Patent Application
20190204310
The disclosure relates generally to biotechnology and more particularly to methods for the quantification of analytes and improved microarray methods for the detection and quantification of multiple analytes in a single sample. It also relates to the simultaneous quantification of isotype immunoglobulin classes Ig G, A, M, E, D and sub-classes including IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 within a single containment vessel. It further relates to microarray methods for the simultaneous detection and quantification of multiple analytes in a single sample. Analytical components may include subunits of therapeutic proteins, including antibody fragments or fusion partners, metabolic products, peptide components, formulation components, bio-similars or potential cross reacting entities.
Current immunoassay methods detect one target per detection test cycle within a single reaction well. It is common for several antigenic substances or bio-markers to be associated with detection and diagnosis for any pathological or physiological disorder. To confirm the presence of multiple markers, each marker within a test sample often requires a separate and different immunoassay to confirm the presence of each target analyte to be detected. This often requires a multitude of tests and samples, increases delay in time to treatment, costs and possibility of analytical error. Current immunoassay methods do not detect antibodies that are of multiple isotypes and subclasses in the same test well/cycle.
Enzyme Linked Immunosorbent Assay (ELISA) was developed by Engvall et al., Immunochem. 8:871 (1971), and further refined by Ljunggren et al., J. Immunol. Meth. 88:104 (1987) and Kemeny et al., Immunol. Today 7:67 (1986). ELISA and its applications are well known in the art.
A single ELISA functions to detect a single analyte or antibody using an enzyme-labelled antibody and a chromogenic substrate. To detect more than one analyte in a sample, a separate ELISA is performed to independently detect each analyte. For example, to detect two analytes, two separate ELISA plates or two sets of wells are needed, i.e., a plate or set of wells for each analyte. Prior art chromogenic-based ELISAs detect only one analyte at a time. This is a major limitation for detecting diseases with more than one marker or transgenic organisms which express more than one transgenic product.
J. N. Macri et al., Ann. Clin. Biochem. 29:390-396 (1992), describe an indirect assay wherein antibodies (Reagent-1) are reacted first with the analyte and then second labelled anti-antibodies (Reagent-2) are reacted with the antibodies of Reagent 1. The result is a need for two separate washing steps which defeats the purpose of the direct assay.
U.S. 2007/141656 to Mapes et al. measures the ratio of self-antigen and auto-antibody by comparing to a bead set with monoclonal antibody specific for the self-antigen and a bead set with the self antigen. This method allows at least one analyte to react with a corresponding reactant, i.e., one analyte is a self-antigen and the reactants are auto-antibodies to the self antigen.
Another method for detecting multiple analytes is disclosed in U.S. 2005/118574 to Chandler et al., which makes use of flow cytometric measurement to classify, in real time, sequential automated detection and interpretation of multiple biomolecules or DNA sequences, each biomolecule having to be separated and independently bound to a specific substrate particle, each particle being specific for separating and concentrating only a single species of analyte. This concentration of analyte is detected when the substrate particle is laser illuminated, as the particles flow, in single file, past the illuminating laser beam.
WO0113120 to Chandler and Chandler determines the concentration of several different analytes in a single sample. It is necessary only that there is a unique subpopulation of microparticles for each sample/analyte combination using the flow cytometer. These bead-based systems' capability is limited to each microparticle, i.e., bead being suspended in a volume of test fluid that contains the analyte to be detected as a separate entity which needs to bind freely and specifically onto the surface of the test bead. Each bead effectively provides a requisite detection signal for only a specific analyte entity. Multiple, different entity binding events onto a single microparticle are not well distinguished or quantified using flow cytometry being restricted to multiple events of the same antibody isotype/subclass.
Simultaneous detection of more than one analyte, i.e., multiplex detection for simultaneous measurement of proteins has been described by Haab et al., “Protein micro-arrays for highly parallel detection and quantization of specific proteins and antibodies in complex solutions,” Genome Biology 2(2):0004.1-0004.13 (2001), which is incorporated herein by reference. Mixtures of different antibodies and antigens were prepared and labelled with a red fluorescence dye and then mixed with a green fluorescence reference mixture containing the same antibodies and antigens. The observed variation between the red to green ratio was used to reflect the variation in the concentration of the corresponding binding partner in the mixes. This method is not suitable for quantitative results.
Mezzasoma et al. (Clinical Chemistry 48, 1, 121-130 (2002)) published a micro-array format method to detect analytes bound to the same capture spot in two separate assays, specifically different auto-antibodies reactive to the same antigen. The results revealed that when incubating the captured analytes with one reporter (for example that to detect immunoglobulin IgG), the corresponding analyte is detected. When incubating the captured analytes with the second reporter in an assay using a separate microarray solid-state substrate (for example to detect IgM), a second analyte (IgM) is detected.
WO0250537 to Damaj and Al-assaad discloses a method to detect up to three immobilized concomitant target antigens, bound to requisite antibodies first coated as a mixture onto a solid substrate. A wash step occurs before the first marker is detected. The presence of the first marker may be detected by adding a first specific substrate. The reaction well is read and a color change is detectable with light microscopy. Another wash step occurs before the second marker is detected. The presence of the second marker may be detected by adding a second substrate, specific for the second enzyme, to the reaction well. After sufficient incubation, the reaction well may be assayed for a color change. Similarly, a wash step may occur before the third marker is detected.
The presence of the third marker may be detected by adding a third substrate, specific for the third enzyme, to the reaction well. After sufficient incubation, the reaction well may be assayed for a color change. Although more than one analyte may be detected in a single reaction or test well, each reaction is processed on an individual basis.
WO2005017485 to Geister et al. describes a method to sequentially determine at least two different antigens in a single assay by two different enzymatic reactions of at least two enzyme labelled conjugates with two different chromogenic substrates for the enzymes in the assay (ELISA), which comprises (a) providing a first antibody specific for a first analyte and a second antibody specific for a second analyte immobilized on a solid support; (b) contacting the antibodies immobilized on the solid support with a liquid sample suspected of containing one or both of the antigens for a time sufficient for the antibodies to bind the antigens; (c) removing the solid support from the liquid sample and washing the solid support to remove unbound material; (d) contacting the solid support to a solution comprising a third antibody specific for the first antigen and a fourth antibody specific for the second antigen wherein the third antibody is conjugated to a first enzyme label and the fourth antibody is conjugated to a second enzyme label for a time sufficient for the third and fourth antibodies to bind the analytes bound by the first and second antibodies; (e) removing the solid support from the solution and washing the solid support to remove unbound antibodies; (f) adding a first chromogenic substrate for the first enzyme label wherein conversion of the first chromogenic substrate to a detectable color by the first enzyme label indicates that the sample contains the first analyte; (g) removing the first chromogenic substrate; and (h) adding a second chromogenic substrate for the second enzyme label wherein conversion of the second chromogenic substrate to a detectable color by the second enzyme label indicates that the sample contains the second analyte.
U.S. Pat. No. 7,022,479 to Wagner, entitled “Sensitive, multiplexed diagnostic assays for protein analysis,” is a method for detecting multiple different compounds in a sample, the method involving: (a) contacting the sample with a mixture of binding reagents, the binding reagents being nucleic acid-protein fusions, each having (i) a protein portion which is known to specifically bind to one of the compounds and (ii) a nucleic acid portion which includes a unique identification tag and which in one embodiment, encodes the protein; (b) allowing the protein portions of the binding reagents and the compounds to form complexes; (c) capturing the binding reagent-compound complexes; (d) amplifying the unique identification tags of the nucleic acid portions of the complex binding reagents; and (e) detecting the unique identification tag of each of the amplified nucleic acids, thereby detecting the corresponding compounds in the sample.
While methods for sequentially detecting and quantifying multiple analytes limited to capturing isotype and subclass for a single analyte are known, these methods require the use of separate assaying steps for each of the analytes of interest and as such, can be time consuming and costly, especially in the context of a clinical setting. A need exists for a method of sequentially detecting and quantifying multiple antibody isotypes and subclasses from a single sample using a single reaction vessel.
Provided is a fast and cost effective method for simultaneous detection and quantifying of multiple analytes in a test sample using a single reaction vessel. The method disclosed herein allows for the simultaneous detection of multiple analytes without the need for separate assays or reaction steps for each target analyte.
In one aspect, provided is a method for simultaneously detecting and quantifying two or more target analytes in a test sample comprising two or more target analytes:
In a further embodiment, the reaction vessel is a well of a multi-well plate where the well has the microarray printed therein.
In a further embodiment, the test sample is a biological sample.
In another aspect, provided is a method for detecting and quantifying biomarkers diagnostic for immunogenicity testing of a therapeutic protein, e.g., insulin, comprising the following steps:
In another aspect, provided is a method for diagnosing multiplex immunogenicity, antibody and insulin factor immunogenicity in a subject, comprising:
In an embodiment, the detection and quantification of predominantly anti-insulin IgM and anti-insulin peptide-IgM antibodies is diagnostic for an early stage of insulin immunogenicity.
In a further embodiment, the detection and quantification of anti-insulin IgA and anti-insulin peptide-IgA antibodies is diagnostic for a transitional stage of insulin immunogenicity.
In a further embodiment, the detection and quantification of anti-insulin IgG, anti-insulin peptide-IgG antibodies as well as their subclasses is diagnostic for a late stage of insulin immunogenicity.
In another aspect, provided is a method for monitoring reactions to immune stimuli by multiplex immuno testing, monitoring development of neutralizing antibodies, including, for example, specific insulin immunogenicity in a subject exposed to various insulin drug immune stimuli, using the method disclosed herein, a plurality of times in the course of treatments.
In another aspect, provided is a method for simultaneously detecting and quantifying two or more different target analytes in a test sample, comprising:
According to yet another aspect, provided is a method for detecting and quantifying biomarkers diagnostic for rheumatoid arthritis, wherein the biomarkers comprise two or more target analytes in a serum sample, comprising:
According to another aspect of the present invention, there is provided a method for simultaneously detecting and quantifying two or more different target analytes in a test sample, the method comprising:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. Preferred embodiments hereof will now be described, by way of example, with reference to the accompanying drawings, in which:
Provided is a method for the detection and quantification of multiple target analytes contained within each test spot or arrays of spots, of a test or test samples, within a single reaction well, per test cycle. The method disclosed herein provides for the simultaneous incubation of an assay device with two or more fluorescently labelled reporters in the same detection mixture as shown in
The methods disclosed herein can be used to detect and quantify biomarkers diagnostic for rheumatoid arthritis. In one embodiment, the method comprises the provision of an assay device having a microarray printed thereon. The microarray may comprise: i) a calibration matrix comprising plurality of spots, each spot comprising a predetermined amount of one of: a human IgA antibody, a human IgG antibody, and a human IgM antibody; ii) a first analyte capture matrix comprising a plurality of spots comprising a predetermined amount of rheumatoid factor; and iii) a second analyte capture matrix comprising a plurality of spots comprising a predetermined amount of cyclic citrullinated peptide. A predetermined volume of a biological sample, preferably a serum sample, is applied to the assay device. A cocktail comprising a first fluorescently labelled reporter compound that selectively binds to IgA antibodies, a second fluorescently labelled reporter compound that selectively binds to IgG antibodies, and a third fluorescently labelled reporter compound that selectively binds to IgM antibodies is then applied to the assay device. The first, second and third fluorescently labelled antibodies are chosen such that each of the antibodies comprise a different fluorescent dye having emission and excitation spectra which do not overlap with each other. A signal intensity value for each spot within the assay device is then measured using a single or multi-channel detector as discussed above. Using the measured signal intensity values, calibration curves are then generated by fitting a curve to the measured signal intensity values for the each of the calibration spots versus the known concentration of the human IgA, IgG and IgM antibodies. The concentration for each of captured rheumatoid factor-IgA, rheumatoid factor-IgG, rheumatoid factor-IgM, anti-cyclic citrullinated peptide-IgG, anti-cyclic citrullinated peptide-IgA, and/or anti-cyclic citrullinated peptide-IgM is the determined using the calibration curves.
In certain embodiments, the method can be used to diagnose or monitor the progress of autoimmune diseases. For example, in the case of rheumatoid arthritis, the detection and quantification of predominantly rheumatoid factor-IgM and anti-cyclic citrullinated peptide-IgM antibodies is diagnostic for an early stage of rheumatoid arthritis whereas the detection and quantification of rheumatoid factor-IgA and anti-cyclic citrullinated peptide-IgA antibodies is diagnostic for a transitional stage of disease progression and the detection and quantification of rheumatoid factor-IgG and anti-cyclic citrullinated peptide-IgG antibodies is diagnostic for a late stage of disease progression. In other embodiments, the method disclosed herein can be used to monitoring the progress of treatment in a subject suffering from rheumatoid arthritis. For example, the concentration levels of rheumatoid factor-IgA, rheumatoid factor-IgG, rheumatoid factor-IgM and at least one of anti-cyclic citrullinated peptide-IgG, anti-cyclic citrullinated peptide-IgA, and anti-cyclic citrullinated peptide-IgM can be measured a plurality of times during the treatment.
When the target analytes of interest are different classes of human antibodies, e.g., hIgG, hIgA, hIgM, hIgE and their respective subclasses are directed to the same antigen (i.e., the Fc region of hIgG), the detection and quantification of each of the target antibodies requires separate assays when conventional methods are employed. With conventional methods, one assay is performed to detect and quantify the amount of hIgG present in a test sample. A second assay must be performed to detect and quantify the amount of hIgM and more assays must be performed to detect and quantify the presence of isoform classes and subclasses. In contrast, the method hereof eliminates the need for multiple detection steps thus reducing costs and time. Using the method hereof, target hIgG, hIgA, hIgM, hIgD and hIgE molecules contained in a test sample can be bound to a single capture spot in an assay device. In the disclosed method, the different classes of antibodies and antibody sub-classes can be detected in a single test by using a cocktail of fluorescently labelled antibodies directed to each of the isoform class hIgG, hIgM, hIgA, hIgE and respective subclass targets. As the antibodies are labelled with different optically excited and emitted fluorescent probes, each of the targets bound to a single capture spot can be detected and quantified using an appropriate calibrator. The use of multi-channel detectors allows for substantially simultaneous detection of multiplex analytes in a single assay. The spot morphology and density of capture molecules is optimized so as to mitigate for steric hindrance. As shown in
The methods employ assay devices useful for conducting immunoassays. The assay devices may be microarrays in 2 or 3-dimensional planar array format.
In one embodiment, the method may employ the use of a multi-well plate and wherein each well has a microarray printed therein. A single well is used as a reaction vessel for assaying the desired plurality of target analytes for each test sample.
The microarray may comprise a calibration matrix comprising a series of calibration spots for each target analyte and an analyte capture matrix comprising one or more of test spots or capture spots which bind the target analytes. A representative microarray is shown in
As used herein, the term “calibration matrix” refers to a subarray of spots printed on and adhering to the reaction vessel, wherein each spot comprises a predetermined amount of a calibration standard. The term “predetermined amount” as used herein, refers to the amount of the calibration standard as calculated based on the known concentration of the spotting buffer comprising the calibration standard and the known volume of the spotting buffer printed on the reaction vessel.
The choice of the calibration standard will depend on the nature of the target analyte. In such embodiments, the microarray will comprise a separate calibration standard for each target analyte. Alternatively, the microarray may comprise a single calibration matrix having calibration spots containing each of the target analytes.
In alternate embodiments, the calibration standard is a surrogate compound. For example, if the target analyte is an antibody, the surrogate compound may be another, different antibody, but of the same class of immunoglobulin (
A person skilled in the art will appreciate that the method of the present invention can be carried out without the use of calibration dots or matrices. Measurements of the intensity of signal from the capture dots can be calculated with reference to known external standards. As such, the determination of the amount of analytes is made without using internal dynamic calibration in alternate embodiments of the method of the present invention.
As used herein, the term “analyte capture matrix” refers to a subarray of spots comprising agents that selectively bind the target analytes. In embodiments where the target analyte is a protein, the agent may be an analyte specific antibody or fragment thereof. Conversely, in embodiments wherein the target analyte is an antibody, the agent may be an antigen specifically bound by the antibody. For example,
A predetermined volume of a test sample is applied to the assay device. Each of the target analytes will bind to their specific capture spot. Thus, in a single capture spot, multiple target analytes may be bound. To detect each of the target analytes, a fluorescently labelled antibody that specifically binds to the target analyte is used. Each fluorescently labelled antibody is coupled to a unique fluorescent dye with a specific excitation and emission wavelength to obtain the desired Stokes shift and excitation and emission coefficients. The fluorescent dyes are chosen based on their respective excitation and emission spectra such that each of the labelled antibodies comprises a different fluorescent dye having emission and excitation spectra which do not overlap with each other. The fluorescently labelled antibodies can be applied to the assay device in a single step in the form of a cocktail.
A signal intensity value for each spot within the assay device is then measured as shown in
The measured signal intensity is directly proportional to the amount of material contained within the printed calibration spots and the amount of analyte from the test sample bound to the printed analyte capture spot. For each calibration compound, a calibration curve is generated by fitting a curve to the measured signal intensity values versus the known concentration of the calibration compound. The concentration for each target analyte in the test sample is then determined using the appropriate calibration curve and by plotting the measured signal intensity for the target analyte on the calibration curve.
The method disclosed herein can be used to detect and quantify multiple clinically relevant biomarkers in a biological sample for diagnostic or prognostic purposes. The measured concentrations for a disease related biomarker can be compared with established index normal levels for that biomarker. The measured concentrations levels which exceed index normal levels may be identified as being diagnostic of the disease. The method disclosed herein can also be used to monitor the progress of a disease and also the effect of a treatment on the disease. Levels of a clinically relevant biomarker can be quantified using the disclosed method a plurality of times during a period of treatment. A trending decrease in biomarker levels may be correlated with a positive and/or negative patient response to treatment.
The method disclosed herein can be used to detect and quantify biomarkers diagnostic for insulin immunogenicity. In one embodiment, the method comprises the provision of an assay device having a microarray printed thereon. The microarray may comprise: i) a calibration matrix comprising plurality of calibration spots, each calibration spot comprising a predetermined amount of one of: a human IgA antibody, a human IgG antibody, a human IgM antibody, a human IgE antibody and respective subclasses; ii) a first analyte capture matrix comprising a plurality of capture spots comprising a predetermined amount of a compound, for example, insulin; and optionally iii) a second analyte capture matrix comprising a plurality of capture spots comprising a predetermined amount of anti-insulin peptide. A predetermined volume of a biological sample, preferably a serum sample, is applied to the assay device. A cocktail comprising a first fluorescently labelled reporter compound that selectively binds to IgA antibodies, a second fluorescently labelled reporter compound that selectively binds to IgG antibodies, a third fluorescently labelled reporter compound that selectively binds to IgM antibodies, a fourth fluorescently labelled reporter which selective binds to IgE and fluorescent labels which bind selectively to immunoglobulin subclasses, is then applied to the assay device. The first, second, third, fourth and selected subclass fluorescently labelled antibodies are chosen such that each of the antibodies comprises a different fluorescent dye having emission and excitation spectra which do not overlap with each other, as shown in
In certain embodiments, the method disclosed herein may be used, e.g., to diagnose or monitor the progress of autoimmune diseases. In other embodiments, the method disclosed herein can be used for monitoring the progress of treatment.
Wherein a therapeutic protein and/or its analytical components are immobilized on a planar microarray surface, analytical components may include subunits of the therapeutic protein, e.g., antibody fragments or fusion partners, metabolic products of the therapeutic protein, peptide components, formulation components, biosimilars, or potential cross reacting entities.
Samples collected from untreated and therapeutic protein treated patients are incubated with the immobilized microarray components. Samples are most likely to be serum or plasma. These samples may be pre-treated or prepared in such a way as to enrich for the availability of any antibodies which the patient may have developed in response to the therapeutic protein or prior exposure to similar entities. Following sample incubation, the microarray surface is interrogated for the presence of patient derived antibodies which have been captured and bound by the immobilized analytes.
The amount of and heavy chain characteristics of the captured patient antibodies are determined by the use of specific anti-human secondary antibodies which have been conjugated to fluorescent dyes.
Secondary reagents can be included to determine the immunoglobulin class Ig G, A, M or E or the sub-classes, including IgG1, IgG2, IgG3, and IgG4. Specific dyes are conjugated to each of the secondary reagents to constitute a reporter and allow differentiation of each of the Ig classes or subclasses. A reporter aliquot is made up of a mix of conjugates as determined by the classes and subclasses that are of interest in the patient study.
As shown in
The intensity of the multiple fluorescent signals when compared to standard curves intensities will allow the qualitative and quantified measurement of a specific immune response to the therapeutic protein or the protein associated analytes.
As one demonstration of the utility of this method, the sensitivity and response of the multiplexed assay for each of the subclasses can be shown to be equivalent to the single plex (one isotype measured at a time) performance.
The signal intensity from multiple isotypes decreases as a sample undergoes serial dilution to develop a coordinated standard curve for quantitation or semi-quantitation of a multiplexed assay, as shown in
The method also interrogates neutralizing effects of a patient's antibodies, i.e.; their ability to directly affect the active mechanism of the therapeutic protein
In cases where the therapeutic protein is a ligand that binds to a receptor, the receptor will be immobilized on the array surface. A fluorescently labeled derivative of the therapeutic protein will be incubated with patient serum in a competitive type immunoassay. A high fluorescent signal in this case indicates an absence of neutralizing antibodies. As the titer of neutralizing antibodies increases in a sample, they will interfere with the ability of the labeled therapeutic protein to bind the receptor and thus decrease the florescent signal on the array surface.
As depicted in
As new insulin variants are developed the need to study the range of immune responses in patients requires the ability to detect, characterize and quantitate anti-insulin antibodies. Regardless of purity and origin, therapeutic insulins continue to be immunogenic in humans. Severe immunological complications rarely occur. Current human insulin and insulin analog therapies result in decreased anti-insulin antibodies levels. Anti-insulin antibody development is also affected by the mode of delivery. For example, use of subcutaneous and implantable insulin pumps or inhaled insulin. Formulation also effects immunogenic potential with regular or semilente insulins being less immunogenic than intermediate or long acting preparations. Aggregation levels also affect immunogenicity.
Anti-insulin antibodies responses consisting of Ig classes and IgG subclasses have been reported. Primarily IgG1-4 but IgA, IgM and IgE have also been reported. IgG is implicated in the most severe cases of insulin resistance. Insulin delivered or inhaled results in a similar distribution of IgG subclasses: IgG1>IgG4>IgG2 and IgG3. IgG1 levels have been reported to decline where IgG4 rises with increased duration of insulin treatment.
The method disclosed is uniquely suited to detect and differentiate the range of anti-insulin antibodies in a single assay, as opposed to running a separate assay for each Ig class or subclass.
In this case, the therapeutic insulin is printed as multiple replicate spots in each well of a 96-well functionalized glass plate. The print conditions, including buffers, concentration, and post print processes are selected to optimize epitope presentation and assay precision. Assay controls including anti-human antibodies or other variants of insulin could be included in each of the 96 wells.
Each well is incubated with patient serum. In cases where the patient has anti-insulin antibodies they are captured by the spotted insulin. Fluorescently labeled anti-human Ig secondary reagents are used to detect the binding anti-insulin antibodies. Secondary reagents include Ig Class specific (IgG, IgA, IgM or IgE) or subclasss specific (IgG1, IgG2, IgG3, IgG4). A fluorescent dye with a different emission spectrum is conjugated to each of the secondary reagents allowing the patient immune response to be characterized based on the intensity of each signal.
In the case where a commercial 3-color array scanner is used to detect the fluorescent signals, the same patient sample is interrogated in multiple wells and different aliquots of labeled reporters are used to fluorescently label each reporter in each well with a specific dye wavelength, e.g., in Well 1, IgA is labeled with dye Cy5 at 667 nm (nanometers), IgG1 with dye FITC at 488 nm and IgG3 with dye PE at 575 nm to measure the IgA, IgG1 and IgG3 in Well 1. In Well 2, IgM is labeled with Cy5 at 667 nm, IgG2 is labeled with FITC at 488 nm and IgG4 is labeled with PE to fluoresce at 575 nm to measure IgM, IgG2 and IgG4. The six immunoglobulins are measured using only three fluorescing labels.
For simultaneous detection of up to six different color fluorescent wavelengths per well, as illustrated in
A method to demonstrate antibody specificity is using a competition assay where increasing amounts of the free drug are added to the sample as the assay signal decreases. In addition to being able to quantitate and isotype ADAs this method can also be applied to simultaneously demonstrate the specificity of each of the isotypes detected.
By adding free drug to the serum all of the isotypes that are specific to the drug will show a decrease in signal. This specificity demonstration can either be carried out at the same time with sample+/−drug being added to two different wells of the assay, or as a follow on assay to confirm specificity in samples showing positive signal. Results are shown in two graphs namely
Drug Tolerance is defined as the maximum amount of free drug that can be present in a sample and still allow detection of ADAs. The presence of high level of free drug may cause anti-drug antibodies to be sequestered in immuno-complexes and unavailable to bind the capture analyte in an immunoassay. Acid dissociation disrupts the immune complexes and improves the drug tolerance of the assay. The ability to form the complexes and also to have binding disrupted by acid or other chemical treatment is independent of isotype. For this application the serum is pre-treated with a disruptive agent. The acid is neutralized before adding the sample to the well. The multiplexed reporter cocktail is used to detect and quantitate each of the isotype or subclasses involved in the anti-drug response.
Various embodiments hereof having been thus described in detail by way of example, it will be apparent to those skilled in the art that variations and modifications may be made without departing from the invention. Also included are all such variations and modifications as fall within the scope of the appended claims.
| Number | Date | Country | Kind |
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| 2475240 | Jul 2004 | CA | national |
This application is a continuation-in-part of U.S. patent application Ser. No. 13/843,297, filed Mar. 15, 2013, pending, which is a continuation in part of U.S. patent application Ser. No. 11/632,746, filed Mar. 26, 2008, which is a national phase entry under 35 U.S.C. § 371 of International Patent Application PCT/CA2005/001147, filed Jul. 20, 2005, published in English as International Patent Publication WO 2006/007726 on Jan. 26, 2006, which claims the benefit under 35 U.S.C. § 119 and under Article 8 of the PCT to Canadian Patent Application No. 2,475,240, filed Jul. 20, 2004, the disclosure of each of which is hereby incorporated herein in its entirety by this reference.
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| Parent | 13843297 | Mar 2013 | US |
| Child | 16230745 | US |
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| Parent | 11632746 | Mar 2008 | US |
| Child | 13843297 | US |
