WO2016154250A1

WO2016154250A1 - Heterophilic blocking agents for immunoassays

Info

Publication number: WO2016154250A1
Application number: PCT/US2016/023682
Authority: WO
Inventors: Sean LUNDQUIST; Tim JENTZ
Original assignee: Surmodics Ivd, Inc.
Priority date: 2015-03-23
Filing date: 2016-03-23
Publication date: 2016-09-29

Abstract

The disclosure is directed to compositions, kits, and methods using a blocking agent that is Protein A, Protein A/G, Protein G, Protein L, or active portions thereof. The blocking agent reduces analyte-independent antibody interactions in an immunoassay, such as an ELISA, and therefore minimizes false positive results.

Description

HETEROPHILS BLOCKING AGENTS FOR IMMUNOASSAYS Cross-Reference to Related Application

The present non-provisional Application claims the benefit of commonly owned provisional Application having serial number 62/136,906, filed on March 23, 2015, entitled HETEROPHILIC BLOCKING AGENTS FOR IMMUNOASSAYS, which Application is incorporated herein by reference in its entirety.

Field

The disclosure is directed to compositions and methods for in vitro immunodiagnostic tests and blocking agents for such tests that reduce false positive results.

Background

Research and diagnostic procedures benefit from rapid, accurate, and qualitative and/or quantitative determinations of substances ("analytes") that are present in biological samples, such as biological tissues or fluids, at low

concentrations. For example, the presence of drugs, narcotics, hormones, steroids, polypeptides, prostaglandins or infectious organisms in blood, urine, saliva, dental plaque, gingival crevicular fluid, or other biological specimens is desirably determined in an accurate and rapid fashion for suitable diagnosis or treatment.

In many cases, an analyte is identified in a sample using a compound that specifically recognizes the chemical features of the analyte. Often, monoclonal antibodies specific for one or more chemical epitopes on an analyte are immobilized on a surface and used to capture the analyte. After the analyte is captured, another antibody can be used to detect the captured analyte. The complex formed between the antibody and analyte can be detected by a variety of known methods.

Commonly used methods employ a signal -generating moiety of some type which is either already attached to the antibody, or becomes attached to the antibody through further reaction. The signal -generating moiety can be used to provide a measurable signal in the immunoassay.

Some problems associated with immunoassays relate to the generation of false positive results. A false positive result occurs when a positive signal is generated in absence of an analyte, wherein the analyte otherwise serves as the chemical moiety that provides the basis for a true positive result. Body fluids have a plethora of compounds capable of reacting with exogenous compounds (such as those used in immunoassays) in unforeseen ways, and the possibility of generating false positive results from such undesired interactions is high.

Summary

Generally, the disclosure is directed to compositions, kits, and methods using a blocking agent that reduces analyte-independent antibody interactions in an immunoassay, such as an ELISA. In particular, a blocking agent selected from the group consisting of Protein A, Protein A/G, Protein G, and Protein L, and active portions thereof, is used to reduce or eliminate an analyte-independent association of an immobilized analyte capture antibody with an analyte detection antibody, which otherwise provides a false positive result. It is thought that the blocking agent is able to prevent interaction of serum antibodies that are cross-reactive with the analyte capture antibody and/or the analyte detection antibody.

In one embodiment, the invention provides a method for reducing non- specific antibody interaction in an immunoassay. The method includes steps of: (a) providing a composition comprising a blocking agent selected from the group consisting of Protein A, Protein A/G, Protein G, and Protein L, and active portions thereof; (b) contacting an immobilized analyte capture antibody, an analyte detection antibody, a biological sample, or any mixture thereof, with the

composition; and (c) performing an immunoassay on the biological sample using at least the antibody and the analyte detection antibody.

In another embodiment, the invention provides a kit for performing an immunoassay. The kit includes (a) a blocking agent selected from the group consisting of Protein A, Protein A/G, Protein G, and Protein L, and active portions thereof and (b) a diluent for the blocking agent, wherein the blocking agent is in dried form separate from the diluent, or is combined with the diluent, wherein the blocking agent is configured for use to reduce analyte-independent antibody interaction in an immunoassay.

In yet another embodiment, the invention provides a non-natural composition comprising (a) a biological sample from a mammalian species or a detection antibody for an immunoassay, and (b) a blocking agent selected from the group consisting of Protein A, Protein A/G, Protein G, and Protein L, and active portions thereof, wherein the blocking agent is present in the composition at a concentration effective to reduce non-specific antibody interaction when the composition is used in an immunoassay.

Detailed Description

Embodiments of the present disclosure described herein are not intended to be exhaustive or to limiting to the precise forms disclosed in the following detailed description. Rather, the embodiments are chosen and described so that others skilled in the art can appreciate and understand the principles and practices of the present disclosure.

All publications and patents mentioned herein are hereby incorporated by reference. The publications and patents disclosed herein are provided solely for their disclosure. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate any publication and/or patent, including any publication and/or patent cited herein.

Kits of the disclosure include those having one or more reagent for use in an immunoassay. In some aspects, an ELISA (enzyme-linked immunosorbent assay) is used for detection of an analyte, and the ELISA kit or method includes a blocking agent of the disclosure.

The kit may also include instructions for performing a blocking step in an immunoassay method, as well as instructions for one or more other steps in an immunoassay method. For example, a kit can include the blocking agent, which can be Protein A, Protein A/G, Protein G, and Protein L, active portions thereof, or mixtures thereof, in solution, or in dried (e.g., lyophilized) form. If in dried form, the blocking agent can be diluted to a desired stock concentration, such as a 2X working concentration or greater (e.g., from 2X to 5 OX), with a diluent supplied in the kit or provided by a user.

Protein A (UniProt P02976) is a bacterial cell surface protein originally discovered in Staphylococcus aureus. Protein A has a molecular weight of 42 kDa and is encoded by the spa gene. It binds immunoglobulins, and has five

homologous Ig-binding domains folded into a three-helix bundle. The domains are able to bind immunoglobulins from various mammalian species, including mammalian IgGs, with binding occurring at the heavy chain within the Fc region of most immunoglobulins. Protein G (UniProt PI 9909) is another Staphylococcus aureus protein like Protein A, that binds to the Fab and Fc region of immunoglobulins. Protein G is a cell surface protein of 65-kDa from G streptococcal strain G148, and a protein of 58 kDa from G streptococcal strain G43 (Sjobring, U., et al. (1991) J. Biol. Chem.; 266:399-405). Active fragments, such as terminal deletions of Protein G, have been shown to have IgG binding activity (e.g., reviewed in Bacterial Immunoglobulin- binding Proteins, Boyle, M.D.P., Academic Press, 2012.)

Protein A/G is a recombinant fusion protein that combines IgG binding domains of both Protein A and Protein G, and displays the additive properties of these two proteins (Eliasson, M., et al. (1988) J. Biol. Chem. 263 :4323-4327).

Protein L (UniProt Q51918) is a 992 amino acid protein from

Peptostreptococcus magnus. Unlike Protein A and Protein G, Protein L binds to the Fc region of immunoglobulins through light chain interactions and binds a wide range of antibody classes, including IgG, IgM, IgA, IgE, and IgD. Protein L binds to antibodies that contain kappa light chains. In humans and mice, most antibody molecules contain kappa (κ) light chains and the remainder have lambda (λ) light chains.

In performing an immunoassay, a composition including a Protein A, G, A/G, or L blocking agent can be added to the immobilized analyte capture antibody, to the analyte detection antibody, to the biological sample, or to any mixture thereof. For example, in some modes of practice the Protein A, G, A/G, or L blocking agent is added to the biological sample which is being analyzed for the presence or amount of an analyte. The Protein A, G, A/G, or L blocking agent, such as in a concentrated stock solution, can be added to the biological sample to provide a working concentration of blocking agent in the biological sample in the range of about 0.1 μg/mL to about 5 mg/mL, or about 1 μg/mL to about 50 μg/mL, such as about 10 μg/mL.

A biological sample can be a material taken from an organism, such as body fluid from a mammal. Biological samples include certain tissues, or body fluid such as blood, sputum, urine, saliva, mucus, vitreal fluid, synovial fluid, semen, cerebrospinal fluid, bone marrow, amniotic fluid, bile, sweat, etc. The biological sample can be one that includes, aside from the analyte, immune compounds.

Biological samples can be obtained from patients and analyzed for the absence or presence of analytes associated with disease states. Quantitation of an analyte can be used to determine the absence, presence, or degree of a disease state. Biological samples can also include sections of tissues, such as frozen sections taken for histological purposes which can also be analyzed for analytes associated with disease states. The biological sample may include the analyte for which

immunoassay is being carried out, and may also include antibodies that are cross- reactive with the analyte capture antibody and the analyte detection antibody.

Generally, analysis can be performed to determine the presence and/or amount of an analyte in a sample. The term "analyte" refers to any substance or chemical constituent of a sample that is being analyzed. The analyte can be a natural compound, such as one that is produced by an organism, or can be a non- natural compound, such as a synthetic compound used as a bio-affecting agent like a drug, a pesticide, or an herbicide. Various types of analytes can be detected and quantified in a sample using the methods of the disclosure. In some aspects, detection of the analyte is facilitated by using an analyte binding member, such as an antibody. Non-limiting exemplary analytes include drugs, drug metabolites, biomarkers, hormones, antibiotics, food supplements, food additives, naturally occurring contaminants, dyes, microorganisms and their toxins, fungi, viruses, pesticides, herbicides, tissue specific markers, tissue specific enzymes, cytokines, chemokines, growth factors, receptor ligands, enzymes, nucleic acids, lipids, and small organic molecules, such as glucose and peroxides.

As other non-limiting examples, in the health care industry ELISAs are used for the detection of various analytes in blood, serum, urine, and other body fluids for the detection of analytes such as erythropoietin (EPO), adrenocorticotropic hormone (ACTH), calcitonin, parathyroid hormone (PTH), thyroid stimulating hormone (TSH), prostate-specific antigen (PSA), human chorionic gonadotropin (HCG), follicle stimulating hormone (FSH), and growth hormone (GH).

Antibody and antibody fragments having specificity towards desired analytes are commercially available or can be prepared by techniques known in the art. For example, monoclonal antibodies (mAbs) can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, for example, the hybridoma technique (Kohler and Milstein, Nature, 256:495-497 (1975)); the human B-cell hybridoma technique (Kosbor et al., Immunology Today, 4:72 (1983); and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, or any subclass thereof. In some embodiments antibody and antibody fragments having specificity towards desired analytes are derived from a mouse (e.g, mouse IgGs having specificity against the analyte).

Fab or Fab '2 fragments can be generated from monoclonal antibodies by standard techniques involving papain or pepsin digestion, respectively. Kits for the generation of Fab or Fab'2 fragments are commercially available from, for example, Pierce Chemical (Rockford, IL).

The kit and method can also include reagents for the detection of an analyte using the blocking agent. For example, the kit can optionally include an analyte- specific binding member (e.g, antibody or antibody fragments). The analyte- specific binding member can be used for the detection of an analyte of interest in a biological sample. The kit can also optionally include an enzyme for promoting the conversion of a substrate, such as a colorimetric substrate, to a product. The enzyme can be, for example, a peroxidase, an oxidase, or a conjugate of a peroxidase or an oxidase. The kit can also optionally include an analyte positive control, an analyte negative control, or mixtures thereof. The kit can also optionally include an analysis plate upon which a colorimetric analysis can be performed.

An ELISA can be performed in any assay vessel that is a suitable receptacle for analyte detection. The assay vessel can be made from material such as glass (e.g., surface modified glass), quartz, or plastic, such as polystyrene, polypropylene, and polycarbonate. Exemplary assay vessels are single and multi-well plates, such as medium and smaller- welled plastic plates such as 6, 24, 96, 384, and 1536 well plates. These are commonly known in the art as microtiter plates, microplates, or microwell plates. Exemplary plates for use in ELISAs in each well hold from microliter to milliliter volumes of liquid. Other types of assay vessels that can be used for analysis include capillary tubes. The assay vessel can optionally be included in a kit, or can be supplied by the user to carry out an ELISA.

Generally, an ELISA uses a solid-phase immunoassay to detect the presence of an analyte in a liquid sample. Various ELISA formats are known in the art, and any of these can be used in conjunction with the blocking agent of the disclosure. An ELIS A method can include an analyte-binding antibody immobilized on the solid phase, along with a detection antibody that will bind specifically to the immobilized analyte and use an enzyme to generate a signal that can be quantified. The particular immunoassay format employed will depend on the particular analyte characteristics, the sample characteristics, the available reagents, and the like.

The blocking agent can be used in a "sandwich" ELISA, in which the analyte binding member (e.g., an analyte "capture" antibody) is first immobilized on a solid substrate, such as a plastic well. Optionally, after the capture antibody is immobilized, a non-specific blocking agent (that is not Protein A, G, A/G, or L) such as albumin is used to prevent adherence of components from the biological sample when added to the well. For example, the non-specific blocking agent can be added to the capture antibody immobilized surface, and then washed off. A nonspecific blocking solution can include a non-specific protein such as bovine serum albumin, which can effectively block vessel binding sites that remain following initial coating steps in ELISA procedures (e.g. 5% BSA-PBS). Washing buffers can include a surfactant such as Tween (an exemplary washing buffer, PBS-T, contains 10 mM phosphate buffer pH 7.4, 150 mM NaCl, and 0.05% Tween 20).

In one mode of practice, the blocking agent that is Protein A, G, A/G, or L, or active portions thereof, or mixtures thereof, can be used along with the non- specific blocking agent. For example, a blocking composition including Protein A, G, A/G, or L, and the non-specific blocking agent can be added to the capture antibody immobilized surface, and then washed off. Alternatively, the Protein A, G, A/G, or L blocking agent can be added to the surface before or after the non-specific blocking agent. Using this approach, the Protein A, G, A/G, or L blocking agent may interact with a constant region of the capture antibody and prevent any interaction of a heterophilic antibody in the biological sample that is directed against the constant region of the capture antibody, to prevent false positive results.

In another mode of practice, the blocking agent that is Protein A, Protein A/G, Protein G, and Protein L, active portions thereof, or mixtures thereof, is added to the biological sample prior to contacting the biological sample to the capture antibody immobilized surface. The blocking agent, such as Protein A, G, A/G, or L can be added to the biological sample to provide a working concentration of blocking agent in the biological sample in the range of about 0.1 μg/mL to about 5 mg/mL, or about 1 μg/mL to about 50 μg/mL, such as about 10 μg/mL. Using this approach, the Protein A, G, A/G, or L blocking agent may interact with heterophilic antibodies directed against the analyte capture antibody in the biological sample and prevent their binding to the analyte capture antibody on the assay surface to prevent false positive results.

Analyte from the biological sample added to the well specifically interacts with the immobilized analyte capture antibody on the plate. The Protein A, etc., blocking agent prevents binding of heterophilic antibodies directed against the analyte capture antibody. After the biological sample is applied and analyte is captured, the plate is washed, and then a solution of a second analyte binding member (such as an antibody conjugated to a peroxidase enzyme, called a detection antibody) is added which interacts with the analyte already immobilized by the plastic bound antibody. The analyte therefore effectively becomes "sandwiched" between the antibody absorbed on the plate, and the enzyme-conjugated antibody.

A detection reaction (e.g., a chromogenic reaction) can then be performed. A reactant, such as hydrogen peroxide, can be added as a substrate for the peroxidase of the detection antibody. A substrate which changes color in the presence of the reaction product of hydrogen peroxide and the peroxidase, such as an aromatic amine chromogenic substrate (e.g., TMB) can be added. A stop reagent compound can optionally be added after that, and colorimetric analysis performed; or a kinetic rate assay method can be performed. Exemplary chromogenic reaction compositions and stop reagent compositions are described in U.S. Pat. No.

8,927,226 (Opperman).

Alternatively, the "sandwich" approach can be performed by first mixing an enzyme-conjugated antibody with a biological sample having an analyte, wherein the biological sample is first treated with the Protein A blocking agent. The enzyme-conjugated antibody is used in excess and analyte-enzyme-conjugated antibody complex forms in the sample. The sample is then transferred to a well of an assay plate that has the analyte capture antibody immobilized thereon. The analyte capture antibody binds the analyte-enzyme-conjugated antibody complex, and then the well can be washed to remove non-bound material. A chromogenic reaction can then be performed.

As noted, kits of the disclosure can include one or more components useful for performing an ELISA, other than the blocking agent that is Protein A, Protein A/G, Protein G, and Protein L, active portions thereof, or mixtures thereof. The kit can include optional components such as analyte-binding members like antibodies, enzymes (e.g., HRP -linked anti-IgG antibodies, etc), and enzyme substrates. The kit can also include vessels, such as multi-welled plates, in which the reaction can take place and be analyzed.

In some methods of analyzing, such as using a direct or sandwich

immunoassay as described herein, and the reagents employed, the absorbance of a chromogenic product as determined by a spectrophotometric method will be directly proportional to the amount of analyte in the sample. Where one is interested in a qualitative result or a semi-quantitative result, such as determining whether the amount of analyte is above a predetermined threshold, versus determining the concentration of analyte, the amount of peroxidase enzyme and/or chromogenic substrate can be selected to provide a clear signal as compared to the absence of analyte or analyte below the predetermined value.

In a kinetic analysis (e.g., kinetic ELISA), absorbance is continuously monitored. For example, a substrate is added to a microplate well, and the optical density increase over time is monitored continuously. In kinetic analysis, analyte concentrations are determined by slope of the reaction curve, rather than by the absolute optical density value reached (as compared to end-point ELISA). Slopes of the reaction curve are desirably determined before enzyme velocity begins to deviate from maximum. Data from kinetic analysis can be expressed as the change in milli- optical density units over time, or AmOD/At. Using the initial absorbance reading of the mixture as its own blank, for example, the time ("reaction time") required for the absorbance to increase by a specific amount (e.g., greater or equal to 0.200 absorbance units), is determined. The reaction time for each sample of each product corresponds to the analyte concentration for that sample. The absolute value of analyte concentration for the sample is determined by comparison of the reaction time for the sample with the reaction time for at least one control solution whose analyte concentration is known. The ratio of reaction times for the unknown relative to the known sample is inversely proportional to the ratio of their concentrations.

For quantitation, absorbance can be accurately measured using appropriate hardware and software. Controls can be employed, where the signal to

concentration of the analyte is determined, so that the signal can be directly related to the concentration of analyte in the immunoassay sample. In this manner, both the presence and the amount of analyte in the sample can be determined. Simple spectrophotometers, such as UV/Vis spectrophotometers for wavelengths between 175 nm and 900 nm capable of determining the absorbance of a sample are commercially available, for example, from Perkin Elmer. In analyzing the sample, a light of a specific wavelength, such as selected by an optical filter or

monochromator, is transmitted through the sample, and a detector measures the percentage of the initial transmitted through the sample. The amount of transmitted light is generally inversely proportional to the amount of analyte in the sample.

In other modes of practice, analysis is performed using a microplate reader.

A variety of microplate readers are also commercially available that are capable of accommodating and analyzing the absorbance of samples in the wells of 96-well plates, are commercially available, from, for example BioTek (Winooski, VT).

Example 1

A 96 microwell plate (VWR, 439454) was coated with a solution of mouse anti-troponin (Abeam, ab 10236). The plate was then blocked with StabilCoat (SurModics, Cat# SCOl). Human anti-mouse antibody (HAMA) positive serum (Bioreclamation) was diluted into diluent buffers containing protein A (Life Technologies, 21184), protein A/G (Life Technologies, 21186), or mouse IgG. All diluent buffers used SMOl (SurModics, Cat# SMOl) as the base. The diluted serum samples were incubated on the plate, washed with phosphate buffered saline with 0.05% tween-20 (PBS-T), and then incubated with Mouse anti-troponin - HRP (Abeam, abl0239). After washing with PBS-T, the plate was developed with tetramethylbenzidine substrate (SurModics, TMBW). The table reports the absorbance readings at 650 nm and the percent of HAMA signal blocked relative to a control sample with PBS as the diluent. HAMA False

Positive % HAMA

Abs 650nm Si nal Blocked

Protein A and Protein A/G provide a false positive decrease as a sample diluent. Protein A/G provided blocking similar to a positive control mouse IgG.

Claims

What is claimed is:

1. A kit for performing an immunoassay comprising (a) a blocking agent selected from the group consisting of Protein A, Protein A/G, Protein G, and Protein L, and active portions thereof and (b) a diluent for the blocking agent, wherein the blocking agent is in dried form separate from the diluent, or combined with the diluent, wherein the blocking agent is configured for use to reduce non-specific antibody interaction in an immunoassay.

2. The kit of claim 1 wherein the diluent comprises one or more of a buffer, a salt, a stabilizer, and polymeric reagent.

3. The kit of claim 1 or 2 comprising an analyte detection antibody.

4. The kit of any of the previous claims further comprising an analyte positive control, an analyte negative control, or mixtures thereof.

5. The kit of any of the previous claims further comprising an immobilized analyte capture antibody.

6. The kit of any of the previous claims further wherein the blocking agent comprises Protein A or an active portion thereof.

7. A method for reducing non-specific antibody interaction in an immunoassay comprising steps of:

(a) providing a composition comprising a blocking agent selected from the group consisting of protein A, protein A/G, protein G, and protein L;

(b) contacting an immobilized analyte capture antibody, an analyte detection antibody, a biological sample, or any mixture thereof, with the composition; and

(c) performing an immunoassay on the biological sample using at least the antibody and the analyte detection antibody.

8. The method of claim 7, wherein the composition comprising the blocking agent is added to the biological sample to form a mixture, and then the mixture is placed in contact with an immobilized analyte capture antibody.

9. The method of claim 7, wherein the composition comprising the blocking reagent is added to an immobilized analyte capture antibody and then the biological sample is placed in contact with the immobilized analyte capture antibody.

10. The method of any of claims 7-9 wherein the biological sample is a serum or blood sample.

11. The method of any of claims 7-10, wherein the blocking reagent is present in the composition in the range of 0.1 μg/mL to 5 mg/mL.

12. The method of claim 11, wherein the blocking reagent is present in the composition in the range of 1 μg/mL to 50 μg/mL.

13. The method of any of claims 7-12, wherein the analyte detection antibody is coupled to a peroxidase or oxidase selected from horseradish peroxidase, soybean peroxidase, glucose oxidase, galactose oxidase, xanthine oxidase, lactoperoxidase, microperoxidase, NADH peroxidase NADPH peroxidase, fatty-acid peroxidase, and catalase.

14. A non-natural composition comprising (a) a biological sample from a mammalian species or a detection antibody for an immunoassay, and (b) a blocking agent selected from the group consisting of protein A, protein A/G, protein G, and protein L, and active portions thereof, wherein the blocking agent is present in the composition at a concentration effective to reduce non-specific antibody interaction when the composition is used in an immunoassay.