limited to SARS-CoV2 or viral or bacterial outbreak at point of care/use.
The present invention generally relates to the field of assay devices and method for performing assays, such as immunoassays. In particular, the present invention relates to an automated quantitative assay device and a method of performing for performing heterogeneous assay suitable for point of care/use applications.
In an aspect of the present invention, an automated device for measuring at least one target analyte is proposed. The system comprises a means to receive the target sample collected from a subject; a means to measure the target analyte possibly present in said target sample; and a process of measuring the target analytes in real time manner. The target analyte includes but is not limited to any biological analyte, microbial entity like those of viral or bacterial sources such as SARS-CoV-2. The invention thus primarily relates to measuring analytes of interest to detect and treat related indications, such as COVID-19. The measuring process includes but is not limited to a competitive or sandwich assays, immunological assays etc. The means to receive the target sample comprises a cassette. The device comprises the cassette, the means or instrument for measuring the analyte and the chemistry of the detection process. The whole device is designed so that sample may be collected in a standard way and injected directly into the cassette, the cassette is then inserted into the instrument, the measurement performed and the result is then reported.
In another aspect, the present invention proposes a method for automated quantitative assay samples containing at least one analyte by injecting the sample in a cassette fully loaded with beads and antibody/conjugate cocktail. The cassette is then inserted into the device for measuring the target analyte quantitatively by the use of measuring instrument. Thereafter the magnetic stirrer is activated to mix the sample and the conjugate/antibody cocktail. A neutral liquid or air is then pumped into the mixing chamber causing the sample/conjugate/antibody mixture to be displaced and to flow round the fluid conduit immersing the beads. The liquid is then heated to approximately 37° C. to allow incubation and pumping the wash through fluid conduit to remove all of the remaining sample not bound to the beads. The wash cycles are repeated and fluid conduit of liquid is purged by pumping air through the conduit between the wash cycles. Chemiluminescence is measured through sensors and result recorded. Thereafter the cassette is ejected from the measuring instrument.
These and other features and advantages of the various embodiments will appear more fully from the following description and the accompanying drawings, which are incorporated in and constitute a part of the specification.
The invention will now be described by way of following reference drawings as exemplary embodiments of the invention:
The present invention is directed toward assay devices for detection of one or more analytes in a sample. The assay devices are constructed in a manner to allow for the real-time interaction of the assay reagents. In a preferred embodiment of the present invention, a device for measuring at least one target analyte is proposed. The device comprises a means to receive the target sample collected from a subject; a means to measure the target analyte possibly present in said target sample; and a process of measuring the target analytes in real time manner. The target analyte includes but is not limited to any biological analyte, microbial entity like those of viral or bacterial sources such as SARS-CoV-2. The invention thus primarily relates to measuring analytes of interest to detect and treat related indications, such as COVID-19. The measuring process includes but is not limited to a competitive or sandwich assays, immunological assays etc. The means to receive the target sample comprises a cassette. The system comprises the cassette, the means or instrument for measuring the analyte and the chemistry of the detection process. The whole system is designed so that body fluid may be collected in a standard way and injected directly into the cassette, the cassette is then inserted into the instrument, the measurement performed and the result is then reported. Further included in this invention is a method for performing the immunoassay.
As will be understood by the ordinarily skilled artisan upon reading the specification, the analyte can be any specific substance or component that one is desirous of detecting and/or measuring in a chemical, physical, enzymatic, or optical analysis. Analytes of interest include, for example, novel coronavirus 2019-nCoV, SARS-CoV2 or viral or bacterial outbreak, antigens (such as antigens specific to bacterial, viral or protozoan organisms); antibodies, particularly those induced in response to an infection, allergic reaction, or vaccine; hormones, proteins and other physiological substances (for example, human chorionic gonadotropin, estrogens, progestins, testosterones, corticosteroids, human growth factors, hemoglobin, and cholesterol); nucleic acids; a variety of enzymes; therapeutic compounds and illicit drugs; contaminants and environmental pollutants; or any number of natural or synthetic substances.
As is appreciated by one skilled in the art, the number of natural and synthetic substances which can be detected by the assay devices and methods of the present invention is extensive, and include, but is not limited to, the following: ACE inhibitors, adrenergics and anti-adrenergics, alcohol deterrents (for example, disulfiram), anti-allergics, anti-anginals, anti-arthritics, anti-infectives (including but not limited to antibacterials, antibiotics, antifungals, antihelminthics, antimalarials and antiviral agents), analgesics and analgesic combinations, local and systemic anesthetics, appetite suppressants, antioxidants, anxiolytics, anorexics, antiarthritics, anti-asthmatic agents, anticoagulants, anticonvulsants, antidiabetic agents, antidiarrheals, anti-emetics, anti-epileptics, antihistamines, anti-inflammatory agents, antihypertensives, antimigraines, antinauseants, antineoplastics, antioxidants, antiparkinsonism drugs, antipruritics, antipyretics, antirheumatics, antispasmodics, antitussives, adrenergic receptor agonists and antagonists, anorexics, appetite suppressants, cardiovascular preparations (including anti-arrhythmic agents, cardiotonics, cardiac depressants, calcium channel blockers and beta blockers), cholinergics and anticholinergics, contraceptives, diuretics, decongestants, growth stimulants, herbal preparations, hypnotics, immunizing agents, immunomodulators, immunosuppresives, muscle relaxants, neurologically-active agents including anti-anxiety preparations, antidepressants, antipsycotics, psychostimulants, sedatives and tranquilizers, sore throat medicaments, sympathomimetics, vasodilators, vasoconstrictors, vitamins, xanthine derivatives, various combinations of these compounds, and the like.
As used herein, the term “reagent’ is used to indicate any liquid, e.g., a solvent or chemical solution which is to be mixed with a sample and/or other reagent in order, e.g., for a reaction to occur or to enable detection. A reagent can be, for example, another sample interacting with a first sample. A reagent can also be a diluting liquid such as, e.g., water. A reagent may comprise an organic solvent or a detergent. A reagent may also be a buffer. A reagent in the stricter sense of the term may be a liquid solution containing a reactant, typically a compound or agent capable, e.g., of binding to or transforming one or more analytes present in a sample. Examples of predefined reactants are, for example, and not limited thereto, enzymes, enzyme substrates, protein reagents, chemical reagents, sera reagents, conjugated dyes, protein-binding molecules, nucleic acid binding molecules, antibodies, chelating agents, promoters, inhibitors, epitopes, antigens, catalysts, etc.
Optionally, dry reagents may be present in the analytical device and be dissolved by a sample, another reagent, or a diluting liquid.
In an embodiment of the present invention,
The automated quantitative assay device is capable of performing heterogeneous competitive or sandwich assays or immunological assays of a target sample across an elevated range in 10-15 minutes.
In another embodiment,
In this particular implementation there are channels shown on the rear of the cassette used to transport fluids from the mixing chamber 106 and reagent port 107. These channels 115 are sealed by an adhesive patch 114 shown in
In another embodiment of the present invention, the beads 103 are pre-labelled immobilized analyte-specific probe are placed individually in a plurality of wells of the cassette device which is sealed using permanent heat seals.
In yet another embodiment of the present invention, the cocktail of conjugate molecules may be pre-packaged in the cassette device in solid form 112 (
In another embodiment, the present invention provides an automated quantitative assay device having a mixing chamber which is filled with a predetermined amount of a cocktail of conjugate molecules tagged with a chemiluminescent molecule, facilitating a competitive assay measurement wherein said conjugate molecules in the cocktail has an individual predetermined concentration.
In still another embodiment of the present invention, the mixing chamber is filled with a predetermined amount of the cocktail of conjugate molecules tagged with a chemical label that binds specifically to the analyte specific probes tagged with the chemiluminescent molecule, facilitating a sandwich assay measurement wherein each analyte specific probe in the cocktail of conjugate molecules have an individual predetermined concentration.
In another embodiment, the present invention provides a mixture of the 2 implementations, viz. a cocktail of conjugate molecules tagged with a chemiluminescent molecule, and a cocktail of analyte specific probe molecules tagged with a chemiluminescent, are possible in the same mixing chamber. At least two chambers may comprise the same type of immobilized analyte-specific probe. Thus, the cassette may be configured for an assay and a confirmatory assay to be conducted. A transparent layer is used to contain the fluid conduit, the beads (used for immobilizing the analyte specific molecule), the mixing chamber and the waste reservoir. The beads, and the antibody cocktail are inserted before the transparent layer is fixed.
In another embodiment
In another embodiment, a small magnet 111 may be placed in the mixing chamber to facilitate magnetic stirring. A motor 113 with a drive magnet attached to its shaft. When the shaft and magnet is rotated by the motor the small magnet in the mixing chamber 111 is caused to rotate causing the contents of the chamber to mix thoroughly.
Once the cassette is inserted, the closure of the insertion slot application of the heating element and the insertion of the injection needles into the cassette is caused by one single action. This may either be done by the machine when the measurement is initiated or manually by the user. At the start of each measurement the heating element is caused to move upwards to make contact with the high thermal conductivity material deposited on the bottom of the cassette, or alternatively a heating element with apertures to allow light to pass will move downwards to press on the top of the cassette. At the same time the injection needles move upwards piercing the rubberized cap 109 forming the seal to the inlet ports.
In still another embodiment, the present invention provides a heating element will be held, pushed against the top of the cassette device 100. This allows the efficient heating of the fluid in the fluid conduit to 37° C. There are holes in the heating element to allow light to pass. Pumps will be used to periodically move the fluid backwards and forwards along the channel by one or two millimetres to ensure all of the fluid in the channel is heated uniformly.
In yet another embodiment, all inlets are on the top of the cassette. All inlets are formed by piercing a membrane adhered to the either the top or bottomeither the top or bottom of the cassette with an injection needle. The membrane may be a sheet of silicone rubber.
In still another embodiment of the present invention, the reservoirs contain wash and the reagents required to trigger the chemiluminescence reaction. The reagents are stored in bottles with septum lids. The bottles are combined and formed into a complete reagent package which is inserted into the instrument. On insertion injection needles pierce all of the septum membranes.
Small bore (0.5 to 1.5 mm inside diameter) silicone, neoprene or bioprene tubing carries the various liquids to their corresponding pumps and forward to the corresponding inlet port of the cassette. The pumps are required to pump up to 200 μL from the reagent bottles to the cassette. The pumps should be either syringe or peristaltic because these are capable of delivering a specific volume. The volume delivered in a particular assay process will be monitored by determining how far the pump mechanism has moved. This is done using optical reflective switches to determine how far the peristaltic pump rotates or how far the plunger has been depressed in a syringe pump. In both cases a geared dc motor is used mainly for simplicity and compactness, alternatively a stepping motor may be used. Because a syringe pump is capable of giving a more accurate and precise dose (the diameter of the peristaltic tubing may change slightly over time) this may be preferred where small precise doses are required to activate one bead at a time.
In yet another embodiment of the present invention, the optical sensor is either a photomultiplier tube or a “multi pixel photon counting detector”, or a “silicone photo multiplier”.
In another embodiment the sensor will be of the silicon photomultiplier type. In order to achieve adequate signal to noise with these devices they must be cooled to reduce the dark current and the temperature must be monitored so that the average dark signal might be subtracted from the actual signal. The proposed system is shown in
In an embodiment of the present invention, the photo-detector (either a photon counting photomultiplier tube or a cooled “silicon PMT”) is placed directly above each well sequentially, geared direct current (DC) motors are used to rotate the light sensor into position. In case when the fluid conduit forms a circular path on the cassette, the motor is moved in a circular path around a fulcrum.
In another embodiment in the present invention, the beads may be placed in a grid pattern and the photomultiplier would be moved in a rectilinear fashion using geared DC motors, lead screws and linear bearings. In both cases mechanical switches or reflective optical sensors are be used to determine the position.
In yet another embodiment, the heating element is an infrared LED emitting at 1500 nm or a radiative infrared emitter. At this wavelength water is quite absorbent and so it may be possible to heat the very small volume (<10 μL) sufficiently using a non-contact 304 method. A non-contact thermometer would be used to monitor the temperature.
In still another embodiment, a heating element 205 along with the injection needles 208 move as a unit to engage with the cassette device 100.
In yet another preferred embodiment of the present invention, a method of automated quantitative assay is disclosed having following steps: obtaining a cassette fully loaded with Beads and antibody/conjugate cocktail,—injecting a sample into a port at or upstream of the mixing chamber and filling the mixing chamber, the sample may be a number of body fluids including blood, saliva, and urine, inserting the cassette into the measuring instrument, activating the magnetic stirrer to mix the sample and the conjugate/antibody cocktail, pumping a neutral liquid or air into the mixing chamber causing the sample/conjugate/antibody mixture to be displaced and to flow round the fluid conduit immersing the beads, heating the liquid to approximately 37° C. to allow incubation, pumping the wash through fluid conduit to remove all of the remaining sample not bound to the beads, repeating the wash cycles and purging the fluid conduit of liquid by pumping air through the conduit between the wash cycles, measuring the chemiluminescence through sensors and recording the result, ejecting the cassette from the measuring instrument and shutting down the device.
There are then 2 alternative assay types that can be utilized in the proposed system—
1. If the chemiluminescence is of a ‘glow’ type. The activating reagents are pumped round the whole fluid conduit and then the light detector is moved to detect light from each of the beads in turn.
2. If the chemiluminescence is of a ‘flash’ type. It must be pumped so that it immerses each bead in turn and the luminescence must be detected before it is pumped to immerse the next bead.
In yet another embodiment of the present invention, the magnetic stirrer is activated by placing a motor comprising a magnet attached to shaft below the mixing chamber and wherein the magnet and a shaft are rotated by the motor thereby resulting in the rotation of the small magnet in the mixing chamber.
In still another embodiment of the present invention said device and the assay is used for the detection of novel coronavirus 2019-nCoV, SARS-CoV2 or viral or bacterial outbreak, antigens; antibodies, particularly those induced in response to an infection, allergic reaction, or vaccine; hormones, proteins and other physiological substances for example, human chorionic gonadotropin, estrogens, progestins, testosterones, corticosteroids, human growth factors, hemoglobin, and cholesterol; nucleic acids; a variety of enzymes; therapeutic compounds and illicit drugs; contaminants and environmental pollutants; or any number of natural or synthetic substances; ACE inhibitors, adrenergics and anti-adrenergics, alcohol deterrents for example, disulfiram, anti-allergics, anti-anginals, anti-arthritics, anti-infectives including antibacterials, antibiotics, antifungals, antihelminthics, antimalarials and antiviral agents, analgesics and analgesic combinations, local and systemic anesthetics, appetite suppressants, antioxidants, anxiolytics, anorexics, antiarthritics, anti-asthmatic agents, anticoagulants, anticonvulsants, antidiabetic agents, antidiarrheals, anti-emetics, anti-epileptics, antihistamines, anti-inflammatory agents, antihypertensives, antimigraines, antinauseants, antineoplastics, antioxidants, antiparkinsonism drugs, antipruritics, antipyretics, antirheumatics, antispasmodics, antitussives, adrenergic receptor agonists and antagonists, anorexics, appetite suppressants, cardiovascular preparations including anti-arrhythmic agents, cardiotonics, cardiac depressants, calcium channel blockers and beta blockers), cholinergics and anticholinergics, contraceptives, diuretics, decongestants, growth stimulants, herbal preparations, hypnotics, immunizing agents, immunomodulators, immunosuppressives, muscle relaxants, neurologically-active agents including anti-anxiety preparations, antidepressants, antipsycotics, psychostimulants, sedatives and tranquilizers, sore throat medicaments, sympathomimetics, vasodilators, vasoconstrictors, vitamins, xanthine derivatives, various combinations of these compounds, and the like.
The following examples further illustrate the invention and its unique characteristics in elaborate manner. However, the example in no way intend to limit the scope of the invention.
A Thyroid stimulating hormone (TSH) assay was developed on the Quantilyte system using Quantilyte beads but washed and read in 96-well plate as a standard ELISA format. The method for each assay was kept as similar as possible. Same conjugate, calibrator and QC preparations were used for each method. Briefly, Quantilyte beads were coated with 20 μg/ml of Goat-anti-TSH Nunc F (US Biologicals). ELISA wells were coated with 5 μg/ml of the same antibody. TSH was spiked into TSH depleted plasma to create a calibration line covering a range of 2905-1.90 IU/ml and quality control samples at 7.5, 15, 50, 300 and 1800 IU/ml. These were aliquoted and stored frozen at −20° C. Samples were analysed by mixing 25 μl of serum sample with 25 μl of conjugate comprising 0.5 μg/ml USB mouse-anti-TSH and 1 μg/ml anti-mouse-HRP. This mixture was incubated with coated beads or wells for 20 minutes before washing. Thereafter pierce supersignal pico or TMB substrate was added and reading was taken using the POLARSTAR plate reader of the Quantilyte reader. Calibrators were analysed in triplicate and average of calibration lines was used to calculate concentration for six replicate quality control samples. % CV and % Bias was calculated for each QC level. Method sensitivity was determined by running six replicate zero calibrators determined mean+three standard deviations and finding a concentration for this value by reading from the averaged calibration lines.
Table 1 shows the Mean Signal, %CV and % Maximum Value for the triplicate calibration lines. Graphs shown in
Table 2 shows the Mean, % CV range and % Bias of calculated TSH concentrations for quality control samples spiked at 50, 300 and 1800 μIU/ml analysed using all three methods additional quality control samples at 7.5 and 15 μIU/ml were analysed in the ELISA only due to the increased sensitivity of this method. The results for each quality control level found using all three methods are shown in
Table 3 shows the mean, % CV range and % Bias calculated for the lyphocheck control samples. Level 3 was analysed using all three methods and levels 1 and 2 were analysed by ELISA only due to increased sensitivity of this method. The results for lyphocheck level 3 found using all three methods are shown in
Table 4 below shows the data and calculation performed to determine minimum detectable TSH concentration for each of the assay methods. Six zero samples were run in each method. Mean and standard deviation of these were found and a mean+3 times standard deviation signal was calculated and read for the calibration lines for each method to give the minimum detectable concentration. It can be seen that the minimum detectable concentration is similar using the ELISA assay and the Bead plate assay. However, the Quantilyte assay has an increased detection limit. This is likely to be due to slightly increased variation from the replicate zero samples in the Quantilyte assay which could reflect a less thorough and consistent wash procedure than the other methods.
A Multiplex assay was developed on the Quantilyte system to detect TT4 and Cortisol in a single cartridge. Results obtained from this assay were compared to separate commercial ELISA assays for TT4 and cortisol obtained from Alpco. Briefly, the Quantilyte assay consisted of Quantilyte beads which were coated at 20 μg/ml with either anti-T4 or anti-Cortisol antibody. T4 and cortisol were spiked into T4 or cortisol depleted serum respectively to create independent TT4 and cortisol calibration lines which were aliquoted and stored frozen at −20° C. Calibrators, Lyphocheck QC's and cat serum samples were analysed by adding 90 μl of serum sample and 90 μl of assay buffer to 20 μl of conjugate concentrate containing 60 μg/ml T4-HRP and 1/10 dilution of cortisol-HRP in an HRP stabilising buffer. This mixture was incubated with coated beads for 20 mins before washing and adding Pierce supersignal pico substrate and reading using the Quantilyte reader. The Alpco ELISAs were conducted according to the kit instructions which being a colorimetric TMB ELISA was read using the POLARSTAR plate reader. Calibrators were analysed in triplicate and averaged calibration lines were used to calculate concentration for four replicates. % CV and % Bias of Lyphocheck quality control samples and 41 cat serum samples was calculated for each QC level.
Table 5 and Table 6 below show the Mean Signal, % CV and % Maximum Signal for the triplicate TT4 and cortisol calibration lines respectively.
Table 7 and 8 below shows the mean calculated concentration, % CV range and % Bias calculated for the Lyphocheck control samples for TT4 and cortisol samples respectively as well as the % difference between the concentrations calculated using the Quantilyte and ELISA methods. The mean concentration with standard deviation error bars at each QC level for both methods are plotted on the graphs shown in
Table 9 below shows the calculated TT4 and cortisol concentrations for 41 cat serum samples found using the both the Quantilyte and ELISA methods as well as the % difference between the result found using the Quantilyte assay and that using the ELISA assay. The graphs shown in
The data shows reasonable correlation of results from Quantilyte and ELISA assay for both the TT4 and Cortisol methods with correlations of 0.945 and 0.912 respectively. While there is considerable variation among individual results along with a few extreme outliers the two methods are broadly in agreement with respect to the relative concentrations of the samples for both analytes. Both analytes show a tendency to increased variation at low analyte concentrations and the TT4 assay shows a pronounced tendency to produce higher value for sample below 20 nmol/L than the ELISA.
150 2 mm polystyrene beads were coated with 3 ml of 20 μg/ml of anti-Cortisol in 100 mM carbonate coating buffer pH 9.6 i.e. 7.5 μl of 7.91 mg/ml anti-Cortisol in dH2O+2992.5 μl coat buffer. Added coated beads to a 5 ml Bijou bottle. Beads were incubated overnight at 4° C. with end-over-end mixing. Beads were washed eight times with 3 ml PBS/0.01% Tween and twice with 3 ml PBS and 3 ml of 50% StartingBlock in PBS+0.25M Trehalose Block buffer was added. Beads were incubated for two hours at room temperature with end-over-end mixing and then washed three times with 1 ml PBS/0.01% Tween and placed in a weigh boat and allowed to air dry at room temperature.
150 2 mm polystyrene beads are coated with 3 ml of 20 μg/ml of anti-Cortisol in 100 mM carbonate coating buffer pH 9.6 i.e. 31.4 μl of 1.91 mg/ml anti-Cortisol in dH2O+2968.6 μl coat buffer. Added coated beads to a 5 ml Bijou bottle. Beads were incubated overnight at 4° C. with end-over-end mixing. Beads were washed eight times with 3 ml PBS/0.01% Tween and twice with 3 ml PBS and 3 ml of 50% StartingBlock in PBS+0.25M Trehalose Block buffer was added. Beads were then incubated for two hours at room temperature with end-over-end mixing and then washed three times with 1 ml PBS/0.01% Tween and placed in a weigh boat and allowed to air dry at room temperature.
Buffer solution is prepared a blocking reagent and the binding release components, 8-Anilinonaphthalene-1-sulfonic acid (ANS) and sodium salicylate.
400 μl of 50 mg/ml ANS+100 μl of 200 mg/ml NaS+9500 μl of AbCam IM Block Final Concentrations=2 mg/ml ANS, 2 mg/ml Sodium Salicylate
Conjugate solution is prepared containing Cortisol-HRP and anti-T4-HRP in an HRP stabilizing buffer
100 μl of cortisol-HRP+10 μl of 6000 nmol/L anti-T4-HRP+890 μl of KPL HRP stabilizer
Final Concentrations= 1/10 Cortisol-HRP+60 nmol/L T4-HRP
T4 is serially diluted into T4 depleted serum as below in Table 10.
Calibrators stored at 4° C. for 24 hours then aliquoted and stored at −20° C.
Cortisol is serially diluted into cortisol depleted serum as below.
Calibrators stored at 4° C. for 24 hours then aliquoted and stored at −20° C.
Instrument wash bottle was filled with PBS/0.01% Tween+1/5000 antifoam wash solution and connected to instrument wash line.
An empty Prime cartridge was placed in the instrument.
The command WASH20 was run, followed by PURGE12, WASH12, PURGE12.
The prime cartridge was removed and emptied, the instrument was then ready for use.
Anti-Cortisol bead was placed in well 1 and Anti-T4 bead was placed in well 2 of an eight well cartridge.
The cartridge was sealed using permanent heat seals using 2 ten second presses of the heat sealer.
A strip of self-adhesive silicon strip was secured over the cartridge injection port.
An air escape hole was pierced in the heat seal in the top right hand corner of the waste reservoir.
90 μl of serum calibrator was mixed with 90 μl of buffer solution and added to 20 μl of conjugate mix.
The serum sample/conjugate mix was injected through the first three wells of the cartridge.
The cartridge was incubated at room temperature for 20 minutes.
The cartridge was placed into the Quantiyte instrument and the wash sequence was run.
150 μl of Pierce supersignal pico substrate solution A was mixed with 150 μl of Pierce supersignal substrate solution B.
Cartridge was removed from the instrument 300 μl volume of mixed pierce supersignal pico substrate was injected into the cartridge.
Cartridge was incubated at room temperature for 2 minutes.
Cartridge was placed into the Quantilyte reader and luminescence signal recorded by running the read sequence.
The assay consisted of beads which were coated at 5 μg/ml with anti-CRP capture antibody. CRP was spiked into CRP depleted serum to create independent CRP serum calibration lines and separately spiked serum quality control samples which were aliquoted and stored at 4° C. Serum calibrators and QCs were diluted 1 in 1000 in PBS and analysed by adding 180 μl of diluted serum sample to 20 μl of detection mix concentrate containing 900 ng/ml CRP detection antibody and 1/20 dilution of streptavidin-HRP in an HRP stabilising buffer. This mixture was incubated with coated beads for 10 mins before washing and adding Pierce supersignal pico substrate and reading using the instrument reader. The R&D systems ELISAs were conducted according to the kit instructions using the same antibodies used for the CRP assay and was a colorimetric TMB ELISA which was read using the POLARSTAR plate reader. A higher sample dilution of 1 in 125000 was required for the ELISA analysis. Calibrators were analysed in duplicate and averaged calibration lines were used to calculate concentration for six replicate quality control samples % CV and % Bias was calculated for each QC level.
2 mm Polystyrene beads were coated by passive adsorption with 5 μg/mg of CRP capture antibody, washed and stored in PBS. Beads were placed in individual wells of an eight well cassette. The cassette is sealed using permanent heat seals. Serum sample containing CRP was diluted and mixed 1:9 with conjugate solution containing CRP detection antibody labelled with streptavidin-HRP. Serum sample mix was injected through each used well of the cassette. Cassette was incubated at room temperature for 10 minutes. Cassette was placed into the measuring instrument and the Wash sequence was run. Pierce supersignal substrate was mixed and injected through each of the used wells and the cassette incubated for 2 minutes. Cassette is placed into the measuring instrument and the Read sequence is run. The luminescence signal from each of the used cassette wells is read in turn and recorded.
One Hundred 2 mm polystyrene beads were coated with 3 ml of 5 μg/ml of anti-CRP in 100 mM carbonate coating buffer pH 9.6. Specifically, 41.6 μl of 360 μg/ml anti-CRP and 2958 μl coat buffer were used to coat the polystyrene beads. Added the coated 100 polystyrene beads in a 5 ml Bijou bottle and incubated overnight at 4° C. with gentle agitation. Beads were then washed four times with 3 ml PBS/0.01% Tween and twice with 3 ml PBS. Thereafter, the beads were stored in PBS buffer.
Conjugate solution was prepared containing Anti-CRP-Biotin detection antibody and streptavidin-HRP in a HRP-stabilising Buffer. Particularly, 55.5 μl of 16.2 μl g/ml Anti-CRP-Biotin, 50 μl of Streptavidin-HRP, 894.5 μl of HRP Stabilizer in a final concentration of 900 ng/ml Anti-CRP, 1 in 20 Streptavidin-HRP Conjugate Mix was stored at 4° C.
CRP is serially diluted into CRP depleted serum as below in Table 11.
Calibrators are stored at 4° C.
CRP is serially diluted into CRP depleted serum as below in Table 12.
QC's are stored at 4° C.
Serum calibrators and QCs are diluted 1/1000in PBS prior to analysis as below:—
1/50-20 μl Calibrator/QC +980 μl PBS
1/1000-50 μl 1/50 Dilution+950 μl PBS
Anti-CRP bead was placed in well 1 and well 2 of an eight well cassette along with 20 μl of PBS. The cassette was sealed using permanent heat seals using 2 ten second presses of the heat sealer. A strip of self-adhesive silicon strip was secured over the cassette injection port. An air escape hole was pierced in the heat seal in the top right hand corner of the waste reservoir. PBS was injected through all wells of the cassette as a storage solution. Air was injected to remove PBS storage solution from the wells. 180 ul of diluted calibrator or QC was mixed with 20 μl of conjugate solution. The serum sample/conjugate mix was injected through the first three wells of the cassette. The cassette was incubated at room temperature for 10 minutes. The cassette was placed into the measuring instrument and the wash sequence was run. 150 μl of Pierce super signal pico substrate solution A was mixed with 150 μl of Pierce super signal substrate solution B. Cassette was removed from the instrument 300 μl volume of mixed pierce super signal pico substrate was injected into the cassette. Cassette was incubated at room temperature for 2 minutes. Cassette is placed into the reader and luminescence signal was recorded by running the read sequence.
Dissolve 80 g of NaCl, 2.0 g of KCl, 14.4 g of Na2HPO4 and 2.4 g of KH2PO4 in 1 L of dH2O. Store at room temperature.
Dilute 100 ml of 10×PBS with 900 ml of dH2O
Add 1 ml of 10% Tween to 990 ml of PBS
Add 20 μl of antifoam to 100 ml of PBS/0.01% Tween 10× Coating Buffer (1M Carbonate)
Dissolve 16.8 g of sodium hydrogen carbonate in 200 ml of dH2O. Store at room temperature.
Mix 50 ml of 10× x coat buffer with 450 ml of dH2O
Preparation: Need supplies on hand:—syringe, heparin tube, sample pipette, conjugate pipette, high-speed centrifuge, untreated sample tube (from kit), conjugate (from kit). Device uses serum or plasma (optimal workflow=plasma)
Dispense 1 cc of sample (freshly collected) into Lithium heparin tube
Invert sample 5 times
Place in centrifuge IMMEDIATELY
Spin for 2 mins (hard spin) or 10 mins (standard Lithium spin)
If using serum, blood must clot for a minimum of 20 mins before spinning
Pipette 20 μl of plasma/serum sample into sample dilution tube containing 20 ml Buffer
Close tube and invert 5 times
Pipette 180 μl of diluted plasma/serum sample into sample tube
Add 20 μl of conjugate
Close tube and invert 5 times
Inject sample into cassette
Start timer
10 min incubation time
Timer alerts when incubation complete
Place cassette in device
Click “Run Full Procedure”
Device runs wash and read sequence CRP result is displayed in application
Conjugate must be stored at 2-8° C.
Table 13 below shows the Signal, % CV and % Maximum Signal for the CRP luminescent values and OD of calibration lines. The graphs in
Covilyte Calibration Line and ELISA Calibration Line are shown in
Table 15 below shows the signal and calculated concentration along with % CV and % Bias calculated for the serum quality control samples using the Covilyte and ELISA methods as well as the % difference between the concentrations calculated using the Covilyte and ELISA methods. The mean concentration with standard deviation error bars at each QC level for both methods are plotted on the three column charts. A correlation plot of Covilyte mean concentration vs ELISA mean concentration and plots comparing the % BIAS and % CV at each QC level for both methods are also shown.
It can be seen that the mean result and % CV are broadly comparable for both methods at all three QC levels although the Covilyte assay does show an increased % BIAS and % CV at the highest QC level an effect likely due to the flatter shape of the Covilyte calibration curve at the higher CRP concentrations.
The assay in accordance with the proposed invention has been developed using the Covilyte (invention) system which can measure CRP in serum across the elevated range of 200-0.781 μg/ml with a total sample processing time below 15 minutes and additionally gives levels of accuracy and precision which are broadly comparable with those achieved using a conventional ELISA technique with total sample processing time of 6 hours. The Covilyte assay has shown acceptable stability for over a period of 9 days, though long term stability has not been assessed.Although a sample dilution step is required, but even that is 100 fold less than that required for a standard ELISA analysis.
While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques, methods, compositions, apparatus and systems described above may be used in various combinations and for the detection of various analytes.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2009823.2 | Jun 2020 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2021/055726 | 6/26/2021 | WO |
