IMMUNOASSAY FOR AN AUTOMATED SYSTEM

Abstract

Disclosed are methods for detecting a target molecule in a sample, including the sequential steps of: (a) incubating at the same time in a buffer solution in a reaction well (i) the sample suspected to contain one or more target molecules, (ii) solid phase-coupled capture molecules suitable for binding to the target molecules, wherein the capture molecules are coupled to beads (capture-beads), (iii) a defined amount of detection molecules suitable for binding to the target molecules and/or the capture molecules, wherein the detection molecules are coupled to beads (detection-beads), wherein the capture-beads are separable from the detection-beads, (b) separating in the reaction well the capture-beads from the buffer solution including unbound detection-beads, and (c) detecting the remaining unbound detection-beads in the buffer solution, which has been separated from the capture-beads, wherein the method does not include a washing step.

Claims

1. A method for detecting a target molecule in a sample, comprising the sequential steps of: a. incubating at the same time in a buffer solution in a reaction well: i. the sample suspected to contain one or more target molecules, ii. capture molecules suitable for binding to the target molecules, wherein the capture molecules are coupled to beads (capture-beads), and iii. a defined amount of detection molecules suitable for binding to the target molecules and/or the capture molecules, wherein the detection molecules are coupled to beads (detection-beads), wherein the capture-beads are separable from the detection-beads, b. separating in the reaction well the capture-beads from the buffer solution comprising unbound detection-beads, and c. detecting remaining unbound detection-beads in the buffer solution, which has been separated from the capture-beads, wherein the detection of remaining unbound detection-beads occurs in the same reaction well as the incubation and separation by means of a label that does not require additional reagents for signal generation, and wherein the method does not comprise a washing step.

2. The method of claim 1, wherein the label that does not require additional reagents for signal generation is a fluorescent tag.

3. The method of claim 1, wherein the method does not require more than one buffer solution.

4. The method of claim 1, wherein the method is performed in an automated system, in some embodiments a microfluidic system, in other embodiments a centrifugal microfluidic system.

5. The method of claim 1, wherein the capture-beads and/or the detection-beads are single compact beads.

6. The method of claim 1, wherein the capture-beads have a higher density than the detection-beads and/or a diameter of at least 0.5 m.

7. The method of claim 1, wherein separating the capture-beads from the buffer solution occurs through magnetic, gravitational, centrifugal or electrophoretic forces.

8. The method of claim 1, wherein a. the capture molecule recognizes a first epitope comprised by the target molecule, and b. the detection molecule recognizes a second epitope comprised by the target molecule (sandwich assay) or c. the detection molecule comprises the first epitope recognized by the capture molecule (competitive assay).

9. The method of claim 1, wherein the capture molecule comprises an antibody or fragments thereof or an antigen, and/or the detection molecule comprises an antibody or fragments thereof or an antigen.

10. The method of claim 1, wherein the detection molecule comprises a label.

11. The method of claim 1, wherein the incubation lasts for no more than 20 minutes and/or separation occurs in no more than 1 minute.

12. The method of claim 1, wherein the assay provides quantitative results for detection of a target molecule.

13. The method of claim 1, wherein two or more different target molecules comprised in the sample are detected in parallel, wherein: a. one or more target molecules are detected by a sandwich assay and one or more other target molecules are detected by a competitive assay, b. all target molecules are detected by a sandwich assay, or c. all target molecules are detected by a competitive assay, and d. one or more of the target molecules are present in a high concentration and one or more other target molecules are present in a low concentration, e. all target molecules are present in a high concentration, or f. all target molecules are present in a low concentration.

14. A kit for detecting a target molecule in a sample, wherein the kit comprises a. capture-beads suitable for binding to the target molecules, b. detection-beads suitable for binding to the target molecules and/or the capture-beads, wherein capture-beads and the detection-beads are single compact beads, the capture-beads are separable from the detection-beads, the capture-beads have a higher density than or equal density to the detection-beads and the capture-beads have a diameter of at least 0.5 m, c. a microfluidic system configured for performing in one reaction chamber the method steps of: i. incubating at the same time in a buffer solution the sample suspected to contain one or more target molecules, the capture-beads and a defined amount of detection beads, ii. separating in the reaction well the capture-beads from the buffer solution comprising unbound detection-beads, and iii. detecting remaining unbound detection-beads in the buffer solution by means of a label that does not require additional reagents for signal generation.

15. The kit of claim 14, further comprising a buffer solution and/or means for detecting the detection-beads in a buffer solution after incubating in the buffer solution the capture-beads, the sample suspected to contain one or more target molecules and a defined amount of the detection-beads, and separating in the reaction well the capture-beads from the buffer solution comprising unbound detection-beads

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0238] This disclosure is further aided by the following figures. These are not intended to limit the scope of the disclosure, but represent preferred embodiments of aspects of the disclosure provided for greater illustration of the methods described herein.

[0239] FIG. 1: Illustration of an immunoassay workflow according to the presently described methods (as example a competitive immunoassay is shown);

[0240] FIG. 2: Process chain with immunoassay steps of an example of the presently described methods;

[0241] FIG. 3: Intensity of remaining fluorescent beads in supernatant (in sandwich format);

[0242] FIG. 4: Intensity of remaining fluorescent beads in supernatant (standard curve in competitive format);

[0243] FIG. 5: Concentration CV range of bound-free phase assay; and,

[0244] FIG. 6: Measurement result of a certified reference material (CRM) here human serum with a certified value of 41.2 mg/l and an uncertainty range of 2.5 mg/l with the immunoassay method presented in this disclosure.

DETAILED DESCRIPTION OF THE FIGURES

[0245] FIG. 1: (1) Magnetic beads (MB) are added to the fluorescent beads (FB) and the sample. (2) The antigens on the FB and in the sample react with the antibodies on the surface of the MB. (3) After the antigen-antibody complex is formed the phases are separated and bound-free phase (supernatant) with the surplus FB is detected. The number of surplus FB and, consequently, the detected signal are dependent on the amount of antigen in the sample due to the competition over the binding sites on the antibodies.

[0246] FIG. 2: This example of an immunoassay contains seven steps. The preparation of the fluorescent and magnetic beads are not included in this schematic as they are not part of the immunoassay workflow itself.

[0247] FIG. 3: Example of application of the described methods in sandwich CRP immunoassay (low concentration ranges). For this experiment fluorescent beads with a diameter of 0.5 m (Merck former Sigma Aldrich, Germany) were used. The protocol principle for the sandwich assay (not described in this document) is the same as described for the competitive assay but two antibodies (R&D systems a biotechne brand, USA) are used to be able to form a sandwich. The capture antibody is immobilized on the magnetic beads (Thermo Fisher, USA). The detection antibody is immobilized on the fluorescent bead. This assay is a one-step sandwich assay.

[0248] FIG. 4: Example of application of the described methods in competitive CRP immunoassay (high concentration ranges). This figure shows a standard curve of the CRP assay conducted with the protocol described in the section EXAMPLES. The standard curve was created with a repetition of three assays for each value.

[0249] FIG. 5: An overview of the concentration CV in % over the CRP concentration range between 30 mg/l and 100 mg/l. The concentration values were calculated from the measured intensity by creating a standard curve with 5 repeated measurements for human serum samples with spiked CRP with the values 20 mg/l, 40 mg/l, 60 mg/l, 80 mg/l and 100 mg/l. The measurement data with their mean values and standard deviations were fitted with a sigmoidal function. This function was used to calculate the measured concentration of 16 repetitions for human serum samples with spiked CRP with the values 40 mg/l, 60 mg/l, 80 mg/l and 100 mg/l.

[0250] FIG. 6: A certified reference material (CRM) (ERM-DA474/IFCC, Joint Research Centre (JRC) European Commission) here human serum with a certified value of 41.2 mg/l and an uncertainty range of 2.5 mg/l of CRP was tested with the CRP assay conducted with the protocol described in the section EXAMPLES. The assay was conducted on 5 different days, each with 5 repetitions. To calculate the concentration from the measured intensity a standard curve created with the same material batch (magnetic beads, fluorescent beads, assay buffer) was used, which was created with the same method as described in the description of FIG. 5, but here with 3 repeated measurements for each value. FIG. 6 shows the mean value of the 5 repeated measurements of the certified reference material with their standard deviation for each day. The line at the value 41.2 mg/l represents the certified value of the sample and the dashed lines at 38.7 mg/l and 43.7 mg/l show the range covered by the certified material considering its uncertainty.

Examples

[0251] The following examples further illustrate the disclosed methods. These are not intended to limit the scope of the disclosure, but represent preferred embodiments of aspects of the disclosure provided for greater illustration of the methods described herein.

[0252] The examples of the described methods relate to a method for an immunoassay, which provides information about an antigen (or antibody) that is assumed to be present in a liquid sample, by detecting it in the bound-free phase (supernatant) of the immunoassay. With this approach it is possible to avoid the washing steps that are needed in other immunoassay approaches known in the art. Combined with a bead-based assay approach this immunoassay requires only a short incubation time and provides the possibility of measuring high as well as low concentration ranges. Furthermore, fewer handling steps compared to other immunoassay approaches are required. Accordingly, the described method is ideal to be performed in an automated or semi-automated system, and/or an automated or semi-automated microfluidic system. The total number of steps for performing the described methods depends for example on the platform that is used. The preparation steps described in this example are for a competitive bead-based assay approach where a microtiter plate well is used only as a reaction chamber with no immobilized antibodies or other reagents on the microtiter place surface.

[0253] The example of the methods provided herein is a one-step, competitive and bead-based immunoassay for the detection of CRP in human serum. The concept is also applicable to different types of immunoassays for example one-step or two-step sandwich assays with different detection methods, such as, but not limited to, fluorescence, chemiluminescence, absorption or colorimetry.

[0254] In this example, the reaction volume consists of a sample containing the analyte antigen, a competitive antigen conjugated with a label entity, the assay buffer (all aforementioned forming the liquid phase), magnetic beads conjugated with antibodies (forming the solid phase). For this assay, the magnetic beads (MB) M280 Dynabeads Tosylactivated (Thermo Fisher Scientific, USA) were used as the solid phase, where the complex is formed. This assay uses polyclonal antibodies, but monoclonal antibodies were also tested and worked as well. The competitive antigen is coupled onto the fluorescent beads (FB) acting as the label entity. For this assay, FluoSpheres Carboxylate-Modified Microspheres with a diameter of 0.2 m and excitation/emission wavelengths of 580 nm/605 nm (Thermo Fisher Scientific, USA) were used and coupled with native CRP antigen (United State Biological, USA). Fluorescent beads from another supplier with different diameters were tested and worked as well.

[0255] These coupled FluoSphere beads are added to the assay buffer together with the sample. In the next step the magnetic beads with the antibodies on their surface are added to the buffer-FB-sample mixture (FIG. 1 (1)). If the to be detected antigen is present in the sample, it will compete with the competitive antigen that is coupled on the surface of the FB and an antigen-antibody complex with either the sample antigen or the FB-coupled detection antigen is formed (FIG. 1 (2)) during the incubation at 37 C. for 15 minutes. The amount of surplus FB-coupled detection antigen (bound-free phase) that has not bound to the MB-capture antibodies is dependent on the amount of antigen that is present in the sample. Thus, with separating the bound-free phase (in liquid phase) from the solid phase (complex that reacted onto the surface of the magnetic particle), the fluorescent signal of the surplus FB-coupled detection antigen can be detected and the concentration of the CRP in the sample can be determined (FIG. 1 (3)). With that the assay also enables color multiplexing. There is no washing step needed, and the separation can be done by, but is not limited to, magnetic forces, centrifugal forces, electrophoretic forces, or biochemical adsorption.

[0256] FIG. 2 summarizes all steps required for the example of the presently described methods. The framework for that can be a bead-based approach like the one presented above. The assay presented herein also allows non-bead-based approaches where the solid phase can be the bottom of a microtiter plate or other suitable surfaces. The signal to be detected is also not limited to derive from fluorescence beads. Fluorescent dyes, or quantum dots, or approaches like chemiluminescence, enzymatic reactions work as well, although some approaches would increase the number of steps (in the detection phase, not in the incubation phase where the described method retains its advantages independently from the detection method) due to additional reagents that have to be added for the signal generation.

[0257] The presently described methods were created for an easy automation, for example but not limited to, a centrifugal microfluidic platform. The assay as described in the present non-limiting example combines short incubation time, only seven steps in total, one liquid reagent that has to be stored (no washing steps and consequently no washing buffer(s) to be stored), small volumes and no additional material like membranes for the detection.

[0258] The presently described methods are described in an example of a CRP-immunoassay.

[0259] There may be four main parameters that can influence the performance of the immunoassay after the reagents like antibodies, antigen and buffers are fixed and optimized: (i) the incubation time, (ii) the sample volume, (iii) the ratio of the amount of magnetic beads to the amount of fluorescent beads, and (iv) the incubation temperature. These parameters can be used to adapt the detection range, linear range and slope of the standard curve and, consequently, the sensitivity. With the incubation temperature it is also possible to adjust the duration of the incubation and also the slope of the linear range.

Materials and Methods of the Examples

Microtiter Plate

[0260] A non-binding black microtiter plate (Greiner Bio-One GmbH, Austria) is blocked with a blocking buffer (PBS/BSA 5%) to prevent non-specific adsorption of the reagents on the microtiter plate surface. The steps are: [0261] Fill well completely with PBS/BSA 5% [0262] Block for 30 min at room temperature (RT) or over night at 8 C. [0263] 3 wash with the wash buffer (PBS) by filling the wells completely, wait for 2 min and carefully shake the plate while waiting [0264] Remove washing buffer and dry the plate carefully on a laboratory paper towel by tapping the plate on it.

Magnetic Beads

[0265] The magnetic beads M280 tosylactivated Dynabeads (14203, Thermo Fisher Scientific, USA) are used as the solid phase. CRP antibodies (A80-125A, Bethyl Laboratories Inc. USA) are coupled on their surface. The protocol is given by the supplier (Thermo Fisher Scientific, USA) [28].

Fluorescence Beads

[0266] The fluorescence beads (F8812, Thermo Fisher Scientific, USA) are used as label of the detection molecule (the competitive antigen). The coupling protocol to immobilize highly purified native CRP antigen (C7907-26, United State Biological, USA) onto the surface of the FluoSpheres is provided by the supplier [29].

Results of the Examples

[0267] For the proof-of-concept, one-step bead-based assays are presented. These assays are able to cover small detection ranges (3 ng/ml to 2000 ng/ml, in sandwich format, FIG. 3) as well as high detection ranges (28.6 mg/l to 140 mg/l in competitive format, FIG. 4) and have fast incubation duration of 15 min or less. Importantly, the exclusion of any washing does not have any negative influence on the reproducibility, as we could show an inter assay coefficient of variation (CV) with 4.3% and an inter assay CV with 4.1% for the example of the competitive CRP assay measured with a certified reference material (ERM-DA474/IFCC, European commission, Reference Materials Unit). It is important to optimize the volumes of the reagents used in the assay protocol. The protocol that is described below is the optimized protocol for a CRP immunoassay with human serum as sample material and a measuring range of 28.6 mg/l to 140 mg/l in a non-binding black microtiter plate (Greiner Bio-One GmbH, Austria).

Protocol for a Competitive Assay with Antigen (Detection Molecule) Coupled on the FluoSpheres (Label) and Antibody (Capture Molecule) Coupled on the Dynabeads (Solid Phase)

Immunoassay (Total Volume 75 l)

[0268] Bring the reagents to room temperature [0269] Add 50 l assay buffer [0270] Add 10 l FluoSphere beads (2 mg/ml) [0271] Add 5 l sample (native human CRP-free serum) [0272] Add 11.5 l Dynabeads (20 mg/ml stock concentration) [0273] Incubate for 15 min at 37 C. on a shaker [0274] Separate the magnetic beads with a magnet from the bound-free phase (supernatant) [0275] Take 50 l of the supernatant [0276] Measure the fluorescence

[0277] This assay shows an inter-assay CV of 4.1% (considered as very good results in the microtiter plate) and an intra-assay CV of 4.35% measured over 5 different days in a non-automated system with the CRM. FIG. 4 shows a standard curve for the CRP. Each measured concentration was repeated three times. The sample material was native human CRP-free serum (HyTest Ltd, Finland) spiked with a known concentration of native human CRP. The read out was done on a microtiter plate reader (Tecan Spark 10M, Tecan Trading GmbH, Switzerland). FIG. 5 shows the calculated concentration CV for 40 mg/l, 60 mg/l, 80 mg/l and 100 mg/l. All mean CV values are lower than 10% with the highest value for 80 mg/l being 5.6% and the lowest value with 3.9% for the 60 mg/l. These values were calculated as described in Detailed description of the figures. This proves that the assay can be considered quantitive between 40 mg/l and 100 mg/l and therefore the absence of washing step does not influence the quality of the assay. FIG. 6 shows the measurement results of the CRM (ERM-DA474/IFCC, Joint Research Centre (JRC) European Commission) with a certified value of 41.2 mg/l and an uncertainty range of 2.5 mg/l with the method described in Detailed description of the figures. The line at the value 41.2 mg/l represents the certified value of the sample and the dashed lines at 38.7 mg/l and 43.7 mg/l show the range covered by the certified material considering its uncertainty. All mean values measured on each day are within the range of the CRM which proves the functionality of the method. It also proves that the method is not in any way influenced by different samples. In conclusion, the assay concept was evaluated and is proven to work. The CRP competitive assay is quantitative with CVs clearly under 10% and was shown to be used with undiluted sample material.

REFERENCES

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[0307] It is understood that the foregoing description is that of the preferred embodiments of the invention and that various changes and modifications may be made thereto without departing from the spirit and scope of the invention as defined in the appended claims.