HK1252561B - Automated immunoanalyzer system for performing diagnostic assays for allergies and autoimmune diseases - Google Patents

Description

Automated immunoassay system for diagnostic testing of allergies and autoimmune diseases

This application is a divisional application of Chinese patent application 201480015969.4 (PCT/US2014/030373) filed on March 17, 2014.

Technical Field

The present teachings relate to a system and method for performing diagnostic assays, and more particularly, to an automated immunoassay system and method for performing diagnostic assays for allergies and autoimmune diseases.

Background Art

The statements in this section merely provide background information related to the present disclosure and are not intended to constitute prior art.

During automated immunochemical analysis, analyte molecules in a patient's biological sample (e.g., serum or plasma) are attached to paramagnetic particles. To remove background signals associated with potential chemical sources that may also be present in the sample, multiple washing steps are typically performed in the process. However, as a result of these washing steps, a portion of the initial particles used in subsequent chemical processes will be lost.

Therefore, there is a need for a method that allows quantification of particles retained after a washing step in order to normalize the luminescence signal from patient samples.The present application aims to improve and address some of these known deficiencies in the art.

Summary of the Invention

According to one aspect of the present application, a quantitative method for performing automated diagnostic assays is provided, the method comprising the following steps: incubating a capture reagent with a streptavidin-coated medium to form a solid phase complex; washing the solid phase complex to remove excess capture reagent; incubating the solid phase complex with a serum sample to form an immune complex; washing the immune complex to remove any unbound sample; incubating the immune complex with a conjugate to generate an immune-conjugate complex; washing the immune-conjugate complex to remove any unbound conjugate; introducing a substrate capable of producing a quantifiable reaction; and calibrating the reaction produced by the introduction of the substrate.

According to yet another aspect of the present application, a method for controlling the incorporation of fluorescent markers into particles within a patient sample is provided. According to this aspect of the disclosure, the method includes incorporating a luminescent marker into the particles and quantifying the particles that remain after a series of wash steps to normalize the luminescent signal from the patient sample. According to this illustrative method, the luminescent marker is bound to the particles in proportion to the number of bound analyte molecules.

According to yet another aspect of the present disclosure, a quantitative method for evaluating allergen-specific immunoglobulin E (IgE) in a serum sample designed for use on an automated platform is provided. According to this method, a biotinylated capture reagent is incubated with a streptavidin-coated solid phase to bind the capture reagent to the solid phase via a biotin-streptavidin interaction. The capture reagent-solid phase complex is then washed to remove excess biotinylated capture reagent. The serum sample is then incubated with the capture reagent-solid phase complex to allow allergen-specific IgE present in the serum to bind to the capture reagent and form an immune complex. The immune complex is then washed to remove unbound IgE and subsequently incubated with a labeled anti-IgE conjugate to allow the conjugate to bind to the allergen-specific IgE component of the immune complex and form an immune-conjugate complex. The immune-conjugate complex is washed to remove unbound labeled anti-IgE and then a substrate capable of generating a quantifiable reaction is introduced. The quantifiable reaction generated by the addition of the substrate is calibrated and the reporting value is adjusted for bead retention.

According to certain aspects of the present invention, the step of incubating a biotinylated capture reagent with a streptavidin-coated solid phase is performed to biotinylate a purified allergen, protein, enzyme, or antibody.

According to other aspects herein, the step of incubating the biotinylated capture reagent with a streptavidin-coated solid phase is derived from the biotinylation of an allergen extract consisting of a plurality of allergens.

According to yet other aspects herein, the step of incubating the biotinylated capture reagent with a streptavidin-coated solid phase is derived from the biotinylation of an allergen extract for in vivo human diagnosis or treatment.

According to certain illustrative aspects of the present disclosure, the biotinylated capture reagent is present as a blend of multiple biotinylated capture reagents from different sources including purified allergens, proteins, enzymes, antibodies, and allergen extracts.

According to yet another specific illustrative aspect of the present disclosure, the streptavidin-coated solid phase is a universal fluorescently labeled magnetic microparticle.

According to certain aspects of the present disclosure, the one or more wash steps comprise washing the solid phase complex by magnetically isolating the complex within a confined area of the reaction cuvette.

According to yet other illustrative aspects of the present disclosure, the step of incubating the capture reagent-solid phase complex with the serum sample includes incubating the suspended capture reagent-solid phase complex with a reaction diluent containing a high concentration of human serum albumin (HSA).

According to yet another illustrative aspect of the present disclosure, the step of incubating the immune complex with a labeled anti-IgE conjugate comprises incubating the suspended immune complex with a conjugate diluent comprising an indicated concentration of polyethylene glycol. According to a specific aspect of the present teachings, the conjugate diluent consists of 100 ng/mL anti-IgE-HRP, 100 μg/mL apo-HRP, 50 mM sodium phosphate, pH 6.7, 150 mM NaCl, 0.05% Tween-20, 1% BSA, 4% (w/v) PEG 6,000, 1% (v/v) ProClin 950, and 0.015% (v/v) antifoam B. According to yet another specific aspect of the present teachings, the conjugate diluent consists of 10 ng/mL anti-IgG-HRP, 10 μg/mL apo-HRP, 50 mM sodium phosphate, pH 6.7, 150 mM NaCl, 0.05% Tween-20, 1% BSA, 4% (w/v) PEG 6,000, 1% (v/v) ProClin 950, 0.015% (v/v) antifoam B.

According to certain specific aspects of the present disclosure, horseradish peroxidase (HRP) conjugated to an anti-IgE antibody can be used as an indirect label when washing the immune complex to remove unbound IgE, particularly because the reaction of PS-Atto with the HRP-labeled conjugate produces long-lasting, high-intensity luminescence for maximum detection sensitivity in solution assays.

According to yet other specific aspects of the present disclosure, adding a substrate to the immuno-conjugate complex comprises adding Lumigen PS-Atto as a substrate capable of generating a quantifiable reaction in the presence of a chemiluminescent signal generated by an HRP-PS-Atto reporter system and detected by an illuminometer in an optical box.

According to certain aspects of the present teachings, the step of adjusting the quantifiable reaction for bead retention includes the following steps: transferring the substrate and the immuno-coupled complex to an optical cartridge, quantifying the fluorescence signal and the chemiluminescent signal therein; using the ratio of the initial to final fluorescence to adjust the quantified chemiluminescent signal for bead retention; and calibrating the adjusted chemiluminescent signal to calculate a reporter value. To transfer the substrate and the immuno-coupled complex to the optical cartridge, an automated pipette arm with a reusable pipette end for aspirating the sample can be used. Within the optical cartridge, fluorescence is measured to determine bead retention, and luminescence is measured to detect the RLU signal generated by the chemical reaction. The measured values are substituted into an algorithm to generate "RLU adjusted for bead retention," which is compared to the calibration curve RLU to determine the IgE concentration.

According to yet other aspects of the present disclosure, the fluorescent label is present as Alexa Fluor 594 biocytin and the universal magnetic particles are present as Thermo Scientific SA-Speed Bead or Bangs Lab BioMag Plus Streptavidin.

Still other objects and advantages of the present invention will become apparent from the following written description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The above aspects of the invention and the manner of obtaining them will become more apparent and the invention itself will be better understood by reference to the following description of embodiments of the invention taken in conjunction with the accompanying drawings, in which:

FIG1 is a schematic diagram of a method for performing an automated diagnostic assay according to the present application; and

2 is a top view of an automated immunochemistry assay and reagent system according to the teachings of the present application.

Corresponding reference characters indicate corresponding parts throughout the several views. Although the exemplification described herein illustrates embodiments of the invention, in several forms, the embodiments disclosed below are not intended to be exhaustive or to be construed as limiting the scope of the invention to the precise forms disclosed.

DETAILED DESCRIPTION

The embodiments of the present application described below are not intended to be exhaustive or to limit the present application to the exact forms disclosed in the following detailed description. However, the embodiments are selected and described so that other persons skilled in the art can recognize and understand the principles and implementation of the present application.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this application, specific methods and materials are now described. Furthermore, the techniques used or contemplated herein are standard methods well known to those of ordinary skill in the art, and the materials, methods, and examples are illustrative only and are not intended to be limiting.

Before describing the illustrative automated immunoassay systems and methods of the present disclosure in detail, it should be understood and appreciated that immunoassays typically require one or more phase separations in the reaction cuvette as a means of minimizing background signal from excess or unbound material. To facilitate the separation or washing process, a variety of techniques can be used, including, but not limited to, well coating, bead coating, or the use of paramagnetic particles. Each of these separation media is coated with a capture reagent that will bind to the analyte molecules of interest in the patient's blood sample. According to certain aspects of the present teachings, the biotinylated capture reagents can be present as a blend or mixture (i.e., capture reagents from similar species but from different genera). It should be understood and appreciated by those skilled in the art that a variety of capture reagents are available and can be used in accordance with the present teachings, including those available with FDA approval, such as Mixed Vespid Venom Protein (mixed yellow wasp, yellow bee, and white-faced yellow bee). It should be understood that the amount and volume of each of the individual capture reagents used in accordance with the present teachings depends on their potency (i.e., their ability to produce a detectable response).

When paramagnetic particles are used as the separation medium, they are pulled toward the cuvette wall by a magnet during the washing process, and any liquid is then aspirated. Those skilled in the art will understand and appreciate that during conventional washing processes, some paramagnetic particles may be aspirated along with the liquid and thus lost to further chemical processing. If the immunoassay procedure involves multiple washing steps, the loss of magnetic particles can be even more pronounced.

One objective of the present teachings is to account for the loss of paramagnetic particles that occurs on immunochemical analyzers during these wash processes. To this end, according to certain aspects of the present teachings, analytes of interest in a patient's blood sample are bound to a capture reagent, which in turn binds to the surface of the paramagnetic particles. A luminescent label then binds to these analyte molecules. When a luminescent reagent or substrate is added to the cuvette, it reacts with the luminescent label to produce light that can be detected using the analyzer's optical detector. Furthermore, if the paramagnetic particles are attached to a fluorescent label, reading the contents of the cuvette fluorescently provides a method for determining the fraction of particles lost during the wash steps.

According to certain aspects of the present disclosure, automated analyzers utilize common paramagnetic particles used in assays, including, but not limited to, magnetic beads or microparticles. For each assay on the analyzer, a capture reagent is incubated and bound to universal particles in a reaction cuvette to produce an assay-specific, particle-based reagent, sometimes referred to herein as a capture-reagent solid-phase complex. According to certain aspects of the present disclosure, a capture reagent useful for performing diagnostic immunoassays consists of biotin-pAb or biotin-allergen, 10 mM sodium phosphate, pH 7.4, 0.9% NaCl, 0.05% Tween-20, 1% (w/v) human serum albumin, 1% (v/v) ProClin 950, and up to 5% (v/v) glycerol. According to yet other aspects of the present disclosure, another capture reagent that can be used to perform a diagnostic immunoassay consists of biotin-Ags, 10 mM sodium phosphate, pH 7.4, 0.9% (w/v) NaCl, 0.05% Tween-20, 1% (w/v) bovine serum albumin, 1% (v/v) ProClin 950, 1% protease inhibitor cocktail, 0.1 mM DTT, 25% (up to 30%) (v/v) glycerol.

After undergoing a washing process, the patient sample, and optionally a diluent (as needed), is added to the particles in the cuvette and incubated. This results in the capture of specific analyte molecules in the patient's blood sample. According to one specific illustrative aspect of the present disclosure, the reaction diluent (sample diluent) consists of 10 mM sodium phosphate, pH 7.4, 500 mM NaCl, 0.02% Tween-20, 1% (w/v) human serum albumin, 1% (v/v) human IgG, 1% (v/v) ProClin 950, 0.005% Antifoam-B v/v, and 2% (w/v) PEG 6,000. According to yet another specific illustrative aspect of the present disclosure, the reaction diluent (sample diluent) consists of 10 mM sodium phosphate, pH 7.4; 500 mM NaCl, 0.02% Tween-20, 25% (w/v) human serum albumin, and 1% (v/v) ProClin 950.

According to these illustrative embodiments, it should be understood herein that the high percentage of HSA (25%) is used, in part, to increase the viscosity of the reaction medium to keep the beads suspended during the incubation step. In addition, high HSA also reduces nonspecific binding during this incubation period and improves relative light unit (RLU) linearity when the patient sample is diluted.

Another wash cycle is then performed to remove any excess or unbound sample, followed by the addition of the luminescent label and conjugate to the cuvette. Upon addition to the cuvette, it is expected that, after an incubation period, a portion of the conjugate will bind to the capture reagent/sample complex on the paramagnetic particles. The particles are then subjected to another wash cycle to remove any unbound conjugate, and then the substrate is added to the cuvette and incubated briefly to allow the chemiluminescent reaction to reach equilibrium.

After reaching equilibrium, luminescence and fluorescence readings are taken of the samples. Because the paramagnetic particles are contained in a common reagent bottle of the analyzer and kept in a uniform suspension before being aspirated into the reaction cuvette, the initial fluorescence measurement of the particles after aspirating them into the cuvette, when combined with the final fluorescence measurement of each test, can be used to determine the fraction of the initial particles that remain in the cuvette after the immunoassay. The retention fraction is given by the following formula:

in

F represents the corrected fluorescence signal (i.e., the measured signal corrected by the counting efficiency of the optical detector). Because optical detectors have a certain temporal resolution, as the number of photons detected per unit time increases, the probability that two photons will reach the detector within the temporal resolution also increases. Because these two photons cannot be distinguished by the detector, they are counted as a single photon. Therefore, the detection efficiency of an optical detector decreases as the incident photon flux increases.

Because of the extremely high flux of fluorescent excitation photons that interact with and scatter from the container walls of the paramagnetic particles, the optical detector will count a certain number of photons even if no fluorescent material is present. The background signal for this calibration is represented by _Fbackground .

The use of fluorescence measurements to determine the percentage of initial paramagnetic particles that remain in the reaction cuvette during an immunoassay is advantageous because the method does not limit system throughput, particularly because the method does not limit the achievable timing or parallel processing. On the other hand, most conventional immunoassay analyzers rely on reproducible processing of paramagnetic particles and samples, which does limit the achievable timing or parallel processing and, therefore, the system throughput. Although changes in processing efficiency over time are not detectable in these conventional immunoassay analyzers, these changes can be detected using fluorescence detection. The teachings of the present disclosure allow the use of parallel processing (e.g., multiple wash arms) whose washing efficiency varies due to slight mechanical alignment or fluid differences. Fluorescence readings taken after each step of the immunoassay are used to verify the equivalent functionality of the parallel processing.

The automated immunoassay apparatus and reagent system for performing diagnostic assays for allergies and autoimmune diseases according to the methods and techniques described above will now be described in greater detail. As described herein, it will be understood and appreciated that the disclosed apparatus for performing the assays can be configured to accept standard or universal collection tubes so that a variety of different assays can be performed using the system. It will also be understood and appreciated by those skilled in the art that a variety of known methods exist for isolating antigens, including allergens and autoimmune antigens from source materials. Because these separation methods are widely understood and accepted in the art, they will not be discussed in detail herein, particularly because those skilled in the art will recognize that any acceptable antigen separation method can be incorporated into the system of the present invention without departing from its spirit or scope. After the allergens or autoimmune antigens are isolated, they can then be coupled to biotin to produce a biotinylated antigen or capture reagent. The biotinylated antigen is then contacted with a streptavidin-linked solid support or membrane. According to certain aspects of the present disclosure, biotinylated capture reagents can be derived from components including, but not necessarily limited to, purified allergens, proteins, enzymes, antibodies, DNA, nuclear extracts, cell extracts, and non-protein antigens (e.g., drugs or materials cross-linked to proteins).

Those skilled in the art will understand and appreciate that standard biotinylation methods and techniques commonly used in diagnostic allergy immunoassays can be used in accordance with the present teachings; however, the biotin/protein ratio of the reaction can be optimized as needed to ensure optimal performance of the various biotinylation reagents used in the chemistry. According to certain aspects of the present teachings, the specifically sized linker arm of the biotin reagent is NHS-PEG ₁₂ -biotin. Furthermore, for non-protein antigens, the material can be cross-linked to the biotinylated protein for coating onto a streptavidin bead solid phase, while for autoimmune antigens such as DNA, the biotinylated dideoxynucleotide can be incorporated into the DNA.

A schematic illustration of an automated diagnostic assay according to certain aspects of the present disclosure is shown in FIG1 . According to this illustrative embodiment, manufactured magnetic beads or microparticles having a streptavidin coating are mixed (incubated) with a known biotinylated allergen or autoimmune antigen (step 10). It will be understood and appreciated by those skilled in the art herein that the well-known affinity binding between streptavidin and biotin facilitates the coating of the antigen onto the bead surface and thus allows the use of universal beads with loaded reagent formulations. It will also be understood and appreciated herein that the amount of time and associated laboratory conditions required to incubate the biotinylated capture reagent with a streptavidin-coated solid phase according to the present teachings may vary depending on the specific experiment being conducted, however, according to certain aspects of the present disclosure, a particularly effective incubation time range is from about 1 minute to about 15 minutes, more particularly from about 5 minutes to about 10 minutes and at a temperature of from about 2° C. to about 40° C., more particularly from about 36.8° C. to about 37.2° C.

As shown in Table 1 below, according to this aspect of the present disclosure, the following beads can be used in the magnetic carrier disclosed herein:

Table 1

While various methods can be used to mix or incubate biotinylated allergens or autoimmune antigens with streptavidin-coated beads, according to certain specific embodiments, the products are mixed in a reaction cuvette to coat the beads with the allergen or antigen due to the biotin/streptavidin interaction. According to one illustrative embodiment, 10 μL of streptavidin (SA)-coated beads are dispensed into a reaction cuvette, followed by 40 μL of biotinylated allergen or autoimmune antigen, with mixing occurring during the dispensing process. The mixture is incubated for 1-15 minutes. Excess biotinylated allergen or autoimmune antigen can then be washed away by pulling the magnetic beads to the side of the reaction cuvette and immobilizing them while the reaction cuvette is washed with a buffer solution (step 20). According to one illustrative embodiment, the buffer solution can consist of 10 mM sodium phosphate, pH 7.4, 0.9% (w/v) NaCl, 0.05% (v/v) Tween-20, 10 mg/mL HSA, and 1% (v/v) ProClin 950. While one skilled in the art may employ any readily adaptable immobilization technique known in the art to retain the magnetic beads on the side of the reaction cuvette, according to certain specific illustrative embodiments, an external magnet is used to immobilize the magnetic beads while performing the wash step.

The streptavidin-coated magnetic beads are then released from the magnetic field and allowed to move freely within the reaction cuvette. A biological sample (serum or plasma) is then added to the reaction cuvette, followed by 40 μL of reaction buffer to resuspend the magnetic beads (step 30). In addition to the biological sample, according to certain aspects of the present disclosure, a high concentration of human serum albumin (HSA) may also be used in the suspension to promote macromolecular binding and maintain the magnetic beads in solution. Human IgG is added to the buffer to maintain reaction linearity.

If the sample contains any antibodies (e.g., IgE, IgG) reactive to any allergen or antigen bead coating, these antibodies will bind during this sample incubation step. The sample incubation is maintained at 37°C for 40 minutes. After any antibodies in the patient sample bind to the beads, a second wash step is performed to remove any unbound patient sample (step 40). 150 μL of wash buffer concentrate (50 mM sodium phosphate, pH 7.4, 4.5% (w/v) NaCl, 0.05% Tween-20, 0.05% (v/v) ProClin 950, 0.02% (v/v) Antifoam-C v/v) is added to resuspend the beads and then the beads are pulled using a magnet for 1.5 minutes. After the solution is removed, the magnet is removed and 200 μL of wash buffer is added to resuspend the beads. The wash is then repeated once more.

After performing the second washing step, the beads are resuspended in antibodies specific for human immunoglobulin E (IgE) in the case of allergy assays or for human immunoglobulin G, M or A (IgG/M/A) in the case of autoimmunity assays. According to certain aspects of the present disclosure, the antibodies are coupled to an enzyme (e.g., horseradish peroxidase) to bind any specific patient antibodies captured by the beads (step 50). The beads are then washed again to remove any excess antibody (step 60), and a highly sensitive light-forming reagent (e.g., a chemiluminescent substrate) is added to maximize the sensitivity of the assay (step 70). According to the teachings of the present disclosure, illustrative reagents that can be used as chemiluminescent substrates include, but are not limited to, PS-atto, ELISA Pico chemiluminescent substrates, or ELISA Femto maximum sensitivity substrates. Those skilled in the art will understand and appreciate that a variety of compounds with various structural species, including xanthene dyes, aromatic amines, and heterocyclic amines, can be used to generate chemiluminescence under these conditions. These compounds are well known in the patent literature and are readily available from a variety of commercial suppliers. Some non-limiting chemiluminescent compounds include, but are not limited to, dioxetane type molecules, fluorescein, PS-2, PS-3, TMA-6, TMA-3.

Once the highly sensitive light-generating reagent is added to the reaction cuvette, light is generated (step 80). According to certain embodiments, the light can be measured by transferring the solution in the pipette tip to a reading station to read both the luminescent and fluorescent signals. However, it should be understood that the light emitted according to the present disclosure can be detected by any suitable known detection method available in the art, including, but not limited to, a luminometer, x-ray film, high-speed photographic film, a CCD camera, a scintillation counter, a chemical photometer, or visual inspection. Those skilled in the art will readily understand and appreciate that each detection average inherently has a different spectral sensitivity; the selected detection device can be dictated by a number of factors, including application and use, cost, and convenience. Furthermore, as used herein, a quantifiable or detectable reaction that can be measured according to the present disclosure refers to a positive sample in which allergen-specific IgE causes binding of anti-IgE-HRP, resulting in luminescence (i.e., RLU = relative light units) upon addition of substrate. Furthermore, it should be understood herein that a quantifiable or detectable reaction can also be applied to a quantifiable reaction generated by a negative sample.

According to certain aspects of the present teachings, the RLU generated by any allergen-specific IgE (sIgE) positive/negative sample is compared to the RLU generated by a whole IgE (tIgE) calibration curve. The calibration curve is generated by evaluating a large number of pre-diluted whole IgE (tIgE) calibrators (generated from WHO standards) using a biotinylated anti-IgE capture reagent.

To better understand the mechanical aspects of the present disclosure, FIG2 illustrates an automated immunochemical analyzer and reagent system 100 that can be used to quantify and normalize the luminescent signal of an analyte sample according to the teachings of the present disclosure. According to this illustrative aspect, the automated immunochemical analyzer 100 begins by dispensing fluorescently labeled paramagnetic particles or fluorescent beads into a cuvette located in a reaction rotator 106. According to one embodiment herein, an exemplary fluorescent bead comprises fluorescent beads (SA-Speed Bead, Atto 590 labeled), 1 mg/mL.

Fluorescent beads are first placed in a vortexer 102 and transferred to a reaction spinner 106 via an R1 pipette 104. The R1 pipette 104 aspirates the desired amount of the fluorescent bead mixture and transfers it to the reaction spinner 106, where it is injected into a cuvette in the reaction spinner 106. After injection into the cuvette, an optical pipette 108 aspirates the test sample from the cuvette in the reaction spinner 106 and transfers it to an optical box 110. Once the sample is placed in the optical box 110, fluorescence and luminescence measurements can be recorded. The initial recording of the fluorescence and luminescence signals can serve as a baseline measurement of the fluorescence signal, which can correspond to the initial concentration of fluorescent beads in the sample. After recording the measurements, a multi-rinse pipette 112 rinses the cuvette with wash buffer.

Next, the fluorescent beads can be transferred from the vortexer 102 to the cuvette in the reaction rotator 106 using the R1 pipette 104. Subsequently, the R1 pipette 104 can aspirate the capture reagent from the reagent rotator 114 and inject it into the cuvette in the reaction rotator 106. After the incubation period, the single-rinse pipette 116 can inject wash buffer to resuspend the fluorescent beads. A magnet can then be used to localize the large number of suspended fluorescent beads in the reaction rotator 106 over a period of time. After the magnet has substantially localized the fluorescent beads in the cuvette, the multi-rinse pipette 112 can aspirate and process a portion of the wash buffer, leaving a portion of the fluorescent beads in the cuvette. The multi-rinse pipette 112 can continue to inject wash buffer into the cuvette in the reaction rotator 106 to resuspend the fluorescent beads. The fluorescent beads can again be concentrated in the reaction rotator 106 using the magnet, and the multi-rinse pipette 112 can then aspirate and discard the unlocalized portion of the sample from the cuvette in the reaction rotator 106.

The patient sample can be placed in a sample tube or sample spinner 118. The patient sample is further partially diluted using a sample diluent. At this point, the sample pipette 120 can aspirate a portion of the patient sample and inject the patient sample into the cuvette of the reaction spinner 106 to resuspend the fluorescent beads. The cuvette containing the patient sample in the reaction spinner 106 can then be incubated at a specific temperature for a specific period of time. After incubation, the single-rinse pipette 116 can inject a rinse buffer to resuspend the fluorescent beads. The reaction spinner 106 then performs another concentration process by allowing the fluorescent beads to essentially collect in the cuvette near the magnet in the reaction spinner 106. After the fluorescent beads are concentrated, the multi-rinse pipette 112 can aspirate and discard a portion of the fluid that did not collect in the cuvette of the reaction spinner 106 during the concentration process.

The sample in the cuvette of reaction rotator 106 is then subjected to several rinse cycles. These rinse cycles may include using a multi-rinse pipette 112 to inject wash buffer into the cuvette to resuspend the fluorescent beads. Another concentration step may allow the fluorescent beads to be collected in the cuvette of reaction rotator 106 using a magnet. After a period of time to allow the fluorescent beads to fully concentrate, the multi-rinse pipette 112 may aspirate and inadvertently discard a portion of the sample, leaving a portion of the fluorescent beads in the cuvette of reaction rotator 106. Another rinse cycle may then be performed using the multi-rinse pipette 112 to re-inject wash buffer into the cuvette and allow the fluorescent beads to resuspend. Another fluorescent bead concentration step may utilize a magnet in reaction rotator 106 to concentrate the fluorescent beads from the remaining sample. Finally, the multi-rinse pipette 112 may aspirate a portion of the sample that was not concentrated by the concentration step.

At this point, the R2 pipette 122 can aspirate the conjugate contained in the cuvette in the reagent rotor 114. The R2 pipette 122 then injects the previously aspirated conjugate into the cuvette in the reaction rotor 106. After the cuvette has been incubated in the reaction rotor 106 for a controlled time and temperature, the single-rinse pipette 116 can inject rinse buffer into the cuvette in the reaction rotor 106. Another fluorescent bead concentration cycle can be performed by allowing the magnet in the reaction rotor 106 to substantially concentrate the fluorescent beads in the cuvette. The multi-rinse pipette 112 can aspirate and discard the portion of the sample in the cuvette that was not concentrated during the concentration cycle.

Two additional rinse cycles can be performed on the sample in the cuvette of reaction rotator 106. The multi-rinse pipette 112 can inject wash buffer to resuspend the fluorescent beads in the cuvette. Another fluorescent bead concentration cycle can be performed by holding the cuvette of reaction rotator 106 close to a magnet for a sufficient period of time to concentrate the fluorescent beads. After the concentration cycle, the multi-rinse pipette 112 can aspirate and discard the portion of the sample that was not concentrated during the concentration cycle. A second wash cycle can then be performed using the multi-rinse pipette 112 to inject wash buffer to resuspend the fluorescent beads. Another concentration cycle can utilize the magnet in reaction rotator 106 to concentrate the fluorescent beads in the cuvette. After the concentration process, the multi-rinse pipette 112 can again aspirate and discard the portion of the sample that was not concentrated during the concentration cycle.

At this moment, R2 pipette 122 can aspirate a part of conjugate from reagent rotator 114 and inject conjugate into the substrate container 124 of mixing, produce mixed substrate sample.Then, R2 pipette can aspirate the substrate sample of mixing from the substrate container 124 of mixing and inject the substrate sample of mixing into the test cup of reaction rotator 106, utilize the substrate sample of mixing to resuspend fluorescent beads.Can utilize optical pipette 108 to aspirate the sample in the test cup of reaction rotator 106 subsequently and put in optical box 110.After optical box observes fluorescence and luminescence, discard sample and rinse pipette repeatedly and rinse the test cup of reactor rotator 106, be ready for next test.

The following are some non-limiting embodiments to further illustrate the present invention.

Embodiment 1: A quantitative method for performing an automated diagnostic assay, comprising:

Incubating a streptavidin-coated medium containing a universal fluorescently labeled magnetic particle with a biotinylated capture reagent derived from an allergen or autoimmune antigen to form a solid phase complex;

washing the solid phase complex to remove excess capture reagent;

incubating the solid phase complex with a serum sample to form an immune complex;

washing the immune complexes to remove any unbound sample;

incubating the immune complex with a conjugate to generate an immuno-conjugate complex;

washing the immuno-conjugate complex to remove any unbound conjugate;

Introducing a substrate that produces a quantifiable response; and

The reaction resulting from the introduction of substrate is calibrated.

Embodiment 2: The method according to embodiment 1, wherein the step of incubating the solid phase complex with the serum sample comprises binding allergen-specific human immunoglobulin E (IgE) present in the serum sample to the biotinylated capture reagent.

Embodiment 3: A method according to embodiment 1, wherein the step of incubating the solid phase complex with the serum sample includes binding autoimmune-specific human immunoglobulin G (IgG), autoimmune-specific human immunoglobulin M (IgM) or autoimmune-specific human immunoglobulin A (IgA) present in the serum sample to the biotinylated capture reagent.

Embodiment 4: The method according to embodiment 1, wherein the biotinylated capture reagent is derived from the biotinylation of a purified allergen, protein, enzyme or antibody.

Embodiment 5: The method according to embodiment 1, wherein the allergen extract is composed of a plurality of allergens.

Embodiment 6: The method according to embodiment 1, wherein the biotinylated capture reagent is present as a mixture of multiple biotinylated capture reagents selected from the group consisting of purified allergens, proteins, enzymes, antibodies and allergen extracts.

Embodiment 7: The method of embodiment 1, wherein the one or more washing steps comprise washing the complex by magnetically isolating the washed complex within a defined area of the reaction cuvette.

Embodiment 8: The method according to embodiment 1, wherein the step of forming a solid phase complex using a streptavidin-coated medium comprises incubating the suspended biotinylated capture reagent with a reaction diluent containing a high concentration of human serum albumin (HSA).

Embodiment 9: The method according to embodiment 1, wherein the step of incubating the immune complex with the conjugate comprises incubating the suspended immune complex with a conjugate diluent containing polyethylene glycol at an indicated concentration.

Embodiment 10: The method according to embodiment 9, further comprising the step of using horseradish peroxidase (HRP) as an indirect label when washing the immune complex to remove unbound sample.

Embodiment 11: The method of embodiment 1, further comprising the step of adjusting the quantifiable response to bead retention by:

transferring the substrate and immuno-conjugate complex to an optical box, wherein the fluorescent and chemiluminescent signals are quantified; and

The ratio of initial to final fluorescence was used to adjust the quantified chemiluminescent signal to calculate the reporter value.

Embodiment 12: The method of embodiment 11, wherein the step of transferring the substrate and immuno-conjugate complex to an optical cartridge comprises utilizing an automated pipette arm with a reusable pipette tip to aspirate the sample.

Embodiment 13: The method according to embodiment 11, further comprising:

measuring fluorescence within the optical cartridge to determine bead retention; and

Luminescence within the optical cartridge is measured to detect a generated relative light unit signal.

Embodiment 14: The method of Embodiment 13, further comprising inputting the fluorescence and luminescence measurements into an algorithm to generate a bead retention adjusted relative light unit signal.

Embodiment 15: The method of Embodiment 14, further comprising comparing the generated bead retention adjusted relative light unit signal to a calibration curve relative light unit signal.

The advantages and improvements of the processes and methods of the present disclosure are illustrated in the following examples. These examples are merely illustrative and are not intended to limit or exclude other embodiments of the present disclosure.

Example 1: Biotinylation of anti-human IgE or allergen extracts:

Add 2 μL of 250 mM NHS-PEG12-biotin (Pierce) in DMSO to 1 mL of affinity-purified anti-human IgE (ImmunoReagents) at 5.0 mg/mL in phosphate-buffered saline (PBS). Alternatively, add 1.6 μL of 250 mM NHS-PEG12-biotin (Pierce) in DMSO to 1 mL of allergen extract at 1.0 mg/mL in phosphate-buffered saline (PBS).

The reagent solutions were mixed and placed on ice for 2 hours. Free biotin reagent was separated from the biotinylated antibody by dialysis against two changes of PBS (antibody to buffer ratio 1:100 by volume) for 4 hours and overnight at 2-8°C.

Example 2: Preparation of fluorescent beads:

5 μL of 1 mM biotin-fluorescent solution (Alexa Fluor 594 biocytin, sodium salt, Life Technologies) in ddH ₂ O was added to 45 mL of PBSTHP buffer (10 mM sodium phosphate, pH 7.4, 0.9% (w/v) NaCl, 0.05% (v/v) Tween-20, 10 mg/mL HSA, 1% (v/v) ProClin 950). Mix thoroughly.

5 mL of SA-Speed Beads (Sera-mag Speedbeads streptavidin-coated magnetic particles, Thermo) 10 mg/mL was added to the biotin-fluorescent solution and mixed thoroughly.

Example 3: Determination of specific IgE levels against allergens

10 μL of fluorescent beads (fluorescently labeled paramagnetic microparticles) were dispensed into a reaction cuvette at a bead concentration of 1 mg/mL; 40 μL of biotin-allergen (e.g., egg white, milk, peanut, etc.) or biotin-anti-IgE antibody was dispensed and mixed into the fluorescent beads and incubated at 37°C for 1-10 minutes. After washing, the allergen- or anti-IgE-coated beads were resuspended in 40 μL of reaction buffer. Serum samples obtained from atopic and non-atopic individuals were assayed for allergens. 10 μL of sample was added to 40 μL of suspended allergen-coated beads in a reaction cuvette. For a six-point standard curve, 10 μL of serum standard (secondary standard calibrated according to WHO IgE standard 75/502) was each added to 40 μL of anti-IgE-coated beads in a reaction cuvette. Although various labeled anti-IgE conjugates can be used according to the present teachings, according to certain teachings, the following anti-IgE conjugates are used: for allergy assays, anti-IgE-HRP is used; for autoimmunity assays, anti-IgA-HRP, anti-IgG-HRP, and anti-IgM-HRP are used; for ECP, anti-ECP-HRP is used; and for tryptase, anti-tryptase-HRP is used. Moreover, as used herein, each conjugate has an optimized HRP incorporation ratio for chemical action. According to certain aspects of the present teachings, the HRP incorporation ratio for the listed conjugates ranges from about 1.2 to about 5.4. In addition, the present teachings also contemplate incorporation of other types of conjugate-reporter systems including, but not limited to, alkaline phosphatase conjugates and b-galactose conjugates.

The solution was mixed and incubated at 37°C for 40 minutes. After washing, the beads were resuspended in 50 μL of anti-human IgE-HRP conjugate and incubated at 37°C for 30 minutes. 50 μL of PS-atto (Lumigen) was added to each cuvette and the beads were resuspended. The bead suspension was transferred to a pipette tip and the fluorescence and luminescence signals were read in an optical box. The standard curve was determined using a four-parameter logistic function equation and allergen-specific IgE levels interpolated from the standard curve.

An illustrative list of reagents and components that can be used according to the present teachings includes, but is not necessarily limited to: Beads: fluorescent beads (SA-Speed Bead, Atto 590 labeled), 1 mg/mL; Capture Reagent Diluent: IgE: 10 mM sodium phosphate, pH 7.4, 0.9% (w/v) NaCl, 0.05% Tween-20, 1% (w/v) human serum albumin, 1% (v/v) ProClin 950, up to 5% (v/v) glycerol; ANA: 10 mM sodium phosphate, pH 7.4, 0.9% (w/v) NaCl, 0.05% Tween-20, 1% (w/v) bovine serum albumin, 1% protease inhibitor cocktail, 0.1 mM DTT, 1% (v/v) ProClin 950, 25% (up to 30%) (v/v) glycerol; Wash Buffer Concentrate (5x): 50 mM sodium phosphate, pH 7.4, 4.5% (w/v) NaCl, 0.05% Tween-20, 0.05% (v/v) ProClin 950, 0.02% (v/v) Antifoam-C v/v; Reagent Diluents (Reaction Diluent and Sample Diluent): IgE - 10 mM sodium phosphate, pH 7.4, 500 mM NaCl, 0.02% Tween-20, 1% (w/v) human serum albumin, 1% (v/v) human IgG, 1% (v/v) ProClin 950, 0.005% Antifoam-B v/v, 2% (w/v) PEG 6,000; ANA - 10 mM sodium phosphate, pH 7.4, 500 mM NaCl, 0.02% Tween-20, 25% (w/v) human serum albumin, 1% (v/v) ProClin 950; Calibrators and Controls : Calibrators: patient samples diluted in sample diluent; Controls: patient sample pool; Conjugate: Conjugate Diluent: 50 mM sodium phosphate, pH 6.7, 150 mM NaCl, 0.05% Tween-20, 1% BSA, 5% (w/v) PEG 6,000, 1% (v/v) ProClin 950; IgE: 100 ng/mL anti-IgE-HRP, 100 μg/mL apo-HRP (in diluent), 0.015% antifoam-B v/v; and Substrate: PS-atto A & B, 0.01% antifoam-B v/v.

Although exemplary embodiments incorporating the principles of this application are disclosed herein, this application is not limited to the disclosed embodiments. Rather, this application is intended to cover any variations, uses, or modifications of this application that utilize its general principles. Furthermore, this application is intended to cover such departures from the present disclosure as come within known or customary means in the art to which this application relates and fall within the limits of the appended claims.

The terms used herein are intended to describe specific illustrative embodiments only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "said" are intended to include the plural forms unless the context indicates otherwise. The terms "comprise," "include," "contain," and "have" are inclusive and therefore indicate the presence of the described features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The method steps, processes, and operations described herein should not be construed as necessarily requiring implementation in the particular order discussed or described, unless an order of implementation is specifically indicated. It should also be understood that additional or alternative steps may be used.

Claims (12)

1. A quantitative method for performing automated immunoassay, comprising: A streptavidin-coated medium is incubated with a biotinylated capture reagent to form a solid-phase complex, wherein the streptavidin-coated medium contains a universally fluorescently labeled magnetic microparticle, and the biotinylated capture reagent is derived from an allergen or an autoantigen. The solid-phase complex was washed to remove excess capture reagent; The solid-phase complex was incubated with a serum sample to form an immune complex. Wash the immune complex to remove any unbound sample; The immune complex was incubated with the conjugate to generate an immune-conjugated complex. Wash the immune-conjugated complex to remove any unbound conjugates; Introducing substrates capable of producing quantifiable reactions; and The reaction produced by calibrating the substrate; and This further includes steps for regulating the quantifiable reaction for bead retention in the following ways: The substrate and the immuno-conjugated complex are transferred into an optical cell, where fluorescence and chemiluminescence signals are quantified; and The ratio of initial to final fluorescence was used to adjust the quantified chemiluminescence signal to calculate the reported value. 2. The method of claim 1, wherein the step of incubating the solid-phase complex with the serum sample comprises binding allergen-specific human immunoglobulin E (IgE) present in the serum sample to the biotinylated capture reagent. 3. The method of claim 1, wherein the step of incubating the solid-phase complex with the serum sample comprises binding an autoimmune-specific human immunoglobulin G (IgG), an autoimmune-specific human immunoglobulin M (IgM), or an autoimmune-specific human immunoglobulin A (IgA) present in the serum sample to the biotinylated capture reagent. 4. The method according to claim 1, wherein the biotinylated capture reagent is derived from the biotinylation of purified allergens. 5. The method of claim 1, wherein one or more washing steps comprise washing the complex by magnetically isolating the complex to be washed within a defined area of the reaction vessel. 6. The method of claim 1, wherein the step of forming a solid-phase complex using a streptavidin-coated medium comprises incubating a suspension-held biotinylated capture agent with a reaction dilution containing a high concentration of human serum albumin (HSA). 7. The method of claim 1, wherein the step of incubating the immune complex with the conjugate comprises incubating the suspended immune complex with a conjugate diluent containing a labeled concentration of polyethylene glycol. 8. The method of claim 7, further comprising the step of using horseradish peroxidase (HRP) as an indirect label when washing the immune complex to remove unbound samples. 9. The method of claim 1, wherein the step of transferring the substrate and the immune-conjugated complex to the optical cartridge comprises using an automated pipette arm having a reusable pipette tip to aspirate the sample. 10. The method of claim 1, further comprising: Measuring fluorescence within the optical cell to determine bead retention; and The emission within the optical box is measured to detect the generated relative light unit signal. 11. The method of claim 10, further comprising substituting fluorescence and luminescence measurements into the algorithm to generate a relative light unit signal that is bead-controlled. 12. The method of claim 11, further comprising comparing the generated relative optical unit signal maintained by the bead with the relative optical unit signal of the calibration curve.