Methods and kits for detecting an analyte

Abstract

The present invention provides kits and methods for detecting certain analytes of interest potentially present in the blood and body fluids of a live mammal. These methods and kits encompass bioassays performed in vivo. Contact of the bioassay reagent with the analyte, if present, provides a response that can be clinically evaluated visually or by reading an instrument or by a biosensor. In one embodiment, the present invention can be used to detect the presence, absence, or amount of a suspected analyte present in a patient test subject. The present invention is particularly suitable for point-of-care (POC) use, self-testing, large-scale implementation, and for patients for whom only a limited sample volume is available or obtainable.

Description

Technical Field

[0001] This application claims the benefit of the following patent applications: earlier filed U.S. Provisional Patent Application Serial No. 62 / 968,648, filed on January 31, 2020; U.S. Provisional Patent Application Serial No. 63 / 040,340, filed on June 17, 2020; U.S. Provisional Patent Application Serial No. 63 / 122,307, filed on December 7, 2020; and U.S. Provisional Patent Application Serial No. 63 / 135,699, filed on January 10, 2021, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to methods, kits, and reagents for determining the presence, absence, or amount of antibodies, receptors, receptor-binding molecules, antigens, drugs, poisons, toxins, pathogens, biomarkers, biochemicals, cell surface markers, and other molecules of interest present in blood and body fluids without sample collection. Background Art

[0004] The global increase in infectious and non-infectious diseases and disease states, combined with the growth of the global population, has created a huge demand for new and innovative diagnostics. Conventional techniques (such as tests performed in centralized laboratories that limit patient access) result in longer sample turnaround times and "lost to follow-up," and may be insufficient to address the health problems of the entire population (Anuj Pathak, BCC Research Report HLC207A (2018)). While "point-of-care" (POC) tests overcome some of these limitations, both conventional and POC assays widely utilize blood to determine the presence of diseases such as HIV / AIDS, malaria, dengue fever, cancer, cardiovascular, viral, and bacterial infections. Notably, blood tests typically require sample collection, sample processing, sample stabilization, and frequent sample transportation. Additionally, pre-analytical sample preparation can significantly increase the cost of laboratory-based and POC tests. Moreover, there is no guarantee that interfering factors potentially present in the sample have been removed or that the sample has been properly processed, either of which can lead to false results.

[0005] Modern diagnostic tests are limited by samples that need to be collected and analyzed outside of a living organism. Pre-analytical errors account for up to 70% of all errors in laboratory diagnosis, and most of these errors are caused by problems in sample preparation, collection, transportation, and preparation for analysis and storage (Plebani, M, Clin Biochem Rev 33:85-87 (2012)). Collecting samples by venipuncture can be painful for some patients, provide opportunities for sample contamination, and pose a biohazard to healthcare workers. Collecting sufficient blood by finger prick can be problematic, as it requires heating and / or massaging the finger, which can in turn lead to problems with sample integrity. The limited amount of sample that can be collected from elderly, pediatric, or frail patients may also impede or hinder the utilization of many blood-based diagnostic tests. Samples can also be lost or mislabeled. Thus, eliminating sample collection would be advantageous for blood-based assays.

[0006] Due to their nature, samples have a fixed volume and a fixed concentration of analytes available for testing, thus constraining the applicability of certain assays due to limitations in their sensitivity thresholds. For example, “rapid diagnostic tests” (RDTs) that utilize finger prick blood sampling provide a means of generating test results anywhere and anytime, but the small volume of blood used in these assays means that there are fewer analytes available for detection, thus limiting sensitivity. RDT devices and other point-of-care (POC) technologies are used worldwide and have various advantages over conventional laboratory tests; however, due to the limited sample size, there are limitations to the accuracy and reliability of these tests. Thus, an assay method in which the sample size does not constrain assay performance would be useful.

[0007] Although the burden of many diseases, such as HIV / AIDS, tuberculosis, and hepatitis, is heterogeneous, their impact on the global poor and "low- and middle-income countries" (LMICs) is particularly severe (for reviews, see Yuen et al., Nature Reviews, Disease Primers DOI: 10.1038 / nrdp.2018.35 (2018); Manns et al., "Hepatitis C Virus Infection" Nature Reviews, Disease Primers DOI: 10.1038 / nrdp.2017.6 (2017); Freiman, J.M., Ann. Intern. Med. 165(5):345 (2016); Pai et al., "Tuberculosis" Nature Reviews, Disease Primers DOI: 10.1038 / nrdp.2016.76 (2016); Sewald et al., Curr. Opin. Cell Biol. 41:81 (2016); Park et al., Journal of Clinical Microbiology 48:2253 (2010)). The diagnostic landscape for these diseases and disease states has historically been dominated by complex, laboratory-based technologies that do not meet all testing needs in resource-limited settings. Complex technologies often require substantial capital investment, sophisticated laboratory infrastructure, and trained technical personnel, which are not available in many settings, often resulting in limited geographic reach. As a result, most patients in LMICs do not have access to this type of testing or the benefits of this type of testing where they receive care. Unfortunately, the fortunate few who can access complex testing rarely receive timely results from conventional laboratories, which often take days, weeks, or even months. This delay often leads to poor patient outcomes or even death. Importantly, patient ignorance of their own health status is considered an important factor in the spread and transmission of infectious diseases (Drain et al., Clinical Microbiology 32(3):1-25 (2019)).

[0008] Although RDTs have transformed the testing landscape, even the simplest RDTs have multiple steps, typically adding and washing away or separating multiple reagents at different points in the assay. This has thus made them difficult to use in the field. As a result, efforts to test hard-to-reach populations have been difficult to implement. In addition, the widespread use of self-testing is limited because patients need to collect their own blood samples or perform oral swabs to obtain results. As such, it would be useful to have an easy-to-interpret, simple-to-operate test that provides real-time, on-site results to patients and healthcare providers without the patient having to collect their own samples for testing (Mugambi et al., BMJ Glob Health DOI: 10.1136 / bmjgh-2018-000914 (2018); Drain et al., Lancet 14(3):239 (2014)).

[0009] In addition to the above, major constraints have been encountered in designing mechanism-of-action studies, such as limitations on the volume of biological specimens available for study. There are no robust and validated assays that can reliably and reproducibly assess immune function, disease state, or therapy efficacy with limited samples. Additionally, the restricted amounts of tissue, cells, and fluids that can be collected from elderly, pediatric, or immunocompromised patients are typically insufficient to apply conventional assays to probe immune function. [[ID=****5]]

[0010] Sample sparing assays that are needed but not available include assays for monitoring or assessing antigen-specific immune responses, identifying the presence of different immune cell populations, measuring markers of T cell turnover, detecting gene expression, and assays for measuring mucosal inflammation and other innate immune responses (Department of Health and Human Services, SBIR, Phase I Program Solicitation PHS 2018-, NHI / NIAID 057). To obtain the maximum amount of information from limited biological materials, novel sample sparing assays are needed.

[0011] In addition to various sample-related issues (such as insufficient sample size and sample collection problems), problems associated with delayed diagnosis due to lack of test results or rapid delivery of test results also pose significant barriers, which are particularly problematic in larger populations potentially exposed to highly contagious and sometimes fatal pathogens. For example, diseases such as Ebola and Zika virus require rapid response times for diagnosis and to contain outbreaks before they spiral out of control. Unfortunately, it can be difficult to diagnose diseases such as Ebola soon after infection. Early symptoms of Ebola such as fever, headache, and weakness are not specific to Ebola virus infection and are often seen in other more common and less fatal diseases such as malaria and typhoid. Given the recent number of disease outbreaks (such as Ebola and listeriosis), there is an urgent need to strengthen health systems and ensure the rapid identification and containment of public health threats. Rapid response tests that can be quickly mass-marketed for existing and future pandemics will clearly provide significant advantages to healthcare providers in the time-sensitive management of such outbreaks.

[0012] Additional problems arise when detecting diseases such as viruses, particularly those marked by latent seroconversion (such as HIV). Millions of people are screened for HIV, HCV, and TB, and only seroconvert negatively after testing. In addition, healthcare workers active in fatal disease outbreaks, first responders reacting to terrorist attacks, or military personnel on the front line are endangered by harmful agents even before symptoms appear. The above misdiagnoses and medical emergencies can be unpredictable, and response measures must incorporate the development of new methods for the rapid detection, accurate diagnosis, and rapid treatment of exposed populations. For example, anthrax (Bacillus anthracis) is a highly toxic agent and can form extremely viable spores that can remain alive for a long time. Once inhaled, the disease can progress rapidly and often leads to death within days without prompt treatment. Therefore, early detection and differential diagnosis are crucial. These groups and others would benefit from sentinel testing that can be applied to healthcare workers before exposure and then detect subsequent exposure days, weeks, or even months later if they come into contact with harmful agents. Such testing can save lives, contain outbreaks, and stop the spread of disease by detecting exposure at the earliest possible moment.

[0013] Even diseases and pathogens that have been around for centuries remain prevalent and problematic, endangering entire populations. Thus, rapid and robust point-of-care tests would also benefit the fight against these health challenges. For example, according to WHO data from 2014, an estimated 9.6 million people developed active TB disease, of whom 1.5 million died. Five to fifteen percent of individuals infected with Mycobacterium tuberculosis will develop (over months to years) active TB disease, while the remaining patients with latent TB remain at continued risk of developing active TB disease throughout their lives. In many settings, up to 50% of all people with culture-positive active TB disease do not have a prolonged productive (sputum- or mucus-producing) cough, and at least 25% have no symptoms (Darin et al., Clinical Microbiology Reviews 31(4) DOI.org / 10.1128 / CMR.00021-18 (2018)).

[0014] Options for low-cost screening tools for TB are limited to detecting latent TB infection (LTBI). There are two tests available for identifying LTBI: the TST and the interferon-gamma release assay (IGRA). The IGRA can also distinguish between BCG-induced and Mycobacterium tuberculosis infection-induced positive TST responses (for a review, see Pai et al., "Tuberculosis" Nature Reviews, Disease Primers D0I:10.1038 / nrdp.2016.76 (2016)).

[0015] The TST, performed using the Mantoux technique, consists of an intradermal injection of 2 to 5 tuberculin units (5TU) of purified protein derivative (PPD). In people with cell-mediated immunity to these antigens, a delayed-type hypersensitivity reaction will occur within 48 to 72 hours. Interpretation of the TST takes into account the size of the induration, the pretest probability of Mycobacterium tuberculosis infection, and the risk of developing active TB disease if the person is truly infected.

[0016] Although the TST has several advantages (especially in low-resource settings), including low reagent and equipment costs and limited technical and laboratory requirements, it has two major limitations. First, its specificity is compromised by later (i.e., after infancy) or repeated BCG vaccination (boosters), and to a limited extent by exposure to nontuberculous mycobacteria. Second, it has limited predictive value. Most individuals with a positive TST result do not develop active TB disease.

[0017] IGRAs were introduced in the early 2000s in the hope of replacing the TST. IGRAs are in vitro blood tests of cell-mediated immune responses: they measure the release of IFNγ from T cells stimulated by antigens encoded by RD1 (i.e., 6 kDa early secretory antigen target and culture filtrate protein). The RD1 antigens are more specific for Mycobacterium tuberculosis than the PPD antigen because they are not encoded in the genomes of any BCG vaccine strain or most non-tuberculous mycobacterial species (except Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium flavescens). However, like the TST, IGRAs have poor predictive value.

[0018] After hundreds of studies, it has become clear that for LTBI, both the TST and IGRAs are acceptable but imperfect tests. The tests can neither accurately distinguish LTBI from active TB disease nor distinguish new infection from reinfection events, a distinction that may be relevant in settings where individuals previously receiving preventive therapy are at risk of reinfection. In summary, none of the currently available LTBI tests meet the need for a highly predictive test that can help identify individuals at increased risk of developing active TB disease and who would therefore benefit most from LTBI therapy (preventive therapy). Notably, because all LTBI tests have low predictive value, widespread screening of low-risk populations is counterproductive.

[0019] Detection of lipoarabinomannan (LAM) antigen in urine has emerged as a potential point-of-care test for detecting HIV-associated active TB disease, with only a modest reduction in mortality in a highly selected group of hospitalized HIV-positive patients (see Sakamuri et al., Tuberculosis 93(3) DOI:10.1016 / j.tube.2013.02.015 (2013); Abd el-Atty et al., Menoufia Medical Journal DOI:10.4103 / 1110-2098.149720 (2014); Choudhary et al., Journal of Immunology

[0020] DOI: 10.4049 / jimmunol.1701673(2018); Youssef et al., Egyptian Journal of Bronchology DOI: 10.4103 / 1687-8426.193639(2016)). WHO now recommends a rapid LAM test of urine to help and speed up the diagnosis of active TB disease in two specific populations: in HIV-positive adult inpatients with pulmonary and / or extrapulmonary TB signs and symptoms and a CD4+ T cell count < 100 cells / μl; or in severely ill HIV-positive patients regardless of their CD4+ T cell count or unknown CD4+ T cell count.

[0021] Given the limitations of available diagnostic screening tests for the larger population for detecting active TB, the development of new and better diagnostic tools for detecting active TB infection remains a priority. Several diagnostic tools are currently under development. Although these tools may seem robust and suitable for field-testing conditions at first glance, the commercial versions of these products are generally designed for laboratory settings and use only proven TB biomarkers: bacterial nucleic acid sequences. Importantly, when considering integration into primary care, tests such as these often fall short in terms of affordability and ease of use requirements. Recently, the World Health Organization (WHO) defined high-priority Target Product Profiles (TPPs) for TB diagnosis. These include rapid non-sputum-based tests for detecting TB, with the aim of starting specific TB therapies on the same day. For these tests to be truly effective, they need to be performed in endemic settings with limited laboratory facilities, at a relatively low cost, using readily available samples that do not require the collection of body fluids and their associated drawbacks. Therefore, there is an urgent need to identify and utilize biomarkers (BMs) that can be used for such tests and to develop novel test formats that enable improved and rapid clinical decision-making, an example being the development of improved tests for more accurately and discriminately diagnosing active TB disease (Goletti et al., Respirology 23:455 (2018); Correia-Neves et al., ERJ Open Res. DOI.org / 10.1183 / 23120541.00115-2018 (2018)). There is currently no general method for adapting newly discovered biomarkers to easily deployable devices.

[0022] Immunity consists of humoral (antibody) and cell-mediated immunity. Cell-mediated immunity involves immune cells that specifically recognize, target, and eliminate infected host cells. The immune status of a patient and the efficacy of a vaccine rely on both humoral and cell-mediated immunity. Efforts to evaluate immune responses have generally focused on humoral immunity (serum antibody responses) because antibody responses can be measured relatively easily by ELISA, neutralization assays, hemagglutination assays, etc. Since cell-mediated immunity plays an important role in defending against infections, cancer, autoimmunity, and many other diseases, new assays are needed. Current methods for analyzing cell-mediated immunity are complex and are generally performed in a research setting in a biocontainment facility.

[0023] T cell responses are sophisticated antigen-specific responses and they are at least as important as antibodies in vertebrate defense against infection. In fact, most adaptive immune responses (including antibody responses) require helper T cells to initiate them. Most importantly, unlike B cells, T cells can help eliminate pathogens that reside within host cells.

[0024] T cell responses differ from B cell responses in at least two key ways. First, T cells are only activated by foreign antigens to proliferate and differentiate into effector cells when the antigen is displayed on the surface of antigen-presenting cells in peripheral lymphoid organs. T cells respond in this way because the form of the antigen they recognize is different from the antigen recognized by B cells. B cells recognize intact antigens, while T cells recognize protein antigen fragments that have been partially degraded within the antigen-presenting cell. The peptide fragments are then carried to the surface of the presenting cell on special molecules called major histocompatibility complex (MHC) proteins, which present the fragments to T cells (Rock, K. L. et al., Trends Immunol. November 2016; 37(11):724 - 737. Doi:10.1016 / j.it.2016.08.010).

[0025] The second difference is that once activated, effector T cells only function within secondary lymphoid organs or within a short distance after they migrate to the site of infection. They directly interact with another cell in the body, which they either kill or signal in some way (we will call this cell the target cell). In contrast, activated B cells secrete antibodies that can function at a greater distance.

[0026] There are two main classes of T cells - cytotoxic T cells and helper T cells. Effector cytotoxic T cells directly kill cells infected with a virus or some other intracellular pathogen. In contrast, effector helper T cells help stimulate the responses of other cells - mainly macrophages, B cells, and cytotoxic T cells.

[0027] Superantigens are a distinct class of antigens that stimulate primary T cell responses (Cheng, M. H. et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS), October 13, 2020, Vol. 117, No. 41, 25254 - 25262). Superantigens bind to MHC and T cell receptor molecules without the need for intracellular processing, which enables them to stimulate a very large number of T cells. Superantigens are produced by many different pathogens, including bacteria, mycoplasmas, and viruses, and the responses they elicit are helpful to the pathogen rather than the host.

[0028] Superantigens differ from other protein antigens in that they are recognized by T cells without being processed into peptides that are captured by MHC molecules. In fact, fragmentation of superantigens disrupts their biological activity, which depends on binding as intact proteins to the outer surface of MHC class II molecules that have already bound peptides. In addition to binding MHC class II molecules, superantigens are also able to bind the Vβ regions of many T cell receptors. Bacterial superantigens mainly bind to the VβCDR2 loop, and to a lesser extent to the VβCDR1 loop and an additional loop called the hypervariable 4 or HV4 loop. The HV4 loop is the main binding site for viral superantigens. Thus, the V region of the α chain and the CDR3 of the β chain of the T cell receptor have little effect on superantigen recognition, which is mainly determined by the germline - encoded V sequences of the expressed β chain. Each superantigen is specific for one or several of the different Vβ gene segments, of which there are 20 to 50 in mice and humans; thus, superantigens can stimulate 2 to 20% of all T cells.

[0029] This mode of stimulation does not initiate an adaptive immune response specific for the pathogen. Instead, it leads to the massive production of cytokines by CD4 T cells, which are the major responding population of T cells. These cytokines have two effects on the host: systemic toxicity and suppression of the adaptive immune response. Both of these effects contribute to microbial pathogenesis. Among bacterial superantigens are the staphylococcal enterotoxins (SEs) that cause food poisoning, and toxic shock syndrome toxin - 1 (TSST - 1), which is the etiological principle of toxic shock syndrome.

[0030] An easily assessable test that detects and / or measures cell - mediated immunity and pathways would be highly beneficial in the diagnosis and treatment of diseases as well as in vaccine production. There is no simple and effective diagnosis to measure cell - mediated immunity and associated pathologies.

[0031] The diagnosis and management of diseases and disease states, whether related to genetics or exposure to pathologic entities, are increasingly facilitated by the detection and measurement of biomarkers present in blood, urine, saliva, and other body fluids. Successive advances in biomarker discovery have enabled early diagnosis, real-time monitoring, and improved disease management. However, urine and saliva are limited by the finite number and variable concentrations of their biomarkers. Interstitial fluid (ISF) is another source of valuable and unique biomarkers, but it is difficult to sample from the body. ISF surrounds the cells and tissues throughout the body, forms by extravasation of plasma from capillaries, and is modified by metabolic and other processes in the tissues. Nutrients and waste products shuttle between the blood vessels and cells via ISF, which is roughly a combination of serum and cellular material (see Kunder et al., Blood 118:5383 (2011); Hanen et al., International Immunology 27:219 (2015); Sewald et al., Current Opinion in Cell Biology 41:81 (2016); Samant et al., Proc Natl Acad Sci USA 115:4583 (2018)).

[0032] Previous studies have shown that 83% of the proteins found in serum are also present in ISF, but 50% of the proteins in ISF are not in serum, indicating that ISF may be a source of unique biomarkers as well as biomarkers found in blood. Many viruses are transmitted through the extracellular fluid composed of interstitial fluid, lymph, and blood. In addition, due to the absence of clotting factors, ISF may be suitable for continuous monitoring, as shown by commercial indwelling sensors for glucose that are close to ISF in the subcutaneous space. As shown by tumor ISF collected from tissue biopsies, ISF is a better indicator of local tissue events. The skin is the most accessible organ and is therefore an attractive source of ISF containing systemic and dermatological biomarkers.

[0033] Currently, ISF can be collected from the skin using suction blisters by applying suction to the skin at an elevated temperature for up to 1 hour to generate blisters filled with ISF. Dermal ISF can also be sampled by implanting a tube in the skin to collect ISF biomarkers by microdialysis or open-flow microperfusion, which requires local anesthesia and expert training. The unique properties of ISF provide fertile ground for assay development, but limited access to ISF has hindered its use in assays. New tools and diagnostic tests are needed to harness the benefits of detecting analytes in this medium.

[0034] Importantly, it will further be noted from the foregoing examples that most diseases and disease states, as well as certain environmental and conflict-based exposures, are typically marked by the presence of certain associated analytes that are directly or indirectly produced or present or caused by a pathological entity or genetic defect. Such analytes provide great opportunities for detection, diagnosis, treatment, and prevention, provided they can be detected in a timely and relatively inexpensive manner. Alternatively, there are other situations where it is advantageous to determine the absence, presence, or amount of substances such as hormones, growth factors, metabolites, etc., in order to confirm or establish a baseline of fitness, health, or health status of a subject. Further still, it is advantageous for healthcare providers, public health organizations, military personnel, and others to be able to perform a sentinel test applied prior to exposure and then detect subsequent exposure days, weeks, or even months later if they are exposed to a harmful agent. Summary of the Invention

[0035] The present invention provides certain novel methods, reagent "constructs", and kits that can be readily used by those skilled in the art to determine the presence, absence, or amount of a broad spectrum of analytes, including but not limited to chemicals, peptides, proteins, lipids, carbohydrates, glycoproteins, nucleic acid sequences, or combinations thereof, as illustrated by, but not limited to, substances such as antibodies, hormones, receptors, receptor-binding molecules, antigens, drugs, poisons, toxins, pathogens, biomarkers, biochemicals, cell surface markers, RNA, DNA, and other molecules of interest.

[0036] More specifically, one embodiment of the present invention provides methods for determining the presence, absence, or amount of a suspected analyte in a live mammalian subject. These methods include a first step of providing at least one affinity reagent "construct" having at least one Fc.ε.R1 receptor-binding domain as defined herein and at least one additional moiety capable of binding the suspected analyte or other analyte of interest. The resulting reagent construct is capable of binding the mast cell Fc.ε.R1 receptor and the analyte in any order and is considered multivalent (also referred to as a "multivalent affinity reagent construct"). The term "construct" is intended to refer to the structure or design of a single resulting unit (i.e., molecule) of the affinity reagent of the present invention. The additional binding moiety may also be referred to herein as an "analyte-binding moiety". For the purposes of this disclosure, we note that determining the presence, absence, or amount of a suspected analyte may be collectively referred to as analyzing the suspected analyte.

[0037] For the purposes of the present invention, those skilled in the art will understand that the terms "affinity reagent", "AR", "reagent construct", "affinity reagent construct", "multivalent affinity reagent", "multivalent reagent construct", "reagent" and "construct" are generally used and interpreted interchangeably, and each is intended to refer to a reagent constructed in accordance with the examples and references incorporated herein by reference.

[0038] Those skilled in the art will also understand that the descriptor "multivalent" is intended to mean that the reagent construct is capable of binding simultaneously to more than one entity. For the purposes of the present disclosure, the term entity is intended to refer to any biological or chemical substance or structure. For example, with respect to the multivalent affinity reagent construct of the present invention, "multivalent" is exemplified as the affinity reagent construct being capable of binding to at least one first entity (e.g., the mast cell FcεRI receptor) at a first region of the construct (e.g., the FcεRI receptor binding domain), and binding to at least one second entity (e.g., an analyte of interest) at a second region of the construct via at least one additional moiety. In other words, the affinity reagent construct includes at least one first binding specificity for the mast cell FcεRI receptor and at least one second binding specificity for the analyte of interest.

[0039] According to the present invention, the binary binding discussed above is of course considered only an example of multivalent binding. It is responsible for the multivalent binding response of the present invention that occurs under the crosslinking conditions discussed below. The multivalent binding of the reagent construct is not necessarily sequence-dependent, and importantly, as further explained herein, the use of the present invention for sentinel monitoring contemplates this aspect. Additionally for this purpose, depending on the type of moiety selected, the analyte binding moiety itself may also possess multiple binding sites (i.e., multiple binding specificities), thereby conferring monovalent, divalent or multivalent binding capabilities to the moiety depending on the number of binding sites.

[0040] The method contemplated by the present invention further includes the step of contacting at least one endogenous mast cell present in situ and within the target tissue of a live mammalian subject with the affinity reagent construct of the present invention to initiate a multivalent binding response in the presence or upon the presence of an analyte of interest. Those skilled in the art will understand that, from a practical perspective, the above step is accomplished by physically delivering multiple affinity reagent constructs (hereinafter these multiple are simply referred to as "affinity reagents" or "ARs") to the target tissue by means of some of the devices discussed below. For the purposes of the present invention, the term "target tissue" refers to the type of tissue that receives the affinity reagent of the present invention, such as the skin.

[0041] In practice, the present invention contemplates selecting an anatomical region and tissue type in a test subject having a localized concentration of endogenous mast cells, which localized concentration should be in the range of about 125 to 20,000 mast cells per cubic millimeter for the mammalian species to which the affinity reagent is to be administered. For most mammals, the skin is an acceptable target tissue and, more specifically, it is the upper dermis of the skin. For the purposes of the present disclosure, the term skin is intended to encompass the superficial epidermal layer composed of epithelial tissue, the deeper dermis or "corium" composed of connective tissue and lymphatic vessels, and the even deeper layer of adipose tissue commonly referred to as subcutaneous tissue. Mast cells are generally found at the highest concentrations directly beneath the epithelial surfaces of the skin and mucosa. In humans, delivery sites include the dermal layers of the upper or lower arm and those skin layers in the lower body extremities (Janssens et al., Journal of Clinical Pathology 58:285 (2005)). As will be further understood by those skilled in the art from the following paragraphs, the method of the present invention can be said to incorporate a bioassay in the sense that, unlike traditional diagnostic assays, the underlying test is actually performed or carried out in vivo.

[0042] It should also be understood that for the purposes of the present disclosure, the terms "analyte", "suspected analyte", "putative analyte" and "analyte of interest" will be used and interpreted interchangeably. The term "analyte" is intended to include chemicals, peptides, proteins, lipids, carbohydrates, glycoproteins, nucleic acid sequences or combinations thereof, as exemplified by, but not limited to, substances such as antibodies, hormones, receptors, receptor-binding molecules, antigens, drugs, poisons, toxins, pathogens, biomarkers, biochemicals, cell surface markers, RNA, DNA and other molecules of interest. An analyte is also intended to refer to an analyte as defined herein, the presence, absence or amount of which is known or suspected to be: 1) an indicator of a disease, disease state or exposure to a substance, or alternatively, 2) an indicator of the overall health, fitness or mood and / or physical state of a subject.

[0043] Diseases and disease states fall within the broad category of "pathology" and they can generally be defined as any harmful deviation from the normal structural or functional state of an organism and typically result in or are marked by certain signs and symptoms, but are etiologically distinct from physically induced injuries. For example, an organism suffering from a disease or disease state will generally exhibit signs or symptoms that are abnormal compared to its normal disease-free state. Such signs and symptoms include the presence or absence of certain substances, or alternatively, abnormally low or abnormally high levels of certain substances, all of which substances are potential analytes of interest and, for the purposes of the present invention, can indicate diseases and disease states.

[0044] The term "body fluid" or "bodily fluid" is intended to refer to any liquid originating from within a mammalian subject, including secreted, excreted, or other flowable substances that are excreted from or contained within the subject, such as blood, plasma, serum, lymph, interstitial fluid, intracellular or extracellular fluid, tears, saliva, urine, and the like. The term "mast cell" is intended to refer to basophils, eosinophils, and any other cells that possess the Fc.ε.R1 receptor. In addition, the terms "Fc.ε.R1 receptor" and "high affinity receptor" are used interchangeably and are intended to refer to the endogenous mast cell receptor for the Fc region of immunoglobulin E (IgE), while the term Fc.ε.R1 receptor binding domain denotes any molecule or entity capable of binding to the mast cell Fc.ε.R1 receptor, whether they are generated by hybridoma technology, recombinant, chemically synthesized from amino acids, or generated by aptamer synthesis produced synthetically.

[0045] The method also includes performing an assessment of the multivalent binding response to determine the presence or amount of an analyte of interest. The multivalent binding response of the present invention is caused by the multivalent binding discussed above and encompasses physiological changes in the target tissue, such as crosslinking of the Fc.ε.R1 receptor across the mast cell membrane of the mast cells and degranulation of the mast cells, as further described below. The assessment of the multivalent binding response, if it occurs, can be accomplished by a variety of qualitative and quantitative methods, including by measuring at least one physiological change in the target tissue or by visually inspecting to identify at least one physiological change in the target tissue caused by the binding response, to make a basic assessment of the target tissue at or near the delivery site. Alternatively, a device capable of measuring at least one physiological change in the target tissue associated with the binding response can be used.

[0046] The term "physiological change" is intended to refer to morphological and biochemical differences that occur in mammalian tissue. For the purposes of the present invention, physiological changes within or in the target tissue will include crosslinking of the Fc.ε.R1 receptor across the mast cell membrane of the mast cells within the target tissue, which are considered to be "localized" in proximity to the site of administration of the affinity reagent, and degranulation of such mast cells and any additional and secondary physiological changes in the target tissue directly or indirectly resulting from such degranulation. The term "localized" is intended to refer to the area starting from within or on the target tissue contacted by the device delivering the affinity reagent up to an area radiating outward approximately 3 cm from that point of contact.

[0047] Mast cell degranulation involves the release of cellular components (also known as granules) from mast cells as part of the degranulation process, as well as the immediate release of stored chemical mediators, followed by newly synthesized mediators. These mediators also have their own primary and secondary effects on cells in the target tissue. These physiological changes that occur within or to the target tissue typically occur together and are often inextricably intertwined. For example, some physiological changes lead to a series of events that induce subsequent physiological changes, such as morphological changes in the tissue that are clearly visible to the naked eye. These changes can generally be evaluated qualitatively or quantitatively.

[0048] As described above, the assessment of the binding response can involve performing a visual inspection to identify or measure physiological changes in the target tissue, where the physiological changes are, for example, the physical presence of the characteristic swelling and erythema of the wheal and flare response induced by biochemical changes occurring according to degranulation. In another embodiment, the assessment of the binding response can involve measuring the mast cell degranulation substances released by mast cells during the mast cell degranulation process, or alternatively, measuring additional and secondary substances released from cells in the target tissue in response to the released degranulation substances during the degranulation process. Alternatively, in yet another embodiment, the assessment can involve using devices such as biosensors or other equipment to identify or measure physiological changes in the target tissue.

[0049] As described above, the present method contemplates an embodiment in which an affinity reagent is delivered into the skin and optimally into the dermis where the mast cell concentration is highest. Generally, any tissue in which the affinity reagent is exposed to approximately 125 to 20,000 mast cells per cubic millimeter is suitable and is exemplified in various publications available to those skilled in the art. The delivery of the affinity reagent can be performed by any suitable means, such as acupuncture, intradermal injection, solid needles, hollow needles, patch methods, or needleless systems.

[0050] Once familiar with the concepts of the present invention, the construction of multivalent affinity reagents can be accomplished using well-known and established laboratory techniques, including hybridoma technology, covalent conjugation, non-covalent binding, genetic engineering methods, and related recombinant techniques, examples of which can be found in the cited references and other relevant related arts. The additional or analyte-binding portion can be shaped using at least one member from the group consisting essentially of: antibodies, recombinant antibodies, engineered antibodies, antibody fragments, synthetic antibodies, engineered non-antibody binding proteins, antigens, chimeric molecules, fusion proteins, aptamers, hormones, receptors, receptor-binding molecules, drugs, poisons, toxins, pathogens, pathogen components, biomarkers, cell surface markers, ligands, RNA or DNA, and other chemical or biological molecules, to the extent that they can confer the analyte specificity necessary for the bioassays taught by the present invention. For example, in one embodiment, the portion is shaped by an antigen such as hepatitis B surface antigen (HBsAg) or an antibody such as anti-hepatitis B surface antigen antibody (HBsAb) or anti-hepatitis B core antigen antibody (HBcAb). In alternative embodiments, it can be shaped by a protein such as interferon γ or troponin. In yet another embodiment, the portion can be shaped by a non-infectious component of a pathogen such as lipoarabinomannan or a non-infectious form of the pathogen itself such as Ebola or Zika virus. These are just some limited examples.

[0051] Additionally, the methods and kits of the present invention provide a method for testing a suspected pathology in a live mammal. For the purposes of the present invention, a suspected pathology can be defined as a specific disease or disease state that is believed to be present in the test subject. It should be understood that an appropriate analyte of interest, known or suspected to be an indicator of such disease or condition, must be selected and thereafter an affinity reagent having at least one Fc.ε.R1 receptor-binding domain and at least one additional portion capable of binding the selected analyte is constructed, wherein the resulting reagent is capable of binding both mast cells and the analyte in any order; contacting the multiple endogenous mast cells present within the target tissue of the mammal with the resulting reagent to elicit a multivalent binding response in the presence of the analyte; and performing an evaluation of the multivalent binding response to determine the presence or amount of the selected analyte.

[0052] The methods and kits of the present invention also provide a method of testing a live mammal for exposure to a particular substance such as a chemical, drug, biological warfare agent, poison, toxin, or pathogen. It should be noted that for the purposes of this application, the term "chemical" is intended to include contaminants such as toxic metals, polycyclic aromatic hydrocarbons, benzene, particulate matter (PM), nitrogen oxides, sulfur oxides, carbon monoxide, ozone, and similar harmful compounds. The method includes selecting an analyte known or suspected to indicate exposure to a particular substance, and thereafter constructing an affinity reagent comprising at least one Fc.ε.R1 receptor-binding domain and at least one additional moiety capable of binding the selected analyte, wherein the resulting reagent is capable of binding both mast cells and the analyte in any order; contacting a plurality of endogenous mast cells present within a target tissue of the mammal with the reagent to elicit a multivalent binding response in the presence of the analyte; and performing an assessment of the multivalent binding response to determine the presence or amount of the selected analyte. Additionally, the methods and kits of the present invention provide a method of monitoring a live mammal for exposure to a particular substance such as a chemical, drug, biological warfare agent, poison, toxin, or pathogen, or alternatively, a suspected pathology caused by such exposure. The method includes selecting an analyte indicative of exposure to a particular substance, and thereafter constructing an affinity reagent comprising at least one Fc.ε.R1 receptor-binding domain and at least one additional moiety capable of binding the selected analyte, wherein the resulting reagent is capable of binding both mast cells and the analyte in any order; contacting a plurality of endogenous mast cells present within a target tissue of the mammal with the reagent to enable a multivalent binding response to occur when the analyte becomes present; and performing an assessment of the multivalent binding response, if it occurs, to determine the presence or amount of the selected analyte.

[0053] Additionally, the methods and kits of the present invention provide a method of testing and monitoring the health status of a live mammal. The method includes selecting an analyte related to a health status parameter of interest, and thereafter constructing an affinity reagent comprising at least one Fc.ε.R1 receptor-binding domain and at least one additional moiety capable of binding the selected analyte, wherein the resulting reagent is capable of binding both mast cells and the analyte in any order; contacting a plurality of endogenous mast cells present within a target tissue of the mammal with the reagent to elicit or enable a multivalent binding response when the analyte is present or becomes present; and performing an assessment of the multivalent binding response to determine the presence or amount of the selected analyte.

[0054] In addition, the methods and kits of the present invention provide a method for monitoring or testing the fitness status of a live mammal. The method includes selecting an analyte relevant to a fitness status parameter of interest and thereafter constructing an affinity reagent comprising at least one Fc.ε.R1 receptor binding domain and at least one additional moiety capable of binding the selected analyte, wherein the resulting reagent is capable of binding both mast cells and the analyte in any order; contacting a plurality of endogenous mast cells present within the target tissue of the mammal with the reagent to enable a multivalent binding response to occur in the presence or when the analyte becomes present; and performing an evaluation of the multivalent binding response to determine the presence or amount of the selected analyte. Additionally, the methods and kits of the present invention provide a method for monitoring or testing physiological changes in a live mammal due to exposure to environmental stress or injury, such as the release of endogenous biomarkers in a soldier involved in battlefield crossfire. The method includes selecting an analyte indicative of the physiological change to be detected, the selection of which should be a function of the environmental stress to which the test subject has been or will be exposed. The method also involves constructing an affinity reagent comprising at least one Fc.ε.R1 receptor binding domain and at least one additional moiety capable of binding the selected analyte, wherein the resulting reagent is capable of binding both mast cells and the selected analyte in any order; contacting a plurality of endogenous mast cells present within the target tissue of the mammal with the reagent to enable a multivalent binding response to occur in the presence or when the analyte becomes present; and performing an evaluation of the multivalent binding response to determine the presence or amount of the selected analyte. It should be understood that there are a variety of environmental stressors, including but not limited to temperature, fire, radiation, terrorist events, war, combat fatigue, and climate stressors, as well as extreme conditions of biological or chemical stressors, all of which can cause certain physiological changes.

[0055] The present invention also provides an affinity reagent for use in an in vivo bioassay for detecting an analyte, wherein the reagent comprises at least one Fc.ε.R1 receptor binding domain and at least one additional moiety capable of binding the analyte. The reagent is capable of binding both mast cells and the analyte in any order.

[0056] Finally, the present invention provides a kit for performing an in vivo bioassay to determine the presence, absence, or amount of an analyte in a live mammal. The kit generally includes an affinity reagent that has at least one Fc.ε.R1 receptor binding domain and at least one additional moiety capable of binding the analyte. The reagent is capable of binding both mast cells and the analyte in any order. The kit also includes a delivery device for delivering the affinity reagent to the target tissue of the mammal. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG. 1 shows a schematic diagram of various affinity reagents to further illustrate the present invention.

[0058] Figure 2 illustrates the basic method of delivering an affinity reagent of the present invention to a target tissue and subsequently detecting a multivalent binding response. Detailed Description

[0059] Although the methods and affinity reagents of the present invention may differ from each other in some aspects, the general methods and procedural steps set forth herein are all designed to determine the presence, absence, or amount of an analyte of interest in a mammalian subject. First, those skilled in the art should understand that, in order to practice the methods and bioassay kits of the present invention, an affinity reagent (AR) must be constructed based on the analyte of interest, as further explained herein. Such analytes will broadly include a wide spectrum of substances and are intended to include chemicals, peptides, proteins, lipids, carbohydrates, glycoproteins, nucleic acid sequences, or combinations thereof, as illustrated by, but not limited to, such substances as antibodies, hormones, receptors, receptor-binding molecules, antigens, drugs, poisons, toxins, pathogens, biomarkers, biochemicals, cell surface markers, RNA, DNA, and other molecules of interest.

[0060] It should be understood that an appropriate analyte of interest must be selected as an indicator for the detection and sentinel monitoring of diseases or disease states and certain environmental and conflict-based exposures that may result from biological warfare and terrorist events. Similarly, selecting an appropriate analyte of interest is also important for determining the absence, presence, or amount of substances such as hormones, growth factors, metabolites, etc., which serve as indicators of the overall health, fitness, or mood and / or physical state of a subject.

[0061] Typically, diseases and disease states and exposure to undesired substances are generally marked by the presence of some associated analyte, the presence or absence of which can be used as an indicator of that analyte. For example, such an analyte can be a pathogen, a component of a pathogen, or a byproduct produced by a pathogen. Such an analyte can also be a biomarker indicative of the presence of a pathogen in a subject to be screened or monitored. Alternatively, such an analyte can be endogenously released by the subject's cells in direct or indirect response to the presence of a pathogen or an acute or chronic disease state such as TB. Such an analyte can also be present due to certain genetic pathologies or latent conditions that develop over time. There are also analytes that can be used to indicate the physical health, emotional well-being, or fitness status of a subject. Exposure of a subject to certain chemicals and other substances can also result in the presence or absence of certain analytes in the subject, whether it is the substances themselves or some analytes endogenously produced and released in the subject in response to such conditions or exposure. Thus, one of ordinary skill in the art should understand that when practicing the present invention, the selection of the appropriate analyte or analytes will depend on the purpose of the test sought to be performed. One of ordinary skill in the art should also understand that there are a large number of readily available and well-known resources in the fields of physiology, medicine, epidemiology, and related health sciences that can be used as a guide when attempting to select the appropriate analyte.

[0062] As a footnote to the foregoing, one of ordinary skill in the art should understand that in the case where the present invention is to be used for sentinel monitoring, it is intended that when the AR delivered to the subject will react with mast cells in the target tissue at the site of administration of the affinity reagent, crosslinking across the mast cell membrane will not occur unless an analyte is present. Once the AR binds to the Fc.ε.R1 receptor on the mast cell, the mast cell is "sensitized" to the analyte and incorporated by the present invention to signal the presence of the analyte in the subject, which is typically based on some event, such as the subject's exposure to a pathogen, toxin, or biological warfare agent, or a change in the subject's physiology, such as a biomarker that is normally absent or actively circulating or only circulating at negligible levels in the blood, interstitial fluid, or lymphatic system. When the analyte of interest in the subject's body fluid reaches the AR delivery site, analyte binding to the AR occurs. This induces a multivalent binding response.

[0063] Once the appropriate analyte has been selected, an affinity reagent (AR) for binding such an analyte should be constructed. As will be understood from the following discussion, the analyte specificity of the test is determined by the appended moiety. Nevertheless, it will be beneficial for one of ordinary skill in the art to be more familiar with the relevant terms and the underlying mechanisms upon which the present invention is based before participating in the construction of the reagent. Thus, for the convenience of the reader, the following sections are provided, although the information can be readily determined from a large number of currently available and well-known resources in the fields of immunology, molecular biology, and biochemistry.

[0064] In immediate (type 1) hypersensitivity, B cells are stimulated (by CD4+ TH2 cells) to produce IgE antibodies specific for antigens such as typical allergens and certain parasites. The difference between a normal infectious immune response and type 1 hypersensitivity is that in type 1 hypersensitivity, the antibody is IgE, rather than IgA, IgG, or IgM produced by B cells. The IgE antibody binds to a substance called the Fc.ε.R1 receptor on the surface of tissue mast cells. This receptor is sometimes referred to as the "high affinity" mast cell receptor. Once these mast cells bind to the IgE antibody, the mast cells are considered to be "sensitized". Subsequent exposure of the sensitized mast cells to the same antigen induces cross-linking of the IgE bound to these sensitized mast cells, leading to allergic degranulation. This degranulation is characterized by the immediate and explosive release of pharmacologically active preformed mediators from storage granules present in the mast cells, as well as the simultaneous synthesis of inflammatory lipid mediators from arachidonic acid. Some of these mediators include histamine, leukotrienes (LTC4 and LTD4), and prostaglandins, which act on proteins (e.g., G protein-coupled receptors) located on surrounding tissues. The main actions of these products are vasodilation and smooth muscle contraction. Typically, a wheal (edema)-and-flare (erythema) response appears at the site of the surrounding tissue, which is commonly referred to as urticaria. As described in the Summary of the Invention of the present invention, the term "mast cell" is intended to refer to any cell that possesses the Fc.ε.R1 receptor on its plasma membrane, an example being basophils and eosinophils to the extent that they possess this Fc.ε.R1 receptor.

[0065] More specifically, the plasma membrane of mast cells is endowed with receptors that bind to the Fc portion of IgE molecules at a site called the "Fc.ε.R1 receptor-binding domain". These receptors bind cyclic IgE with very high affinity and hold it at the mast cell surface for long periods of time. Activation of mast cells is produced by crosslinking between bound IgE molecules, which induces fusion of granules with the cell surface membrane. This results in exocytosis of granule contents and the initiation of an immunological cascade of events typically associated with allergy. (Aalberse, R.C., Journal of Allergy and Clinical Immunology 106:228 (2000); Al-Muhsen et al., Canadian Medical Association Journal 168(10):1279 (2003); Scholl et al., The Journal of Immunology 175:6645 (2005); Handlogten et al., The Journal of Immunology 192:2035 (2014); Handlogten et al., Chemistry & Biology 21(20):1445 (2014); Matsuo et al., Allergology International 64:332 (2015)).

[0066] In humans and other mammals, mast cells are found directly beneath the epithelial surfaces of the skin and mucosa at the highest concentrations. Their location at the host-environment interface suggests a central role for these cells in immune surveillance, which has been supported by numerous studies examining the response of mast cells to various pathogens. One of the earliest observations of mast cells, made by their discoverer Paul Ehrlich, was that they often adopt a perivascular location within tissues. Mast cells are also in close proximity to lymphatic vessels in connective tissue (Ovary, Z., Japanese Journal of Allergology 43 1375 (1994); Turner et al., Nature 402B24 (1999); Marcelino da Silva et al., Journal of Histochemistry and Cytochemistry 62 698 (2014)).

[0067] Constructing affinity reagents

[0068] The affinity reagent constructs for the methods and biometric assay kits of the present invention comprise at least one Fc.ε.R1 receptor binding domain and at least one additional moiety selected and shaped to specifically bind to an analyte of interest. As described above, the additional moiety or "analyte binding moiety" determines the analyte binding specificity of the present invention, while the Fc.ε.R1 receptor binding domain binds the AR to the mast cell Fc.ε.R1 receptor. As described above in the Summary of the Invention of the present invention, this binary binding may also be referred to as "multivalent binding" and is responsible for the process herein referred to as "multivalent binding response" that occurs in the target tissue. The multivalent binding response occurs in the target tissue, starting from the cross-linking at the Fc.ε.R1 receptor present on mast cells, and results in mast cell activation and other physiological changes or responses in the test subject that can be detected or measured.

[0069] Generally, any molecule having analyte binding specificity for an analyte of interest can be shaped to be incorporated as an additional moiety into the reagent construct during the construction of the affinity reagent of the present invention. For the purposes of this disclosure, the terms "shaped" or "shaped to" are intended to refer to the preparation of an analyte binding moiety for incorporation into the reagent construct of the present invention and include, but are not limited to, removing any foreign materials that are unnecessary for incorporating the binding moiety into the reagent construct or for binding to the analyte of interest, so as to fully retain the desired analyte binding specificity of the moiety. For example, a protein antigen may only require the molecular portion (i.e., short amino acid sequence) necessary for the AR to bind the antibody of interest. Limiting unnecessary materials can generally help reduce the cross-reactivity of the AR. Shaping or shaping to may also include adding materials that are not normally present on the analyte binding moiety but are necessary for constructing the AR. For example, medicinal chemistry conjugation to the F1.ε.R1 binding domain may require adding a linker to the drug. Some examples of suitable types of additional moieties for constructing the affinity reagent of the present invention include antibodies, recombinant antibodies, engineered antibodies, antibody fragments, synthetic antibodies, engineered non-antibody binding proteins, antigens, chimeric molecules, fusion proteins, aptamers, hormones, receptors, receptor binding molecules, drugs, poisons, toxins, pathogens, pathogen components, biomarkers, cell surface markers, ligands, RNA and DNA, and biochemicals and chemicals of specific binding substances. Those skilled in the art should understand that the selected moieties will be compatible in type and specificity to bind the analyte of interest.

[0070] Turning now to the drawings, Figure 1AAn AR molecule 1 of a homogeneous group is shown, both having an antibody F(ab)2 fragment 2 and an Fc-ε-R1 receptor binding domain 3. The antibody F(ab)2 fragment 2 is shaped to serve as an additional part, and the Fc-ε-R1 receptor binding domain 3 is shown as binding to the mast cell Fc-ε-R1 receptor 4. Those skilled in the art should readily understand that each arm of the antigen-binding fragment (Fab)2 2 is capable of binding the same epitope 5 on the antigen 6. Mast cell activation is accomplished by the analyte binding to at least two of the AR molecules that bind to mast cells. Figure 1B An AR molecule of a heterogeneous group is shown, where the first AR molecule 7 and the second AR molecule 8 each have an (Fab)2 fragment that binds to a first epitope 5 and a different second epitope 9 on the antigen 6.

[0071] Alternatively, Figure 1C An AR molecule 10 of a homogeneous group is shown, both containing an Fc-ε-R1 receptor binding domain 3 and an antigen molecule 11 that is shaped to serve as an additional binding part. The analyte of interest is an antibody 12. Mast cell activation is accomplished by the (Fab)2 region of the antibody binding to at least two of the AR molecules that bind to mast cells upon crosslinking.

[0072] Figure 1D An AR molecule 13 is shown, which contains an Fc-ε-R1 receptor binding domain 3 and a single-stranded DNA or RNA molecule 14 that is shaped to serve as an additional binding part. In this example, the AR is constructed by covalently attaching streptavidin 15 to the Fc-ε-R1 receptor binding domain 3 and then allowing it to react with a biotinylated 16 nucleic acid molecule 17, thereby providing the AR molecule. The streptavidin-biotin forms one of the strongest known non-covalent bonds and is commonly used for the purpose of attaching one molecule to another. Those skilled in the art should understand that the streptavidin-biotin model is merely exemplary, and various other attachment methods are applicable to the present invention. It should also be understood that the method of envisioning incorporation into the AR as exemplified herein is designed to detect circulating DNA or RNA 18 having a nucleic acid sequence complementary to the DNA or RNA of the AR molecule.

[0073] In Figure 1E an AR molecule 19 is shown as containing an Fc-ε-R1 receptor binding domain 3 and a TC cell receptor binding protein 20 that serves as an additional binding part. When a T cell 21 is in close proximity to a sensitized mast cell, the T cell binds to the AR through a cell surface receptor 22 and activation occurs upon crosslinking. Similar to Figure 1EIn the examples shown, viruses and bacteria recognize their hosts by binding to specific receptors on the surface of host cells through receptor-binding molecules. ARs containing receptor-binding molecules as appendages can also be used to directly detect viruses or bacteria. For example, AR molecules in which the analyte-binding portion consists of the CD4 or CD48 (or TLR2) receptor can be used to detect HIV virus or Mycobacterium tuberculosis bacteria, respectively. Similarly, AR molecules in which the analyte-binding portion consists of the angiotensin-converting enzyme 2 (ACE2) receptor (active or inactive) can be used to detect SARS-CoV-2 virus.

[0074] Importantly, the figures are merely exemplary of a limited number of embodiments for constructing the affinity reagents of the present invention. The analyte-binding portion of the present invention can, for example, comprise peptides, proteins, nucleic acids, lipids, carbohydrates, or any combination thereof. Additionally, the AR can be homogeneous, where the AR molecules delivered to the target tissue consist of the same AR molecules; or a heterogeneous combination of AR molecules that recognize different binding sites on the analyte of interest. Alternatively, the analyte-binding portion of the AR can be monospecific, requiring the analyte to possess two or more identical epitopes or binding sites for crosslinking. Alternatively, the binding portion can be bispecific, multispecific, or a mixed population of ARs that allow crosslinking by binding to similar and dissimilar epitopes or binding sites on the analyte of interest. Importantly, it should be readily understood by those skilled in the art that the AR libraries for each analyte can be constructed in a multitude of ways to optimize the performance of the assay and identify analyte variants or isotypes.

[0075] There are numerous available in vitro techniques and recombinant methods that can be used by those skilled in the art to construct the Fc.ε.R1 receptor-binding domain and the analyte-binding portion to form an AR as contemplated by the present invention. Examples of these in vitro techniques and recombinant methods include hybridoma technology, covalent conjugation, non-covalent binding, genetic engineering methods, recombinant techniques, or combinations thereof. For example, genetic engineering methods use recombinant DNA methods to form proteins that are not normally produced by cells and provide a means for the large-scale expression of these proteins in various expression vectors. For the purposes of the present invention, the terms "in vitro construction" and "constructed in vitro" are intended to have the ordinary and customary meanings ascribed to them by those skilled in the art. Similarly, the terms "recombinant construction" and "constructed recombinantly" are also intended to have the ordinary and customary meanings ascribed to them by those skilled in the art.

[0076] Additionally, those skilled in the art will note that the Fc.ε.R1 receptor-binding domain of human IgE, which includes the C2, C3, and C4 regions, is sufficient to bind to the alpha chain of the high-affinity receptor (Fc-ε-R1) mast cell receptor. Studies with the human IgE Fc domain have demonstrated that the minimal fragment showing Fc-ε-R1 binding activity spans amino acids 329 to 547 and lacks the entire C2 domain. In addition, studies have also shown that the Fc fragment 315-547 is an S-S-linked dimer, and Fc fragments 329-547 that form dimers without S-S bonding all bind to the Fc-ε-R1 receptor with high affinity. The presence of N-linked sugars does not appear to be necessary for high-affinity binding (Basu et al., The Journal of Biological Chemistry 268:13118 (1993)). The Fc.ε.R1 receptor-binding domain can be purified directly from proteolytic digests of IgE molecules or can be produced by recombinant methods. The Fc.ε.R1 receptor-binding domain from IgE has been recombinantly produced and purified from various expression vectors (Kamiya et al., Exp. Med. 180:297 (1996); Liu et al., Proceedings of the National Academy of Sciences of the United States of America 81:5369 (1984)). Accordingly, construction of the Fc.ε.R1 receptor domain of AR will generally include the C2, C3, and C4 regions. In alternative embodiments, such construction will include at least the C3 and C4 regions and minimally include amino acids 329 to 547.

[0077] Those skilled in the art will appreciate that Fc.ε.R1 receptor-binding domains derived from or modified from any species, recombinantly synthesized, or otherwise constructed will generally be sufficient to bind to the mammalian F1.ε.R1 mast cell receptor. Thus, the methods and kits of the present invention can be readily modified to test any mammal possessing mast cells, examples of which include humans, dogs, cats, horses, farm animals, and livestock, provided that the design and adaptation of the methods and kits for each species incorporate appropriate considerations regarding sensitivity, cross-reactivity, etc. between the different species being tested. To this end, there are various humanization protocols available to address those situations where portions derived from another species may be modified for use in humans.

[0078] As described above, one embodiment of the present invention includes an antibody or antibody fragment that can be shaped to serve as an attachment moiety. Those skilled in the art should readily understand that antibodies contemplated for use in the present invention in the art can be shaped using standardized tools and techniques readily available for the design and development of research products, diagnostics, and therapies. Generally, antibody molecules can be engineered to include monospecific, bispecific, or trispecific antigen-binding domains and further manipulated to provide non-humanized, humanized, or chimeric molecules. Whether used in whole or in part, such antibodies exemplify suitable analyte-binding moieties that can be shaped for use in AR molecules. Over the past 30 years, antibodies have been initially cleaved by proteolysis and subsequently by genetic engineering into smaller antigen-binding fragments to generate monovalent or multivalent fragments, including but not limited to Fab, F(ab)2, monospecific Fab2, bispecific Fab2, trispecific Fab3 fragments. Antibody fragments (e.g., single-chain variable fragments (scFv) and V.sub.HH domains) and artificial affinity binders (e.g., affibodies, antibody mimetics (Monobodies), DARPins, etc.) have been generated and developed by screening large gene libraries of potential binders with various selection techniques. Such antibody fragments also create suitable analyte-binding moieties that can be shaped for use in AR molecules.

[0079] Additionally, various techniques have provided for the development of a large number of protein scaffolds with unique affinity interaction domains that bind to target epitopes (Groff et al., Biotechnology Advances 33:1787 (2015); Marx, V., Nature Methods 10(9):829 (2013)). Antibody-antigen binding diversity can also be accessed through unorthodox mechanisms, which represent a third layer of immunoglobulin repertoire diversification beyond the variability introduced by recombination and mutagenesis (as described by Kanyavuz et al., Nature Reviews, Immunology 19:355 DOI:org / 10.1038 / s41577-019-0126-7 (2019)). Additionally, additional specificities can be accessed by using phage display technology, which has been widely used to generate large libraries of antibody fragments by exploiting the ability of phages to express and display biofunctional protein molecules on their surface. Combinatorial libraries of antibodies have been generated in the λ phage expression system, which can be screened as plaques or as colonies of lysogens (Marks et al., Biotechnology 10 779 (1992)). Thus, any of the foregoing or their derivatives will constitute suitable analyte-binding moieties for constructing the affinity reagents of the present invention.

[0080] Aptamers are similar to antibodies in that they can bind to proteins and modulate their functions, and because of their synthetic production, they are often referred to as chemical antibodies (Groff et al., Biotechnol. Prog. 33:1787 (2015); Marx, V., Nat. Methods 10:829 (2013)). Aptamers also make suitable analyte-binding moieties for AR molecules. Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with high specificity and affinity through van der Waals forces, hydrogen bonds, salt bridges, and hydrophobic and other electrostatic interactions. Aptamers have the ability to fold into complex and stable three-dimensional shapes, which allows them to fold within or around their targets. DNA aptamers have greater chemical stability, while RNA aptamers generate more structural shapes due to their greater flexibility. Aptamers have been identified for targets that include small organic and inorganic molecules such as dyes, nucleotides, amino acids, and drugs; biopolymers such as peptides, proteins, and polysaccharides; ions; phospholipids; nucleic acids; viruses; bacteria; cell fragments; and whole cells. Thus, aptamers are additional examples of analyte-binding moieties that can be incorporated into the ARs of the present invention.

[0081] There are multiple molecules that can participate in molecular binding and include proteins, nucleic acids, carbohydrates, lipids, and small organic molecules such as drugs. Types of complexes formed due to molecular binding include: protein-protein, protein-DNA, protein-hormone, and protein-drug. These include proteins that form stable complexes with other molecules. Cellular receptors are structures composed of proteins that receive and transduce signals, which are typically integrated into biological systems. These signals are usually chemical messengers that bind to the receptor and cause some form of cell / tissue response, such as a change in cell electrical activity. Molecules that bind to receptors are commonly referred to as ligands. Ligands can be proteins or peptides, or other small molecules such as hormones, drugs, toxins, or parts external to viruses or microorganisms. Both receptors and receptor ligands are additional examples of analyte-binding portions that can be incorporated into the AR molecules by those skilled in the art. Circulating nucleic acids (CNA) have been reported in a variety of clinical conditions such as cancer, stroke, trauma, myocardial infarction, autoimmune disorders, and pregnancy-related complications. The term CNA refers to segments of genomic, mitochondrial, or viral DNA, RNA, and microRNA (miRNA) found in the bloodstream. Scientists have now discovered disease-specific genetic aberrations, such as mutations, microsatellite alterations, epigenetic regulation (including abnormal methylation), and viral DNA / RNA from nucleic acids in plasma and serum. CNA has received particular attention due to its potential application as a non-invasive, rapid, and sensitive tool for the molecular diagnosis and monitoring of acute pathologies and for the prenatal diagnosis of fetal genetic diseases (Suraj et al., Biomedical Reports 6:8 (2017)). Adapting CNA complementary sequences associated with diseases and disease states would provide yet another analyte-binding portion for constructing the AR of the present invention.

[0082] The above analyte-binding portions can be shaped and incorporated into the AR of the present invention by a variety of methods known in the art, including covalent conjugation and non-covalent binding methods. Additionally, molecular techniques can be used to construct and generate AR molecules as fusion proteins, where the nucleic acid sequences of both the Fc.ε.R1 receptor-binding domain and the analyte-binding portion are recombinantly ligated and inserted into an expression vector. A fusion protein or chimeric protein (literally, made up of parts from different sources) is a protein produced by ligating two or more genes originally encoding for different proteins. Translation of this fusion gene produces a single or multiple polypeptides with functional properties derived from each of the original proteins. Recombinant fusion or chimeric proteins are artificially produced by recombinant DNA technology for biological research or therapy. The term chimeric or chimera generally refers to a hybrid protein made up of polypeptides with different functions or physicochemical patterns. Thus, both fusion proteins and other chimeric molecules also provide suitable options to be incorporated as additional binding portions of the present invention.

[0083] Additionally, monoclonal and polyclonal IgG, IgM, IgA, and IgD antibodies can of course be sequenced and recombinantly produced, thus providing simple routes for isotype switching from Fcγ, Fcμ, Fcα, and Fcδ to Fcε, respectively. AR molecules can also be generated by in vitro methods using hybridoma technology, thus essentially producing IgE molecules having an Fc.ε.R1 binding domain and an F(ab)2 region specific for the analyte of interest. These techniques are well known in the art. Pathogens sometimes recognize host cells through receptor-binding proteins such as CD4, CD48, ACE2, or TLR2. These receptors and other pathogen components can be used as analyte-binding moieties on affinity reagents to assess disease states. For example, the number of CD4 cells is routinely measured in HIV patients as an indicator of advanced HIV disease.

[0084] Based on the foregoing, one of ordinary skill in the art should readily understand that one AR can be readily modified and used to replace another AR to produce different kit embodiments. Therefore, it should also be understood that the standardized manufacturing SOPs of the present invention can be rapidly modified to allow for the rapid production of new AR molecules and novel bioassay kits, thus providing a broad platform for diagnostic testing. New AR bioassays can be carried out through a similar continuous product development cycle for rapid and large-scale deployment in foreseeable and unforeseeable healthcare emergencies. Patient implementation procedures can also be standardized, thus reducing the uncertainties typically associated with the introduction of new methods.

[0085] Contacting mast cells with an AR

[0086] As described above, the methods and kits of the invention further include the step of sensitizing mast cells by contacting an affinity reagent in vivo with a plurality of endogenous mammalian mast cells via delivery of an AR into mast cells present within a target tissue of a live mammalian subject to initiate and enable a multivalent binding response in the presence or upon becoming present of an analyte of interest; one skilled in the art will appreciate that such delivery also contemplates selecting an anatomical region and tissue type having a certain concentration of endogenous mast cells in the test subject, which concentration ranges from about 125 to 20,000 mast cells per cubic millimeter for the mammalian species to which the delivery is to be performed. For example, in most mammals, the skin is an acceptable target tissue, and more specifically, the upper dermis of the skin. In humans, delivery sites include the skin of the upper arm or forearm and the lower limbs, at a depth and in a manner substantially the same as a typical allergy prick test or tuberculin skin test, the details of which are well known and established in the clinical art. Although as described above, any tissue in which the affinity reagent is exposed to about 125 to 20,000 mast cells per cubic millimeter is generally suitable, provided that the method for detecting the multivalent binding response can be effectively utilized and is reasonably practical for clinical purposes. Physical delivery of the affinity reagent can be performed by any suitable means, including but not limited to acupuncture, intradermal injection, solid needles, hollow needles, microneedles, patch methods, or needleless systems. In a simple embodiment, the AR is delivered into the skin by a needle. The needle can be solid or hollow. The AR is injected into the skin by a hollow needle, or the needle can be solid and coated with an AR coating, where the AR is released from the needle when the needle contacts body fluid.

[0087] For example, Figure 2A Shown is a multi-tipped applicator 23 having four solid needles 24a to 24d, which solid needles can be coated with four different AR molecules in a multiplex format, as further discussed below. By way of example only, one needle can be used for a positive control 24a. Another needle can be used for a negative control 24b. Yet another needle can be coated with an AR that recognizes an analyte 24c, while another needle can be coated with an AR that recognizes a different analyte 24d. The positive control can be, for example, an AR molecule having an analyte-binding moiety specific for serum albumin or an endogenous molecule that is always present systemically and circulates continuously in the subject being tested. Additionally, the positive control can include histamine to ensure that the patient has an effective wheal and flare / erythema response. The negative control can be, for example, an AR molecule that exhibits binding activity to any substance not present in the species being tested. Figure 2B Shown is the delivery device or applicator 23 being applied to the skin and penetrating the skin, rehydrating and releasing the AR molecules coated on the delivery end of the device, and then the AR molecules can freely bind in situ to mast cells in addition to the circulating analyte present in the target tissue. As Figure 2CAs shown, the patient tested positive only for one analyte and the positive control. The negative control and one analyte were negative, with no visible response. The positive control and the other analyte were positive, showing two wheals and a flushing reaction. In the above example, the described assay method is multiplexed. A multiplex assay measures multiple analytes in a single experiment. There are many ways to position or label multiplex tests to track and confirm the location of each analyte or control.

[0088] Where applicable, the tests, together with the positive and negative controls, can be applied together or separately, and in any order and at any time. For example, when using a commercially available or custom-designed allergy skin test applicator, a single applicator with a single tip (which includes one or more lancet members to penetrate the skin) can be used to apply the tests, as well as the positive and negative controls, in the desired order. Similarly, a dual-tip applicator (where each tip has one or more lancet members) can be used to apply the tests and controls in parallel. In addition, a multi-tip applicator (where each tip has one or more lancet members) can be used to apply various tests and controls in parallel. Each tip can also have a touch activator to reduce pain, which is well known to practitioners using allergy test devices in practice.

[0089] The multiplex use of the present invention provides a way to detect the characteristics of active diseases, thus preventing unnecessary tests and treatments. For example, the present invention can be used to simultaneously detect viral particles, DNA, RNA, and other components of a virus, rather than detecting antibodies in patients who have been previously exposed to HBV and have cleared the virus, without the need to collect multiple samples to prove the presence of an active disease. (Liang, T.J., Hepatology 49(5 Suppl): S13-S21 (2009); Gerlich, W.H., Virology Journal 10(239) http: / / www.virologyj.com / content / 10 / l / 239 (2013); Venkatakrishnan et al., Annu. Rev. Virol. 3:451 (2016); Jiang et al., Journal of Virology 90(7):3330 (2016); Hu et al., Viruses 9 56 (2017)).

[0090] As shown below, in the information from the Centers for Disease Control in Atlanta in Table 1, the use of multiplexed applications of AR to simultaneously detect viral particles (HBsAg) and antibodies (HbsAb and HBcAb) can provide a definitive diagnosis and treatment plan that is not currently commercially available for hepatitis B patients (Diepolder et al., Gastroenterology 116:650 (1999); Kimura et al., Journal of Clinical Microbiology 41(5):1901 (2003); Tong, S., Int. J. Med. Sci. 2(1):2 (2005); y et al., Journal of Clinical Microbiology 44:2321 (2006); Gallagher et al., Virology 502:176 (2017)).

[0091] Table 1. Hepatitis B Test Panel

[0092] Explanation and Required Actions HBsAg HBsAg HBcAb Unimmunized - Vaccinate Negative Negative Negative Immunized - Protected Negative Positive Positive Immunized - Protected Negative Positive Negative Infected - More Tests Needed Positive Negative Positive Possibly Infected - More Tests Needed Negative Negative Positive

[0093] As discussed above, AR can be delivered in situ, either alone or multiplexed, to areas of the skin (or other suitable mast cell-rich areas) by acupuncture or intradermal injection or patch methods (including needle-free systems). Recent advances in intradermal delivery needle design have reduced the pain associated with injection. Smaller gauge and sharper needles reduce tissue damage and thus the amount of inflammatory mediators released (see references Zehrung et al., "Intradermal Delivery of Vaccines: A Literature Review and Potential for Use in Low- and Middle-Income Countries" PATH August 27, 2009; Ita, K., Pharmaceutics 7:90 (2015); Larraneta et al., Materials Science and Engineering R 104:1 (2016); Martin et al., Safety 3:25 DOI:10.3390 / safety3040025 (2017); Shrestha et al., Scientific Reports 8:13749 DOI:10.1038 / s41598-018-32026-9 (2018)).

[0094] Microneedles typically have a width of less than 0.2 mm and a length of less than 2 mm. They are usually made of silicon, plastic, or metal and can be hollow for delivering or sampling substances through a lumen, or the needles can be solid and coated with a substance. By choosing an appropriate needle length, the penetration depth of the microneedles can be controlled to avoid the cutaneous peripheral nerve network and reduce or eliminate the sensation of pain. The extremely small diameter and sharpness of the microneedles also contribute to reducing the sensation during injection.

[0095] For some time, the advantages of needle-free injection devices have been recognized. Some of the advantages of needle-free devices and methods include the absence of needles that can startle patients and pose a hazard to healthcare providers. The diameter of the injection jet generated by a needle-free device is typically smaller than that of a subcutaneous injection needle, and thus, in some cases, needle-free injection is less painful than an injection provided by a subcutaneous injection needle device. Because of these and other advantages of needle-free injection, many variants of pneumatically, electronically, or spring-actuated needle-free injection devices have been designed to deliver a single injection or alternatively a series of injections to one or more patients. Most known needle-free injection devices operate by driving an injectable liquid through a nozzle with a power piston to generate a fine, high-pressure liquid jet that penetrates the skin.

[0096] Additionally, there are prefillable delivery devices available for administering intradermal injections, which include a prefillable container adapted to store a substance. The substance is expelled through a needle cannula that has a front tip adapted to administer an intradermal injection. A restrictor surrounds the needle cannula and has a generally flat skin-engaging surface that extends in a plane generally perpendicular to the needle cannula. An insert is centered within the skin-engaging surface and can be pierced by the front tip. A sleeve having a first end and a second end surrounds the prefillable container. The restrictor is inserted through the first end, and the second end is attached to a depressible plunger for expelling the substance from the container. The restrictor can move between a first position and a second position to expose the front tip and can selectively move between a third position and a fourth position to hide the front tip. These and other devices can be used for painless and safe intradermal application of substances, and the methods described and other methods are well known to those skilled in the art.

[0097] Those skilled in the art should understand that AR can be applied alone or in combination with any of several well-known drug carriers that are suitable as vehicles for adding ingredients that can be used to affect AR binding kinetics, promote chemotaxis, or elicit an evaluable and measurable substance from nearby cells. The vehicle can be added simultaneously with AR or separately.

[0098] It should also be understood that there are currently a variety of techniques in the clinical laboratory and medical manufacturing fields that can be used to provide a suitable vehicle to carry the AR to the target tissue or deposit it on the delivery end of the device until the AR is delivered to the target tissue using the device. In one embodiment, the AR can be suspended in a liquid medium of suitable viscosity that is used, for example, to deposit the AR on the delivery end of the device such as the tip of a solid needle, and then dried to deposit an AR layer on the device until it is used on a subject. Alternatively, the AR can be delivered to the target tissue, for example, in a liquid medium that is delivered through a hollow needle or a needleless system and injected into the target tissue. The selection of such a medium should incorporate factors such as storage conditions and shelf life. A variety of suitable substances are commercially available for use as coating vehicles or media for carrying or depositing the AR, provided that they are correctly selected for the AR to be delivered, the details of which are further elaborated below.

[0099] The vehicle or medium for application, transport, storage, use of the AR, etc. can depend on the AR construct itself. The vehicle can be liquid or dry and can include buffers, salts, protease or nuclease inhibitors, bacteriostatic agents, chemotactic agents, detergents, stabilizers, etc., or can consist of proprietary reagents provided by third-party suppliers. The list of additives is extensive and well-known to those skilled in the art. The AR is delivered in vivo and thus, once delivered, resides in body fluids. It optimally performs within a physiological pH range of 6 to 9.

[0100] As described above, the AR can be applied alone without additional components or in a vehicle suitable for adding components that can be added to affect AR binding kinetics, promote chemotaxis, or can elicit substances from nearby cells that can be measured as contemplated by the methods of the present invention. In addition, the vehicle for delivery can be optimized to disrupt the balance between the endogenous pre-existing IgE bound to mast cells and the newly available AR molecules in order to provide a method for increasing the concentration of AR molecules on mast cells. The shift in the AR binding equilibrium can be accomplished by various methods known in the art. For example, adding an excess of AR, changing the pH, changing the salt concentration, and adding detergents can be used for equilibrium shifting.

[0101] The delivery step of the method of the present invention may also include a vehicle consisting of chemotactic or other compositions that attract cells of interest to the target tissue. While theoretically the multivalent binding response can be localized to many body regions, the most common sites will consist of AR-sensitized mast cells located in the interstitial fluid of the target tissue. This provides an opportunity to detect cells expressing specific cell surface markers such as different immune cell populations. Detection or measurement of receptor-binding ligands such as LAG3 cleavage, CD8 / regulatory T cell ratio, cytokines, chemokines can be used to select and manage patients receiving immunotherapy. Chemokines can be introduced simultaneously or synchronously with the AR to attract cells of interest to the target tissue.

[0102] The delivery vehicle may also allow the method of the present invention to initiate and detect analytes secreted or released from surrounding cells. Delivery of the AR may optionally include a vehicle consisting of components that cause the release of analytes from adjacent cells, such as T lymphocytes known to be physically close to endogenous mast cells. ARs specific for analytes released from T cells will detect their presence. For example, the interferon gamma release assay (IGRA) is an in vitro test used to diagnose some infectious diseases, particularly tuberculosis. The interferon gamma (IFN-γ) release assay relies on the fact that when exposed to specific Mycobacterium tuberculosis (MTB) antigens, T lymphocytes will release IFN-γ. When patient samples are mixed with certain MTB antigens, T cells from most people infected with MTB will release IFN-γ. Therefore, in addition to ARs specific for IFN-γ, MTB antigens can also be delivered to the target tissue. MTB antigens included in the AR delivery medium will stimulate the release of IFN-γ from adjacent pre-exposed T cells. Then, methods in which the AR binds to IFN-γ to trigger mast cell degranulation can be used to detect IFN-γ. The presence of IFN-γ is associated with MTB infection. Additionally, the methods and kits of the present invention reduce the complexity of tests similar to the IGRA test, but eliminate the need to draw patient blood samples and the necessity of performing the test on a bench top under tissue culture conditions.

[0103] In addition, there are a large number of molecules released from mast cells during and after threshing, any of which can be used to measure the response; alone, in combination, or through enhanced secondary reactions and cascades. Thus, in yet another embodiment of the present invention, the probe placed in the target tissue can remain there as a physicochemical detector. Biosensitive detectors can be generated using bioengineering techniques known in the art. The transducer or detector element deployed in the target tissue can be readily designed to transform a signal or change that occurs as a function of the multivalent binding response into a second signal for ease of measurement and quantification. Alternatively, the delivery device can be used to deliver the AR to the target tissue while thereafter remaining positioned to operate as a detector as discussed above.

[0104] Delivery of active molecules into or through the skin (transdermal delivery) can offer significant advantages compared to more conventional delivery routes. Use of chemical penetration enhancers such as dimethyl sulfoxide which interacts with skin lipids to facilitate transmembrane transport of the AR into the dermis of the skin.

[0105] Dyes that can be detected in the dermis of the skin visually or by means of a device include, but are not limited to, methylene blue, fluorescein, and fluorescent peptides and can be used to enhance the detection / evaluation of the multivalent binding response of the present invention. For example, a dye such as methylene blue can be included in the application vehicle to provide an indication that the affinity reagent has been correctly applied to the dermis of the skin. There are many conceivable adaptations where dyes or fluorescent compounds and other reagents that are visible to the naked eye or detectable by a device can be added to the application vehicle or the AR. Attachment of a dye (such as fluorescein) to the AR will allow monitoring of the positive reaction by an external fluorescence detector. Monitoring dyes can be used to determine parameters such as the mast cell density at the application site, the number of AR molecules occupying the mast cell Fc.ε.R1 receptor, and the reaction rate. Measurement of these and other parameters can be used alone or in combination to quantitatively or semi-quantitatively measure the analyte using the present invention.

[0106] Evaluating the multivalent binding response

[0107] The method of the present invention further includes performing an evaluation of the multivalent binding response to determine the presence, absence, or amount of an analyte of interest. It should be understood that such an evaluation of the binding response presumes sufficient time for the binding response to occur. The binding response time depends on the purpose or intended reason for performing the method and the concentration of the analyte in the target tissue at the time of delivery of the AR.

[0108] The multivalent binding response of the present invention is a function of the multivalent binding discussed above and encompasses cross-linking of the Fc.ε.R1 receptor across the mast cell membrane, as well as degranulation of the mast cell and any additional and secondary physiological changes in the target tissue directly or indirectly resulting from such degranulation. Evaluation of the multivalent binding response, if it occurs, can be accomplished by a variety of qualitative and quantitative methods, including performing a basic clinical evaluation of the target tissue at or near the delivery site by visually inspecting for at least one morphological change in the target tissue associated with the binding response (e.g., wheal and flare dermal responses). Alternatively, the tissue can be evaluated using a device capable of measuring at least one change in the target tissue associated with the binding response.

[0109] Because there are a large number of molecules released from mast cells during degranulation, in theory any of these molecules can be used to measure the response, either alone, in combination, or through enhanced secondary reactions and cascades. As such, evaluation of the performance of the binding response can be accomplished by measuring biochemical changes in the target tissue (such as mast cell degranulation substances released from mast cell degranulation) or by measuring endogenous substances released in response to mast cell degranulation substances. Alternatively, the wheal and flare response is a visible and easily measurable physiological tissue change. Devices can also be used to measure parameters associated with the wheal and flare response, such as width, density or intensity, stretching changes in the skin, or heat generated by the positive reaction. In addition, enhancement techniques such as secondary reagents and their conjugates provide additional secondary modes of signal enhancement and measurement.

[0110] A sensor or biosensor can also be used to evaluate the binding response through a transducer or similar device. As earlier mentioned in this disclosure, the probe can be placed near the AR after delivery to the target tissue and remain there as a physicochemical detector. The biocompatible element can be generated using techniques commonly known in the field of biomedical engineering and related fields. The transducer or detector element deployed in the target tissue can be easily designed to transform the signals or changes that occur due to mast cell degranulation. Three main techniques are based on their transduction mechanisms, namely, electrochemical, optical, and acoustic sensors. Electrochemical sensors are further classified into impedance, amperometric, potentiometric, and conductometric sensors. Optical sensors include fluorescence- and chemiluminescence-based biosensors, surface-enhanced Raman spectroscopy (SERS), and surface plasmon resonance (SPR)-based sensors. Acoustic wave-based sensors have two subclasses: quartz crystal microbalance (QCM)-based sensors and surface acoustic wave (SAW)-based sensors. A sensor reader device with an associated electronic device or signal processor will result in the display of the results in a user-friendly manner suitable for connection to networks, smartphones, and similar display and communication devices, as well as user interface mechanisms. Sensor technology is a rapidly evolving field, and many variations known to those skilled in the art can be used. The applicator can both introduce the AR into the mast cells and act as a detector element providing a single-step assay and result (Upasham et al., Advanced Health Care Technologies 4:1 https: / / doi.org / 10.2147 / AHCT.S138543 (2018); Turner et al., Oxford, UK: Oxford University Press p770 (1987); Banica et al., UK: John Wiley & Sons p576 ISBN97811*18354230 (2012); Dincer et al., Advanced materials 31(30) doi:10.1002 / adma.201806739 (2019)).

[0111] In cases where the method is used to test a population or group of individuals suspected of exposure to substances such as pathogens, toxins, etc., the expected binding response time will typically occur immediately or within a few seconds, provided that the test subject has been exposed to the substance and the analyte selected to label that exposure is circulating actively in the test subject. Generally, if the analyte is circulating actively, it can be expected to be found in the interstitial fluid at the AR delivery site within the target tissue. In contrast to the foregoing, there are cases where the method is used as a sentinel test to perform an ongoing monitoring function to detect future exposures, as further described in the following sections. As expected, the multivalent binding response does not occur until the subject has been exposed to the substance and the analyte selected to label that exposure has had time to circulate to the target tissue delivery site. In short, the evaluation of the multivalent binding response is secondary to providing sufficient opportunity for a detectable multivalent binding response to occur in the test subject. In cases where the binding response occurs immediately, the evaluation can then be performed immediately.

[0112] It should also be understood that the multivalent binding response time can also vary depending on the analyte of interest to be detected and / or the characteristics of the analyte binding moiety used for the reagent construct. The binding response time can be adjusted based on the construction and concentration of the AR, the vehicle used to bring the mast cells into contact with the AR, and the detection method. Despite the foregoing, sufficient assay times can generally be classified as follows: rapid assay methods range from immediate (T 1 = 0) to approximately 2 hours (T 2 = 2 hours); sensitive assay methods range from 2 hours (T 1 = 2 hours) to approximately 48 hours (T 2 = 48 hours); and the AR sentinel test will have a validity period ranging from approximately 12 weeks to approximately 6 months from the time of AR delivery to the target tissue.

[0113] In the following example section, depending on the concentration of HBsAg in the circulatory system, the binding response and the resulting tissue changes are in the range of 30 seconds to 8 hours. As an example of a sensitive method, mice challenged with 20 μg or more of HBsAg were positive after 20 minutes. However, mice challenged with 10 μg could only be detected after 8 hours. Unlike all blood assays, the bioassay performed by the method of the present invention exposes the assay continuously to the analyte as the analyte circulates through the body. This continuous exposure enables very low detection levels and confers significant sensitivity to the assay. Thus, it should be understood that bioassays incorporating the method of the present invention are generally not limited by the amount of analyte in the sample as in, for example, in vitro assays.

[0114] In additional embodiments, the methods and kits of the invention can be designed or adapted to monitor immunotherapy agents, immune function, disease states, and the effectiveness of treatments and therapies. While the AR can be delivered to target tissues residing in various regions of the body of the test recipient, optimal delivery sites will include those sites that are populated with endogenous mast cells located within the interstitial fluid of the target tissue. This provides the opportunity to detect cells expressing specific cell surface markers such as different immune cell populations. Detection or measurement of receptor-binding ligands such as LAG3 cleavage, CD8 / regulatory T cell ratio, cytokines, chemokines can be used to select and manage patients receiving immunotherapy. Chemokines can be introduced simultaneously or synchronously with the AR to attract cells of interest. As described above, the delivery medium can include chemotactic compositions or other compositions that will attract cells of interest to the vicinity of the AR once they are delivered.

[0115] In addition to the above, another embodiment of the invention provides a tool for probing interstitial fluid by providing methods for collecting key information present in interstitial fluid and lymph fluid, and for the development of specialized assays or treatments. It is nearly impossible to collect large volumes of interstitial fluid. The time-consuming nature of currently available procedures for removing and testing interstitial and lymph fluid poses risks to patients, and the need for medical experts and specialized equipment limits the use of these procedures to basic research. Interstitial fluid and lymph fluid contain unique biomarkers that can be used for detecting and diagnosing diseases and disease states, monitoring treatment and recovery, and for tracking the health of patients. By shaping the analyte-binding portion from appropriate biomarkers, the invention can be adapted to ideally accomplish these purposes.

[0116] In additional embodiments, the bioassays of the invention can be used for point-of-care and self-testing, where the AR bioassay is applied topically (or to other suitable areas) of the skin as a homogeneous assay, singly or multiplexed, by means of a needle prick or any intradermal injection method, including needleless systems. No blood sample is required, and the results can be read visually, by means of a device or a sensor, within a few minutes.

[0117] In yet another embodiment, the present invention can be used for the immediate measurement of troponin, providing a real-time measurement that will allow first responders to provide immediate and life-saving care to patients suffering from a potential myocardial infarction at the scene or en route. Such a troponin test can be designed for visual interpretation or for interpretation by a biosensor or other detection procedure. Additional aspects of this embodiment can include a transmitter having a delivery needle and / or sensor, the delivery needle and / or sensor being separate or formed integrally together, which is applied to the patient. The test results corresponding to the presence or amount of troponin present in the subject are sent in real time via the transmitter to a communication device such as a cellular phone, providing the ability to give immediate results and monitor results (if they change over time) to first responders or telemedicine personnel. Many tests that are considered STAT (where immediate results can affect the health of the patient) can be adapted to the present invention in this manner. The use of sensors and other means for detecting multivalent binding responses is discussed in more detail below.

[0118] An additional embodiment of the present invention relates to testing for Mycobacterium tuberculosis (Mtb), where the test directly detects Mtb-activated T cells. An affinity reagent is constructed that contains at least one Fc.ε.R1 receptor-binding domain and at least one MHC class I molecule. The MHC molecule portion of the affinity reagent is paired with an Mtb antigen to form a complex. The MHC molecule can be complexed with any suitable Mycobacterium tuberculosis antigen. Preferably, the antigen is selected from any one of the secreted ESAT-6 protein, the 10-kDa culture filtrate protein (CFP-10), or the proteins present in the PPD preparation (Hodapp, T. et al., European Respiratory Journal (Eur Respir J) 2012; 40:152-160. doi:10.1183 / 09031936.00175611; Urdahl, K.B. et al., Nature Reviews, Volume 4, Issue 3, May 2011). The affinity reagent is applied to the subject by any of the methods described herein. The affinity reagent binds to the T cell receptor on T cells that have been activated by the presence of Mtb, resulting in mast cell degranulation and a wheal and flare response corresponding to the presence of Mtb.

[0119] Another embodiment of the invention is a test for detecting superantigens. Superantigens are implicated in T cell-driven cytokine release syndrome (CRS) associated with multiple organ dysfunction (MOD). Superantigens bind directly to the TCR and MHC-II receptors outside of the conventional antigen-binding site and stimulate large numbers of T cells. Superantigens are implicated in multisystem inflammatory syndrome in children (MIS-C), COVID-19 cytokine storm syndrome, sepsis, toxic shock syndrome, chimeric antigen receptor T cell (CART) therapy, administration of certain T cell-activating antibodies, and immune checkpoint inhibitors. An affinity reagent is constructed that contains at least one Fc.ε.R1 receptor-binding domain and at least one MHC class II molecule. A second affinity reagent is constructed that has at least one Fc.ε.R1 receptor-binding domain and at least one T cell receptor (TCR) molecule. The paired affinity reagents are applied to the skin. The presence of a superantigen will result in crosslinking of the MHC and TCR portions of the affinity reagent, leading to mast cell degranulation and subsequently a measurable wheal and flare response. A biometric assay kit

[0120] Minimally, the biometric assay kit of the invention includes an AR and means for contacting in vivo mast cells with the AR. After reading the detailed description of the method of the invention, the use of the kit will be apparent to those skilled in the art. The kit can optionally include sensors or other means for determining the presence, absence, or amount of an analyte of interest. The means for contacting mast cells with the AR component of the kit can be any suitable means as described herein. The AR component of the kit can include any affinity reagent constructed according to the methods set forth herein. Also as described above, the AR component will be pre-coated onto or pre-loaded into a delivery device. It is contemplated herein that in some cases, the AR can be included in the kit in lyophilized form for reconstitution with a suitable sterile carrier medium and subsequently applied to the delivery end of the device immediately prior to commencing delivery of the AR to the target tissue of the subject. Optionally, the kit can include means for detecting the multivalent binding response as described above.

[0121] Sentinel surveillance

[0122] The present invention also provides a method for sentinel monitoring of an individual, wherein an affinity reagent is applied to the subject before the subject has been exposed to an analyte of interest or when the analyte is at an undetectable concentration. In this embodiment, even if the AR is delivered into the subject and reacts in situ with mast cells to sensitize them to the analyte, cross-linking does not occur in the absence of the analyte. Mast cells that have been sensitized by the AR will remain sensitized for an extended period of time. Resident mast cells are long-lived cells that can survive in the skin of Wistar rats for up to 12 weeks or longer (Kiernan J.A, Journal of Anatomy 128:225-238 (1979)). If the sensitized mast cells are subsequently exposed to the analyte of interest over the next few days, weeks, or months, cross-linking that induces degranulation of the sensitized mast cells and other additional physiological changes in the target tissue will occur, thereby constituting a multivalent binding response that can be evaluated to determine the presence or amount of the analyte of interest. For example, an AR molecule specific for Ebola virus is delivered into the skin on the forearm of a healthcare worker before the healthcare worker enters the scene to treat patients. If the healthcare worker is exposed to Ebola virus at any time, the Ebola virus or particles from the virus will bind to the sensitized mast cells, triggering a multivalent binding response, which can in turn be evaluated by assessing the tissue at the delivery site, where a wheal and flare reaction appears to warn the healthcare worker. Modifications and variations of the present invention can be made without departing from the spirit and scope of the invention, which will be apparent to those skilled in the art. The specific examples described below are provided only to illustrate some embodiments of the present invention, and the present invention is intended to be limited only by the appended claims and the full scope of the equivalents of these claims.

[0123] Example

[0124] Example 1

[0125] CD-1 mice

[0126] Female CD-1 mice (non-breeding) (approximately 30 grams) were purchased from Charles River (251 Ballardvale St., Wilmington, MA 01887). Mice have played a historically important role in the detailed investigation of human allergic reaction mechanisms. Cross-linking of antigen-induced mast cell-associated IgE results in mast cell degranulation and release of mediators, which is highly conserved in mammals. Those skilled in the art will appreciate that allergy-based examples as covered by the present invention and conducted in mice will translate to humans and other mammals (Ovary Z., "Arerugi" 43(12):1375 (1994)).

[0127] Preparation of HBsAg Affinity Reagent (HBsAg-AR)

[0128] AR was constructed by recombinantly converting the Fcγ region of an anti-HBsAg IgG antibody into the Fcε region. Specifically, HBsAg-AR was prepared from the anti-HBsAg 5C3 murine IgG2a Fc-silent antibody to contain the Fl.ε.Rl receptor domain by converting the IgG isotype to IgE (mouse), and was performed by Absolute Antibody Ltd (Wilton Centre, Redcar, Cleveland TS10 4RF, UK) by proprietary methods. Anti-HBsAg 5C3 IgE is supplied in PBS with 0.2% Procline 300 and recognizes the antigenic determinant on HBsAg (Wands et al., "Proceedings of the National Academy of Sciences of the United States of America" 79:1277 (1982); Wands et al., "Proceedings of the National Academy of Sciences of the United States of America" 81:2237 (1984); Ben-Porath et al., "Journal of Clinical Investigation" 76:1338 (1985)).

[0129] The recombinant full-length HBsAg antigen (subtype adw) of hepatitis B surface antigen (HBsAg) produced in Saccharomyces cerevisiae (containing plasmid pCGA7) was purchased from Fitzgerald Industries International (30 Sudbury Rd., Suite 1A North, Acton, MA 01720 (30 Sudbury Rd., Suite 1A North, Acton, MA 01720)). HBsAg was supplied in 0.05 M phosphate, 0.2 M NaCl, pH 7.2. Expression of HBsAg in yeast results in spherical and particulate forms that are identical in size and shape to those particles (20 to 22 nm) found in the sera of HBV-infected patients (Miyanohara et al., Proceedings of the National Academy of Sciences of the United States of America, Vol. 80, pp. 1-5, January 1983). HBsAg itself has a molecular weight of approximately 24 kDa, and the glycosylated form has a molecular weight of 27 kDa, while the 22 nm particles have a molecular weight of 2.18 MDa (Ono et al., Nucleic Acids Research 11(6):1747 (1983); Miyanohara et al., Proceedings of the National Academy of Sciences of the United States of America 80:1 (1983); Gilbert et al., Proceedings of the National Academy of Sciences of the United States of America 102(41):14783 (2005)).

[0130] Avertin anesthetic

[0131] Prepare the stock solution from 99% 2,2,2-tribromoethanol (Acros Organics) and reagent-grade tert-amyl alcohol (Fisher Scientific). Add 10 ml of tert-amyl alcohol to 10 g of Avertin (2,2,2-tribromoethanol). The bottle in which Avertin arrives facilitates mixing. Place a stir bar and stir on a magnetic stirrer until Avertin is completely dissolved. This step takes approximately 2 hours. Store the stock solution in a dark bottle and cap tightly. Store it at room temperature. It should be noted that the Avertin stock solution is photosensitive and hygroscopic. The stock solution should be stable for 6 to 12 months. The working solution (1.2% Avertin) is prepared as follows: Add 0.240 ml of the stock solution dropwise to 19.76 ml of sterile water while stirring the water with a stir bar. Stir until the stock solution is completely dissolved. Refrigerate the stock solution and protect from light for use within 2 weeks. Administer Avertin IP at 0.800 ml / 30 g of mouse.

[0132] Evans blue

[0133] Due to its water solubility, slow excretion, and tight binding to serum albumin, Evans blue has been widely used in biomedicine to determine vascular permeability. When histamine and other vasodilators are released from mast cells, causing barrier disruption and increased vascular permeability, the albumin bound to Evans blue will extravasate from the circulation into the adjacent tissues. The leakage of the dye across the blood vessels means the disintegration of the barrier, and the accumulation of Evans blue dye can be quantified. A stock solution is prepared by adding 1 gram of Evans blue to 30 ml of water.

[0134] Example 2

[0135] Method for detecting an analyte

[0136] Contact of the AR with the analyte results in a visible and measurable localized inflammatory response that is proportional to the amount of analyte present in the subject. To demonstrate this, HBsAg-AR was injected intradermally into mast cells in mice, and then the mice were challenged intravenously with different amounts of HBsAg. Wheal and flush measurements can be performed separately in humans, while the skin of mice is very thin, and measuring the leakage of Evans blue around the positive wheal and flush responses provides a simple way to visualize the positive response.

[0137] 100 ng (10 μl) of HBsAg-AR in phosphate-buffered saline (PBS) was injected intradermally into the right ear pinna of mice under avertin anesthesia; mice that received 10 μl of PBS intradermally in the left ear pinna served as controls. The next day, the mice were challenged intravenously with 100 μl of HBsAg in 1% Evans blue. The mice were challenged with 100 μg, 50 μg, 20 μg, and 10 μg of HBsAg. The HBsAg challenge was administered by retro-orbital injection. The response was measured at 20 minutes. The mice were classified based on the surface area and density of the Evans blue dye in the ear (Table 2).

[0138] Table 2.

[0139] Attack 100 μg 50 μg 20 μg 10 μg Reaction +++++ ++++ ++ Negative

[0140] Example 3

[0141] Enhanced sensitivity

[0142] As the analyte circulates through the body, the AR delivered to the target tissue is continuously exposed to the analyte until a measurable response occurs, thereby enhancing the assay sensitivity. To demonstrate this, the mice were challenged with 10 μg of HBsAg as described in Example 2, and the mice were observed for the next 8 hours. The mice were recorded as negative 20 minutes to 4 hours after exposure to the analyte, but were clearly positive after 8 hours (Table 3).

[0143] Table 3.

[0144] Time 20 minutes 1 hour 2 hours 4 hours 8 hours Reaction Negative Negative Negative Negative ++

[0145] Example 4

[0146] Ease of use and simplicity

[0147] The method of the present invention is performed in one step without processing the sample through separation or rinsing steps, thereby providing a homogeneous type format that is easy to use. To demonstrate this, mice with circulating HBsAg were tested according to the method of the present invention, where AR was delivered by both intradermal and acupuncture. As described above, mice under avertin anesthesia were challenged with 100 μg of HBsAg diluted in 1% Evans blue in 100 μl. The HBsAg challenge was administered by retro-orbital injection. As described above, the injected HBsAg was allowed to pre-circulate for 10 minutes before intradermal application of HBsAg-AR to the right ear pinna. HBsAg-AR was also pipetted onto the surface of the left ear, and the surface layer of the skin was punctured with a needle. The reaction time was observed and recorded in Table 4. Intradermal application of AR resulted in a dense blue patch covering 30% to 40% of the ear. Acupuncture resulted in small discrete and dense blue patches.

[0148] Table 4.

[0149] Time 30 seconds 2 minutes 3 minutes 5 minutes Intradermal + / - +++ ++++ +++++ Needle prick + / - +++ ++++ ++++

[0150] Example 5

[0151] Sentinel testing To demonstrate this, 100 ng (10 μl) of HBsAg-AR in PBS was intradermally injected into the right ear pinna of mice under avertin anesthesia as described above; mice that received 10 μl of PBS intradermally in the left ear pinna served as controls. Seven days later, mice under avertin anesthesia were challenged with 100 μg of HBsAg diluted in 1% Evans blue in 100 μl as described above. Sixteen days later, anesthetized mice were challenged with 30 μg of HBsAg diluted in 1% Evans blue in 50 μl as described above. The HBsAg challenge was administered by retro-orbital injection. The reaction was measured at 20 minutes. The results demonstrated that the method can be used to detect exposure to the analyte 7 days and 10 days after in situ exposure of AR mast cells to AR (Table 5).

[0152] Table 5.

[0153] Time 7 days 16 days Reaction ++++ ++++

[0154] Example 6

[0155] Efficacy and sentinel testing in humans

[0156] Recombinant-produced affinity reagents for detecting Ara h 2 were made by Absolute Antibody using proprietary methods. In this case, the affinity reagent is essentially an IgE antibody specific for an epitope on the Ara h 2 allergen. The reagent has the required Fc.ε.R1 binding domain and an additional part for detecting Ara h 2. The affinity reagent (1 mg / ml) is supplied in PBS with 0.2% Procline 300.

[0157] Intradermal injection (Protocol #1) was performed on the applicant's forearm with 100 ng (10 μl) of the Ara h 2 affinity reagent in PBS. Additionally, using two different procedures, the affinity reagent was applied using a Unitest PC skin test applicator (Lincoln Diagnostics, Inc., Decatur, IL 62526). In the first procedure, approximately 20 μl of the affinity reagent in PBS was pipetted onto the skin, and the Unitest PC applicator was applied through the affinity reagent into the dermal layer of the skin (Protocol #2). In the second procedure, the affinity reagent was suspended in 50% glycerol in PBS, allowing the affinity reagent to adsorb onto the tip of the Unitest PC by capillary action. Then the Unitest PC applicator was applied to the dermal layer of the skin in one step (Protocol #3). An affinity reagent at a concentration of 50 μg / ml was used with the Unitest device. Peanuts were consumed at 30-minute intervals for 6 hours. The assays using Protocols #1, #2, and #3 were applied to the forearm, and the reaction was measured 20 minutes later. Each protocol included a control consisting of the application vehicle without the affinity reagent. With all three protocols using the applied affinity reagent, distinct wheals and flushing were clearly visible, while the negative control showed no sign of reaction (Table 6).

[0158] After waiting 24 hours after consuming the last peanut, Ara 2h was eliminated from the applicant's blood. Then the assay was applied using Protocol #2, and the reaction was measured 20 minutes later. To demonstrate the feasibility of the sentinel test, the assay was applied to the applicant before consuming peanuts. The test was negative and remained negative for 24 hours. Due to the lag period between peanut consumption and the appearance of Ara 2h in the blood, the test results were measured 20 minutes after the initial signs of the reaction (Table 7).

[0159] Table 6

[0160] Protocol #1 (mm) Protocol #2 (mm) Protocol #3 (mm) Control 0.0 0.0 0.0 + Ara h 2 12.00 8.5 7.0

[0161] [[ID=1,5]]

[0162] Protocol #2 (mm) - Ara h 2 0.0 + Ara h 2 (24 hours later) 7.0

[0163] ​Example 7

[0164] Human Sentinel Testing

[0165] The affinity reagent (anti - Ara h 2, IgE) was prepared as described above, and approximately 5 to 10 μl of saline was applied by intradermal injection to the applicant's forearm to produce a bleb - like area. The bleb - like area disappeared within a few hours, and all traces of the injection site had disappeared by the next day. One week later, the applicant consumed peanuts. Within 30 minutes of ingestion, a pricking sensation occurred at the site, followed by a mild stinging pain. A few minutes later, an obvious wheal and flushing reaction occurred and persisted for approximately 2 hours. The wheal was measured at 20 mm within 20 minutes of the initial signs of the reaction (Table 8).

[0166] Table 8.

[0167] Reaction - Ara h 2 0.0 mm + Ara h 2 (1 week later) 20.0 mm

[0168] Example 8

[0169] Application of the Affinity Reagent Dried onto the Applicator

[0170] The affinity reagent (anti - Ara h 2, IgE) was dried onto the tip of a Unitest PC applicator. A 1:10 dilution of a 50 μl aliquot of the stock affinity reagent in PBS was placed in a 1.5 ml microcentrifuge tube. The Unitest PC applicator was placed in the microcentrifuge tube, where the tip was immersed in the affinity reagent for 1 or 2 minutes. The applicator was removed and allowed to dry for 4 hours. The applicator was applied to the subject's arm for 30 seconds. There was no visible blood, but there was an imprint of the applicator, indicating that the applicator had been correctly applied to the forearm as per the manufacturer's user guide instructions. All marks of the applied test disappeared within 30 minutes. Twenty - four hours later, the subject consumed peanuts. Within 20 minutes, a mild pricking sensation occurred, followed by a feeling of low - grade stinging pain, at which time a wheal and flushing reaction was observed. At 40 minutes after ingestion, the wheal and flushing were at maximum intensity. There was an obvious 8 mm wheal and flushing reaction, and it persisted for more than 2 hours (Table 9). Two hours later, the wheal and flushing decreased, and only remnants of the reaction remained in the next 6 hours.

[0171] Table 9

[0172] Reaction - Ara h 2 0.0 mm + Ara h 2 (Applied dry) 8.0 mm

[0173] While the foregoing disclosure, together with the references incorporated herein, relates to and describes certain embodiments which are provided to implement and illustrate the concepts of the invention and certain exemplary guidance regarding putting it into practice, those skilled in the art should understand that changes and modifications can be made to the foregoing without departing from the spirit of the invention and without undue experimentation. Accordingly, all such changes and modifications that fall within the true scope of the invention are intended to be claimed.

Claims

1. Use of an affinity reagent for in vivo bioassays in the preparation of a detection reagent for determining the presence, absence or amount of an analyte in a live mammal, said determination comprising the steps of: providing at least one affinity reagent construct comprising at least one Fc.ε.R1 receptor binding domain and at least one additional moiety capable of binding said analyte, wherein the resulting construct binds both mast cells and said analyte in any order, said analyte comprising a chemical, peptide, protein, lipid, carbohydrate, glycoprotein, nucleic acid sequence or combination thereof, contacting at least one endogenous mast cell present in situ and within the target tissue of said mammal with said reagent construct to elicit a multivalent binding response in the presence of said analyte; said multivalent binding response is a function of multivalent binding and encompasses cross-linking of the Fc.ε.R1 receptor across the mast cell membrane, degranulation of the mast cell and any additional and secondary physiological changes in the target tissue directly or indirectly resulting from such degranulation, and Performing the evaluation of the multivalent binding response to determine the presence or amount of the analyte, wherein, performing said assessment further comprises assessing said target tissue in the vicinity of the site of administration of the affinity reagent radiating outwards 3 cm from the point of contact.

2. The use according to claim 1, wherein said contacting further comprises delivering said affinity reagent construct into the skin of said mammal.

3. The use according to claim 1, wherein said contacting further comprises delivering said affinity reagent construct into the tissue of said mammal, wherein said affinity reagent is exposed to 125 to 20,000 mast cells / mm³.

4. The use according to claim 3, wherein said delivery further comprises performing the delivery with at least one device selected from the group consisting of: acupuncture, patch method or needleless system; wherein said acupuncture includes intradermal injection, solid needle, hollow needle.

5. The use according to claim 2, wherein said contacting step further comprises delivering said affinity reagent construct into the dermal layer.

6. The use according to claim 1, wherein performing said assessment further comprises evaluating said target tissue for at least one physiological change in said target tissue.

7. The use according to claim 6, further comprising evaluating morphological changes in said target tissue to provide an assessment of said multivalent binding response.

8. The use according to claim 7, further comprising using a device to measure morphological changes.

9. The use according to claim 6, further comprising using a sensor to evaluate said target tissue to provide an assessment of said multivalent binding response.

10. The use according to claim 7, further comprising performing a visual inspection of the wheal and flare response.

11. The use according to claim 8, wherein said device measures the wheal and flare response.

12. The use according to claim 1, wherein said step further comprises providing at least one affinity reagent construct comprising at least one Fc.ε.R1 receptor binding domain and at least one additional moiety capable of binding said analyte using at least one technique selected from the group consisting of: hybridoma technology, covalent conjugation, non-covalent binding and genetic engineering methods.

13. The application according to claim 1, wherein the step further comprises shaping the additional part using at least one member selected from the group consisting of: antibodies, antibody fragments, engineered non-antibody binding proteins, antigens, chimeric molecules, fusion proteins, aptamers, hormones, receptors, receptor binding molecules, drugs, poisons, toxins, pathogens, pathogen components, biomarkers, ligands, RNA, or DNA; wherein the antibodies include recombinant antibodies, engineered antibodies, and synthetic antibodies; and the biomarkers include cell surface markers.

14. The application according to claim 1, wherein performing the assessment further comprises measuring substances released during mast cell degranulation.

15. The application according to claim 1, wherein the analyte is Mycobacterium tuberculosis, and the additional part comprises at least one major histocompatibility complex protein complexed with at least one Mycobacterium tuberculosis antigen.

16. The application according to claim 15, wherein the major histocompatibility complex protein is selected from the group consisting of MHC class I and MCH class II proteins.

17. The application according to claim 15, wherein the Mycobacterium tuberculosis antigen is the Mtb secreted antigen target ESAT-6 or the 10-kDa culture filtrate protein CFP-10.

18. The application according to claim 1, wherein the analyte is selected from SARS-CoV-2 antibodies, and the additional part comprises an antigen derived from the SARS-CoV-2 virus.

19. The application according to claim 18, wherein the antigen is derived from a protein selected from the group consisting of: the SARS-CoV-2 receptor binding domain, the spike protein, and the SARS-CoV-2 nucleocapsid protein; wherein the spike protein includes the spike S1 protein and the spike S2 protein.

20. Use of an affinity reagent for in vivo bioassay in the preparation of a detection reagent for testing a suspected pathology in a live mammal, the test comprising the following steps: Selecting an analyte indicative of the suspected pathology; Providing at least one affinity reagent construct comprising at least one Fc.ε.R1 receptor binding domain and at least one additional part capable of binding the analyte, wherein the resulting construct binds both mast cells and the analyte in any order; Contacting at least one endogenous mast cell present in situ and within the target tissue of the mammal with the reagent construct to elicit a multivalent binding response in the presence of the analyte; the multivalent binding response is a function of multivalent binding and encompasses cross-linking of the Fc.ε.R1 receptor across the mast cell membrane, degranulation of the mast cell, and any additional and secondary physiological changes in the target tissue directly or indirectly resulting from such degranulation, and Performing the evaluation of the multivalent binding response to determine the presence or amount of the analyte, wherein, Performing the assessment further includes assessing the target tissue in the vicinity of the administration site of the affinity reagent radiating outwards 3 cm from the point of contact.

21. Use of an affinity reagent for in vivo bioassays in the preparation of a detection reagent for testing exposure of a live mammal to at least one substance selected from the group consisting of: chemicals, drugs, biological warfare agents, poisons, toxins, and pathogens, said testing comprising the steps of: selecting an analyte indicative of exposure to said substance; providing at least one affinity reagent construct comprising at least one Fc.ε.R1 receptor-binding domain and at least one additional moiety capable of binding said analyte, wherein the resulting construct binds both mast cells and said analyte in any order; contacting at least one endogenous mast cell present in situ and within the target tissue of said mammal with said reagent construct to elicit a multivalent binding response in the presence of said analyte; said multivalent binding response being a function of multivalent binding and encompassing cross-linking of Fc.ε.R1 receptors across the mast cell membrane, and degranulation of the mast cell and any additional and secondary physiological changes in the target tissue directly or indirectly resulting from such degranulation, and Performing an evaluation of the multivalent binding response to determine the presence or amount of the analyte, wherein, performing said assessment further comprising assessing the target tissue in the vicinity of the affinity reagent administration site radiating out 3 cm from the point of contact.

22. Use of an affinity reagent for in vivo bioassays in the preparation of a bioassay kit for determining the presence, absence, or amount of an analyte in a live mammal, said kit comprising at least one affinity reagent construct comprising at least one Fc.ε.R1 receptor-binding domain and at least one additional moiety capable of binding an analyte of interest, said reagent construct being capable of binding both mast cells and said analyte in any order; a delivery device for delivering the affinity reagent construct to the target tissue of the mammal, a device for detecting a multivalent binding response, said determining comprising contacting at least one endogenous mast cell present in situ and within the target tissue of said mammal with said reagent construct to elicit a multivalent binding response in the presence of said analyte; said multivalent binding response being a function of multivalent binding and encompassing cross-linking of Fc.ε.R1 receptors across the mast cell membrane, and degranulation of the mast cell and any additional and secondary physiological changes in the target tissue directly or indirectly resulting from such degranulation, and Performing the evaluation of the multivalent binding response to determine the presence or amount of the analyte, wherein, performing said assessment further comprising assessing the target tissue in the vicinity of the affinity reagent administration site radiating out 3 cm from the point of contact.

23. The use according to claim 22, wherein said additional moiety is selected from the group consisting of: antibodies, antibody fragments, engineered non-antibody binding proteins, antigens, chimeric molecules, fusion proteins, aptamers, hormones, receptors, receptor-binding molecules, drugs, poisons, toxins, pathogens, pathogen components, biomarkers, ligands, RNA, and DNA; wherein said antibodies include recombinant antibodies, engineered antibodies, synthetic antibodies; and said biomarkers include cell surface markers.

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