MULTIPLEX MEASURE OF ISOTYPE ANTIGEN RESPONSE

Abstract

Described are methods for simultaneous detection and quantifying multiple target analytes, including immunoglobulin isotypes and sub-classes, single and multiple protein antibodies within a test sample contained in a single reaction vessel. Such methods use reaction wells as on a multi-well plate, each single well comprising microarrays of calibration spots, each having a predetermined quantity of a target analyte; and capture spots, each having multiple agent antibodies, including isotypes and subclasses that specifically bind the target analytes. The captured analytes and the calibration spots are detected with fluorescently labeled antibodies specific for each different target analyte. Calibration spots generate calibration curves for quantitative determinations of different target analytes. Also described are methods for detecting and quantifying biomarkers, therapeutic proteins and patient derived antibodies; the use of secondary reagents to determine immunoglobulin classes Ig G, A, M, E and sub-classes including IgG1, IgG2, IgG3, IgG4 and IgA. The intensity of each fluorescent signal allows measurement of a specific immune response to a therapeutic protein and associated analytes; interrogates neutralizing effects of patient antibodies on therapeutic proteins, e.g., insulin therapy.

Claims

1.-17. (canceled)

18. A method of determining an amount of an analyte in a sample, the method comprising: introducing the sample into a solution comprising a first reagent able to specifically bind the analyte, wherein the first reagent is labeled with a detectable marker, thereby forming a sample solution, wherein the first reagent binds the analyte in the sample solution to form a first binding reagent-analyte complex; introducing the sample solution onto an assay device having a surface, wherein the surface comprises printed thereon multiple arrays of calibration dots, each of the calibration dots including a different pre-determined quantity of the analyte, wherein the surface further comprises printed thereon a multiple arrays of test dots, each of said multiple arrays of test dots corresponding to a respective multiple array of calibration dots, each of the test dots including a second reagent able to bind the analyte, the test dots within each array being printed at predetermined X-Y locations; wherein the first reagent is present in the solution in excess compared to the total amount of analyte present in the sample and the plurality of calibration dots, wherein the arrays of test dots and the arrays of calibration dots are present together in a single well structure of the assay device, and wherein the test dots and the calibration dots are in fluid contact with each other through the sample solution; binding the first reagent in the solution to the analyte in each of the calibration dots, and binding the first binding reagent-analyte complex in the solution to the second reagent in the test dots in a single discrete fluid flow of the sample solution; measuring an intensity of the detectable marker in each of the calibration dots; preparing a calibration curve correlating the amount of analyte in each of the calibration dots to the measured intensity of the detectable marker in each of the calibration dots; measuring an intensity of the detectable marker in the test dots simultaneously with the measurement of the intensity of the detectable marker in each of the calibration dots; and calculating an amount of analyte present in the test dots within each array of test dots by comparing the measured intensity of the detectable marker in the test dots to the amount of analyte corresponding to said intensity in the calibration curve.

19. The method according to claim 23, wherein the first reagent is an antibody that binds specifically to the analyte, and the second reagent is a capture antibody.

20. The method according to claim 23, wherein the assay device comprises: a loading portion for receiving the sample solution; and a reading portion comprising the plurality of calibration dots printed thereon and the test dot printed thereon.

21. The method according to claim 23, wherein the calibration dots are printed on a portion of the assay device having a length that is between 1 picometer and 12 millimeters.

22. The method according to claim 23, wherein the calibration dots are printed on a portion of the assay device having a length that is between 25 micrometers and 300 micrometers.

23. The method according to claim 23, wherein the calibration dots are printed in arrays at predetermined X-Y co-ordinates on the surface of the assay device.

24. The method according to claim 23, wherein the surface of the assay device is substantially planar.

25. The method according to claim 23, wherein the different pre-determined quantities of the analyte in the calibration dots correspond to a dynamic range for measuring the analyte.

26. The method according to claim 23, wherein the calibration dots are arranged in at least three replicated arrays.

27. The method according to claim 23, wherein the calibration dots are arranged in at least three arrays for preparing at least three calibration curves.

28. The method according to claim 23, wherein an amount of the analyte in a single sample is measured on a single assay device.

29. The method according to claim 23, wherein an amount of the analyte is measured in a plurality of arrays on the assay device.

30. The method according to claim 23, wherein amounts of a plurality of different analytes are measured contemporaneously on the assay device.

31. The method according to claim 23, wherein amounts of a plurality of different analytes in a single sample are measured contemporaneously on the assay device.

32. A method of determining an amount of an analyte in a sample without utilizing externally-derived calibration standards, the method comprising: introducing the sample comprising the analyte into a solution comprising an excess of a fluorescently-labeled antibody that binds specifically to the analyte, thereby forming a sample solution comprising fluorescently-labeled antibody-analyte complexes and free fluorescently-labeled antibody; introducing the sample solution onto an assay device, wherein the assay device comprises: multiple arrays of calibration dots, the calibration dots comprising multiple different pre-determined quantities of the analyte, and multiple arrays of test dots, each of said multiple arrays of test dots corresponding to a respective multiple array of calibration dots, each of the test dots, each of the test dots comprising a capture antibody against the fluorescently-labeled antibody-analyte complex, wherein the free fluorescently-labeled antibody is comprised in the sample solution in excess compared to the total amount of the analyte present in the calibration dots, wherein introducing the sample solution onto the assay device results in binding of the free fluorescently-labeled antibody in the solution to the analyte in each of the calibration dots, and wherein introducing the sample solution onto the assay device results in binding of the fluorescently-labeled antibody-analyte complexes in the solution to the capture antibody in the test dots in a single discrete fluid flow of the sample solution; and wherein the test dots and the calibration dots are together in a single compartment of the assay device, measuring a fluorescence intensity in each of the calibration dots; preparing a calibration curve correlating the amount of analyte in each of the calibration dots to the measured fluorescence intensity in the calibration dot; measuring fluorescence intensity in the test dots simultaneously with the measurement of the intensity of the detectable marker in each of the calibration dots; and calculating an amount of analyte present in the test dots by comparing the measured fluorescence intensity in the test dots to the amount of analyte corresponding to said intensity in the calibration curve.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0074] 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:

[0075] FIG. 1 is a schematic illustration of the multiplex analyte detection method hereof;

[0076] FIG. 2 is a schematic illustration of the multiplex analyte immunogenicity detection content per spot, in a single well, including IgG, IgA, IgM, IgE and subclasses for method of the disclosed invention, defined as Ig_plex;

[0077] FIG. 3 is a schematic illustration of a microarray printed on an assay device hereof;

[0078] FIG. 4 illustrates typical 4 level multiplex microarray spots contained in a single well of an assay device hereof;

[0079] FIG. 5 is a plot to illustrate composite fluorescent intensity detection for six fluorophores at non-interfering wavelengths, at 457 nm (nanometers), 488 nm, 575 nm, 615 nm, 667 nm and 767 nm hereof;

[0080] FIG. 6 is a table showing the results 17 celiac patients and 7 normal patients to demonstrate overall agreement to single plex predicate;

[0081] FIG. 7 is a schematic illustration of the multiplex analyte immunogenicity detection content per spot, in a single well, including IgA, IgG.sub.1, and IgG.sub.3;

[0082] FIG. 8 is a bar graph showing MFI from CEL serum on analyte singleplex (S) versus multiplexed (M);

[0083] FIG. 9 is a graph showing a plot of MFI versus sample dilution for Anti-Ig1-FITC, IgGA-APC and Anti-IgG3-RPE in a first multiplex well for CEL0058 on Analyte 3;

[0084] FIG. 10 is a graph showing a plot of MFI versus sample dilution for Anti-Ig1-FITC, IgGA-APC and Anti-IgG3-RPE in a second multiplex well for CEL0167 on Analyte 3;

[0085] FIG. 11 is an illustration depicting the effect of neutralizing antibodies;

[0086] FIG. 12 is a graph showing a plot of raw fluorescence units versus inhibitor concentration for three samples showing IgG response;

[0087] FIG. 13 is a graph showing a plot of raw fluorescence units versus inhibitor concentration for three samples showing IgA response;

[0088] FIG. 14 is a schematic illustration demonstrating multiplexed serum antibody specificity for serum IgG and IgA antibody for a drug molecule analog demonstrated in a single inhibition assay; and

[0089] FIG. 15 is a graph showing a plot of raw fluorescence units versus acid concentration in terms of guanidine-HCl concentration.

DETAILED DESCRIPTION

[0090] 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 FIG. 2. The method disclosed herein can detect a plurality of multiplexed analytes per test spot or capture spot, using a single reaction vessel instead of separate reaction vessels to detect each analyte. The terms test spot and capture spot can be used interchangeably for the purposes of the present specification.

[0091] FIG. 1 illustrates the capturing of six different antibodies that selectively bind to two different antigens. The six different antibodies fall into three different antibody classes. In this example, an IgG is included that specifically binds to antigen A while a separate IgG is included that specifically binds to antigen B. Similarly, an IgA is included that specifically binds to antigen A while a separate IgA is included that specifically binds to antigen B. Finally, an IgM is included that specifically binds to antigen A while a separate IgM is included that specifically binds to antigen B. In such embodiments, only one calibration matrix may be required for each of the three different classes of immunoglobulins.

[0092] 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.

[0093] 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.

[0094] 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 FIG. 2, the target analytes are IgG, IgA, IgM, IgE, IgG1, IgG2, IgG3, and IgG4. Capture spots include an antigen that binds to these antibody isotypes and subclasses. Once the target analyte antibodies bind to the capture spots, fluorescently labeled anti-IgG, anti-IgA, anti-IgM, anti-IgE, anti-IgG1, anti-IgG2, anti-IgG3, and anti-IgG4 bind to the respective target analyte antibodies so that the amount of bound IgG, IgA, IgM, IgE, IgG1, IgG2, IgG3, and IgG4 can be detected.

[0095] The methods employ assay devices useful for conducting immunoassays. The assay devices may be microarrays in 2 or 3-dimensional planar array format.

[0096] 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.

[0097] 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 FIG. 3. The microarray 1 includes capture spots 2 and calibration spots 4. In addition, internal control spots 6 are included to ensure that the microarray is functioning properly.

[0098] 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.

[0099] 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.

[0100] 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 (FIG. 4). The calibration matrix may be printed on the base of the individual reaction vessel in the format of a linear, proportional dilution series. The predetermined concentrations of calibration standards are selected to include lower and upper expected detection limits to define the dynamic range. The mid-point of dynamic calibrated concentration range approximates the diagnostic critical concentration of the detection system used to read the microarray.

[0101] 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.

[0102] 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, FIG. 4 illustrates the capturing of five different antibodies that selectively bind to two different antigens.

[0103] 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.

[0104] A signal intensity value for each spot within the assay device is then measured as shown in FIG. 5. The fluorescent signals can be read using a combination of scanner components such as light sources and filters. A signal detector can be used to read one optical channel at a time such that each spot is imaged with multiple wavelengths, each wavelength being specific for a target analyte. An optical channel is a combination of an excitation source and an excitation filter, matched for the excitation at a specific wavelength. The emission filter and emission detector pass only a signal wavelength for a specific fluorescent dye. The optical channels used for a set of detectors are selected such that they do not interfere with each other, i.e., the excitation through one channel excites only the intended dye, not any other dyes. Alternatively, a multi-channel detector can be used to detect each of the differentially labelled antibodies. The use of differential fluorescent labels allows for substantially simultaneous detection of the multiple target analytes bound to a single capture spot.

[0105] 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.

[0106] 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.

[0107] 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 FIG. 5. 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 each of the calibration spots versus the concentration of the human IgA, IgG, IgM, IgE and subclass antibodies. The concentration for each of the captured insulin analytes is determined using the calibration curves.

[0108] 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.

Example 1Multiplex Immunogenicity Testing

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] As shown in FIGS. 5, 6 and 7, this assay can be configured to use (i) a three color fluorescent scanner by including the same patient sample in multiple interrogated wells and adding a three color constituent reporter blend to each of the wells; or (ii) increased to multiples of up to six colors per well as determined by selecting fluorescent dyes which have separable emission peaks used in conjunction with a scanner equipped with appropriate excitation and emission filters.

[0114] 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.

[0115] 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. FIGS. 5 and 7 show the results.

[0116] 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 FIGS. 8 and 9.

Example 2Neutralizing Antibodies

[0117] 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

[0118] 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.

[0119] As depicted in FIG. 10, in cases where the mechanism of the therapeutic protein is to block a ligand/receptor interaction, the receptor is immobilized on the microarray surface. A fluorescently labeled derivative of the appropriate ligand, and the therapeutic protein is incubated with patient serum. In the absence of neutralizing antibodies the therapeutic protein will block the binding of the labeled ligand to the receptor and little or no fluorescent signal will be detected. As the titer of neutralizing antibodies increases, they will interfere with the ability of the therapeutic protein to block the ligand/receptor interaction, and the fluorescent signal will increase.

Example 3Insulin Immunogenicity

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] For simultaneous detection of up to six different color fluorescent wavelengths per well, as illustrated in FIG. 5, the excitation source and emission filters are coordinated with a set of dyes with compatible spectra; IGg1-FITC dye at 488 nm, IgG2-PE dye at 575 nm, IgG3-Cy5 dye at 667 nm, IgG4-Pe-Cy dye at 767 nm, IgA-Pac Blue dye at 457 nm, IgM-Alexa fluor dye at 594 615 nm. If more than six fluorescent labels are required to report and measure multiple antigens, the identified set, or compatible sets of six wavelengths are applied in each separate well. It is to be understood that more than six different color fluorescent wavelengths per well can be detected in alternate embodiments hereof.

Example 4Anti Drug Antibody (ADA) Specificity Confirmation

[0127] 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.

[0128] 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 FIGS. 12 and 13.

[0129] FIG. 14 is an illustration showing as the objective of this example to demonstrate multiplexed serum antibody specificity. In the example, 3 IgG/A positive serum samples pre-incubated with serial dilutions of drug molecule analog before microarray assay are depicted. Serum IgG and IgA antibody specificity for a drug molecule analog is demonstrated in a single inhibition assay.

Example 5Multiplexed Compatibility with Acid Dissociation

[0130] 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.

[0131] FIG. 15 graphically depicts an example of this assay showing that bioanalytic assays may require a dissociation step prior to measurement of anti-drug antibodies. IgG positive serum plus analog plus two-fold dilutions of guanidine are neutralized with IN NaOH. The graph shows IgG signal on printed drug analog. In conclusion the multiplex assay is compatible with immune complex dissociation.

[0132] 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.