Agglutinants, devices and methods for whole blood assays

Abstract

Devices, methods, and kits are provided that include means for trapping red blood cells. Such means include physical means for trapping red blood cells and agglutinating agents, including hemagglutinating agents, for retaining red blood cells in specific regions of the devices described herein. The physical means and agglutinating agents separate the red blood cells from the whole blood sample. Analysis of the separated and / or agglutinated whole blood sample for the presence or absence of an analyte of interest can be performed without interference from the red blood cells. Devices such as lateral flow assays with sample receiving zones that include physical means and / or agglutinating agents are also described. Specific agglutinating agents include lectins and antibodies, such as red blood cell binding antibodies.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 242,754, filed September 10, 2021, which is incorporated by reference herein.

[0002]

[0002] The present disclosure relates generally to devices and methods including agglutinants, including hemagglutinants, for immunoassays such as lateral flow assays for analysis of whole blood, and means for capturing red blood cells, such as physical means. [Background technology]

[0003]

[0003] Various devices, assays, methods, and techniques can be used to analyze bodily fluids such as whole blood for the presence or absence of a particular analyte of interest. For example, tests are available to detect glucose, uric acid, or protein in urine, or to detect pathogens, antibodies, glucose, triglycerides, potassium ions, or cholesterol in blood. For example, devices and methods that process whole blood by removing red blood cells prior to testing are used to separate the liquid sample into distinct parts or fractions, such as a red blood cell fraction and a plasma fraction. For example, the cellular components of whole blood can be separated from the plasma or serum for use in an assay to test for a particular analyte of interest. The resulting plasma or serum can be tested for accurate detection of the analyte of interest without the interference of red blood cells present in the whole blood sample.

[0004]

[0004] The cellular components of whole blood, especially red blood cells, are major interfering substances in assays such as devices including lateral flow immunoassays or strip tests for analytes of interest present in whole blood. Many blood tests are chromogenic, where the analytes present in whole blood interact with a specific reagent to form a complex or derivative of a unique color as a quantitative or qualitative indicator of the presence or absence of the analyte, or form colored complexes or derivatives of various color intensities as a quantitative indicator of the presence of the component. The deep red color of a whole blood sample substantially interferes with these chromogenic tests, so the highly colored red blood cells are usually separated from the plasma or serum before the blood sample is assayed for the specific analyte of interest. The presence of red blood cells can also interfere with various non-chromogenic blood assays, resulting in inconsistent or consistent but inaccurate assay results. Summary of the Invention

[0005]

[0005] The subject technology is illustrated, for example, according to various aspects described below.

[0006] According to some embodiments, a device is provided for detecting an analyte of interest in a whole blood sample. In some aspects, the device provided herein includes an immunoassay such as a lateral flow assay or a strip test. In some aspects, the device includes a sample receiving zone that includes a means for capturing red blood cells. In some embodiments, the means is a physical means such as a net or mesh present in the sample pad of the lateral flow assay or strip test, the net / mesh physically interacting with the red blood cells to retain them in the sample pad without lysis of the red blood cells. In some embodiments, the sample pad may also include a non-lytic hemagglutinating agent, which captures red blood cells from a whole blood sample added to the sample receiving zone without lysis of the red blood cells.

[0007] In other aspects, the device comprises a labeling zone including a means for specifically labeling an analyte of interest. In other aspects, the device also comprises a capture zone having a means for specifically binding and immobilizing the labeled analyte of interest. In some aspects, the device comprises a sample receiving zone, a labeling zone, and a capture zone disposed in a liquid flow path.

[0008]

[0008] In some embodiments, the sample receiving zone including the sample pad includes a means for physically interacting with red blood cells, which means comprises a structure such as a net, mesh, screen or lattice having an appropriate mesh size for physically retaining and / or capturing red blood cells in the sample pad of the lateral flow strip test device described herein, and the red blood cells are captured and / or retained in the sample pad by interaction with the physical structure.

[0009] In some embodiments, the hemagglutinating agent is an antibody, such as a monoclonal or polyclonal antibody. In some embodiments, the antibody binds to red blood cells, such as human red blood cells. In some embodiments, the antibody binds to the H antigen of human red blood cells, and thus binds to essentially all human red blood cells, regardless of ABO type. In some embodiments, the antibody is biotinylated.

[0010]

[0010] In some embodiments, a hemagglutinating agent such as an antibody may be self-immobilized within a device, such as a sample receiving zone of a device comprising an immunoassay such as a lateral flow assay or a strip test.

[0011] In some embodiments, the device can include an immunoassay such as a lateral flow assay having a sample receiving zone (i.e., a sample pad), the sample receiving zone comprising streptavidin. In some embodiments, the streptavidin is associated with or on particles immobilized in the sample receiving zone of the lateral flow immunoassay.

[0012]

[0012] In some embodiments, the hemagglutinating agent is a lectin. In some aspects, the lectin is any lectin, including but not limited to wheat germ agglutinin, Ulex europaeus agglutinin I, Pisum sativum agglutinin, Lens culinaris agglutinin, or Phaseolus vulgaris hemagglutinin. In some embodiments, the lectin may be self-immobilized in a device, such as a sample receiving zone of a device including an immunoassay, such as a lateral flow assay or a strip test. In some embodiments, the lectin is biotinylated.

[0013] In some embodiments, the means for specifically labeling the analyte of interest is a detectable antibody, such as a detectable monoclonal antibody or a detectable polyclonal antibody.

[0014] In some embodiments, the means for specifically labeling the analyte of interest is a detectable antigen, such as a protein, peptide, macromolecule, or small molecule.

[0015] In some embodiments, the means for specifically binding and immobilizing the labeled analyte of interest is an antibody, such as an immobilized antibody. In some embodiments, the immobilized antibody is a monoclonal antibody or a polyclonal antibody.

[0016] In some embodiments, the means for specifically binding and immobilizing the analyte of interest is an antigen, such as an immobilized antigen. In some embodiments, the immobilized antigen is an immobilized protein, an immobilized peptide, an immobilized macromolecule, or an immobilized small molecule.

[0017]

[0017] In another embodiment, a method is provided for detecting the presence or absence of an analyte of interest in a whole blood sample. In some embodiments, the method includes providing a device as described herein, placing a whole blood sample into the device where red blood cells are retained in a sample receiving zone, and determining the presence or absence of a labeled analyte immobilized in the capture zone. In some embodiments, the method also includes providing an instrument for collecting the whole blood sample, and collecting the whole blood sample into the instrument for addition to the device, such as a sample receiving zone of a device including a lateral flow immunoassay.

[0018]

[0018] In another embodiment, a kit is provided that includes a device as described herein, an apparatus for collecting a whole blood sample, and instructions for use. In some aspects, the apparatus for collecting a whole blood sample includes a capillary tube.

[0019] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by understanding the following descriptions.

[0020]

[0020] Additional embodiments of the present devices, assays, methods, and compositions will become apparent from the following description, drawings, examples, and claims. As can be understood from the foregoing and following description, every feature described herein and every combination of two or more of such features is included within the scope of the present disclosure, provided that the features included in such combinations are not mutually inconsistent. In addition, any feature or combination of features may be specifically excluded from any embodiment described herein. Additional aspects and advantages of the present disclosure are described in the following description and claims, particularly when considered in conjunction with the accompanying examples and drawings. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 illustrates a human hemagglutination test using various lectins as agglutinating agents. [Diagram 2]

[0022] FIG. 2 illustrates a human hemagglutination test using anti-human red blood cell antibodies as the agglutinating agent. [Figure 3A]

[0023] 3A illustrates the capture of human red blood cells in a lateral flow immunoassay where the sample receiving zone comprises control condition 1. In this configuration, the sample pad treatment included a blank control that did not contain any antibody, and retention of red blood cells on the sample pad was achieved by physical means, such as the red blood cells being trapped in the reticulated mesh of the sample pad. [Figure 3B]

[0024] 3B illustrates the capture of human red blood cells in a lateral flow immunoassay where the sample receiving zone contains a hemagglutinating agent under condition 2. In this configuration, sample pad treatment with mAb H101 and retention of red blood cells on the sample pad was achieved by antibody-induced agglutination of red blood cells in addition to physical means such as capture of agglutinated red blood cells on the reticulated mesh of the sample pad. [Figure 3C]

[0025] 3C illustrates the capture of human red blood cells in a lateral flow immunoassay where the sample receiving zone contains a hemagglutinating agent under condition 3. In this configuration, treatment of the sample pad containing mAb H101 with streptavidin and retention of the red blood cells on the sample pad was achieved by agglutination of the red blood cells by the antibody and / or streptavidin in addition to physical means such as trapping of agglutinated red blood cells on the reticulated mesh of the sample pad. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022]

[0026] Various aspects will now be described in more detail below. However, such aspects may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey its scope to those skilled in the art.

[0023]

[0027] The various devices, assays, methods, and techniques provided herein can be used to analyze bodily fluids, such as whole blood, for the presence or absence of a particular analyte of interest without the interference of red blood cells. The techniques provided herein, such as agglutinating agents and devices and methods comprising same, provide for the analysis of whole blood samples without the interference of red blood cells and without the need for additional, costly, cumbersome, complicated, or time-consuming steps.

[0024]

[0028] Conventionally, plasma or serum can be separated from the cellular material of whole blood by centrifugation or clotting. The cellular material collects at the bottom of the centrifuge or sample tube, and the supernatant plasma or serum is decanted. Thus, the interfering cellular components of whole blood are removed such that substantial background interference, such as that of red blood cells, is avoided. However, the centrifugation method requires a large volume of blood sample, long centrifugation times, and appropriate equipment. Furthermore, the centrifugation method requires several operation steps. The clotting method to obtain serum also requires a long time (30-60 min) for clotting to occur, and centrifugation may be necessary after clotting has occurred.

[0025]

[0029] Other technologies may include test strips for lateral flow of a sample for analysis. However, in some designs, red blood cells form aggregates or blockages within the chemical strip, which can disrupt the lateral flow and / or discolor the test strip, making the results difficult to interpret. Such blockages often produce inaccurate and / or invalid results.

[0026]

[0030] The lateral flow immunoassay devices described herein comprise a sample receiving zone including a sample pad configured to physically interact with red blood cells so that the red blood cells are retained or trapped within the pad material. The sample pad comprises a means for physical interaction with red blood cells so as to maintain, retain, trap and / or otherwise slow the flow of red blood cells from the sample pad to a downstream portion of the lateral flow strip test device. For example, the sample pad can comprise a means for physical interaction with red blood cells having a structure such as a net, mesh, screen or lattice having an appropriate mesh size to physically retain and / or trap the red blood cells in the sample pad of the lateral flow strip test device described herein.

[0027]

[0031] In addition, some lateral flow immunoassay devices and methods described herein include an agglutinating agent that efficiently separates and accurately assays whole blood samples without the need for additional processing steps performed by the end user. The agents, devices, and methods provided protect users, such as home testers or laboratory assistants, from unnecessary contact with the blood sample, avoid time delays, and provide accurate and reproducible results.

[0028]

[0032] Techniques according to the present disclosure allow for safe, accurate, and economical assay of whole blood samples for specific soluble components, such as analytes of interest, and can achieve essentially complete separation of red blood cells of whole blood from plasma or serum. The systems, devices, methods, and kits described herein are contemplated for use with a variety of fluid samples, and although the description herein uses blood as a model sample, it will be understood that the systems, devices, and methods can also be used with other fluid samples.

[0029] Flocculant

[0033] The embodiments described herein include agglutinating agents, such as hemagglutinating agents, that agglutinate and / or cause red blood cells to clump together. As used herein, "agglutination" refers to cells clumping together and "hemagglutination" refers to the clumping of red blood cells. In some cases, "agglutination" and "hemagglutination" are related terms that can be used interchangeably. For example, agglutinated or hemagglutinated red blood cells can be clumped together by an agent described herein and then captured or removed so as not to interfere with the analysis of the remainder of the sample for the presence or absence of an analyte of interest.

[0030]

[0034] In some embodiments, the agglutinating agent is "non-lytic," i.e., it does not cause lysis or disruption of cells by rupture of cell walls or membranes. Instead, in some embodiments, the agglutinating agent provides hemagglutination of intact red blood cells without rupture or lysis of the aggregated cells. This feature ensures that all red blood cell components are captured or retained upon agglutination of intact red blood cells, so that internal cellular components, including heme groups, are not released and therefore cannot interfere with subsequent and / or downstream sample analysis, as the internal cellular components are retained in the agglutinated and trapped intact red blood cells.

[0031] Lectins

[0035] In some embodiments, the agglutinating agent is a lectin. Lectins have excellent affinity and multivalency for specific binding to complex carbohydrates, such as surface antigens of red blood cells, and have a long history as medical diagnostic reagents and tools for many applications. Lectins are typically composed of four subunits, i.e., tetravalent, for efficient agglutination / capture of red blood cells. Thus, certain lectins can be used to greatly facilitate separation of plasma fluid from various red blood cells. Thus, devices and methods including lectins specific for all red blood cells, such as all types of human red blood cells, can be used to separate, capture, and retain human red blood cells from whole blood samples of any blood type. Suitable lectins include, but are not limited to, those listed in Table 1, which are suitable for agglutinating and capturing human red blood cells of all blood types (ABO). Furthermore, in some embodiments, biotinylated lectins can be used in combination with streptavidin (SA) to provide improved hemagglutination activity for more efficient and effective binding, capture, and retention of human red blood cells, such as capturing red blood cells on a lectin-impregnated sample pad in the sample receiving zone of a lateral flow device as provided herein.

[0032] [Table 1]

[0033]

[0036] In some embodiments, lectins can be recombinantly expressed for large-scale production. Recombinant expression of lectins with native or tandem repeats has been systematically studied. In some embodiments, lectins can include streptavidin and be biotinylated for use in combination with devices and methods that advantageously utilize biotin-streptavidin. In some embodiments, lectins can include tandem repeats that can increase the efficiency of large-scale lectin production in bacterial expression systems (Hwang et al., Biomolecules 2018, 8, 146). Additionally, fusion techniques are widely used for proteins, and such fusion techniques can be applied to produce lectins with higher capture efficiency.

[0034]

[0037] In some embodiments, lectins can be conjugated to bead nanoparticles. In this situation, lectin-conjugated beads or nanoparticles provide efficient agglutinants because they can agglutinate and capture / retain a large number of blood cells on their multivalent surface. In some embodiments, recombinant fusion technology allows lectins with fusion tags to be directly attached to nanoparticles with higher efficiency and lower cost for production and use as agglutinants.

[0035] Agglutinated antibody

[0038] In other embodiments, the agglutinating agent is an antibody, such as an antibody with binding specificity for red blood cells. Human ABO blood group discrimination is based on the presence of non-reduced αGal and αGalNAc termini, while αFuc is the determinant for H(O) blood group. Thus, these carbohydrate antigens provide desirable targets for binding of agglutinating agents, such as lectins and / or antibodies. Because antibodies are bivalent in nature, antibodies with red blood cell specificity provide effective agglutinating agents for retaining / capturing red blood cells.

[0036]

[0039] Polyclonal and / or monoclonal antibodies with red blood cell binding affinity can be used as the agglutinating agent. For example, polyclonal or monoclonal antibodies with binding specificity for the H antigen of human red blood cells can be used as the agglutinating agent according to certain embodiments. In some embodiments, monoclonal antibodies against H(O) blood type are provided as the agglutinating agent. Such monoclonal antibodies demonstrate desirable hemagglutination activity with pooled human red blood cells (hRBCs) of mixed blood types. Additionally, in some embodiments, biotinylated antibodies can be used in combination with streptavidin (SA) to provide improved hemagglutination activity for more efficient and effective binding, capture, and retention of human red blood cells, such as capturing red blood cells on an antibody-impregnated sample pad in a sample receiving zone of a lateral flow device as provided herein.

[0037]

[0040] In some embodiments, the antibodies can be recombinantly expressed for large-scale production. Recombinant expression of antibodies, either native or with tandem repeats, has also been investigated. In some embodiments, the erythrocyte-specific antibodies can be biotinylated for use in combination with devices and methods that include streptavidin and advantageously utilize biotin-streptavidin. In some embodiments, the antibodies provided can also include tandem repeats that can increase the efficiency of large-scale antibody production in bacterial expression systems (Hwang et al., Biomolecules, 2018, 8, 146). Additionally, fusion techniques are widely used for proteins and antibodies, and such fusion techniques can be applied to the production of antibodies with improved efficiency of agglutination and capture of erythrocytes.

[0038]

[0041] In some embodiments, the antibody can be conjugated to a bead nanoparticle. In this situation, the antibody-conjugated bead or nanoparticle provides an efficient agglutinating agent because it can agglutinate and capture / retain a large number of blood cells on its multivalent surface. In some embodiments, recombinant fusion technology allows antibodies with fusion tags to be directly attached to nanoparticles with high efficiency and low cost for production and use as agglutinating agents.

[0039] Lateral flow device, method, and kit using agglutinant Device

[0042] In some embodiments, an agglutinating agent is present in or on a device, such as a lateral flow immunoassay or strip test. As used herein, a "sample receiving zone" refers to a portion of an assay or device, such as a lateral flow device or strip test, that is configured for sample addition. In some embodiments, the sample receiving zone may contain an agglutinating agent or may comprise a sample pad that contains or is impregnated with one or more of said agglutinating agents.

[0040]

[0043] As used herein, "label zone" refers to a portion of an assay or device, such as a lateral flow device or strip test, configured to contain a mobilizable reagent for labeling an analyte of interest, such as an antibody or antigen that binds to the analyte of interest. In some embodiments, the mobilizable binding agent is specific for the analyte of interest and is detectable, e.g., optically or visually detectable.

[0041]

[0044] As used herein, "capture zone" refers to a portion of an assay or device, such as a lateral flow device or strip test, configured to contain immobilized reagents with specific binding for capturing a labeled analyte of interest, such as a capture of a mobile detectable agent conjugated to the analyte of interest. In some embodiments, the capture zone may contain an immobilized antibody or antigen that binds to the analyte of interest and a conjugate of the analyte of interest and a mobile detectable agent. In some embodiments, the agent in the capture zone causes the labeled analyte of interest, if present in the sample being analyzed, to accumulate at a test line where it can be visually or optically inspected and / or detected.

[0042]

[0045] As used herein, "liquid flow path" refers to the path of an assay, such as a lateral flow immunoassay, in which an applied sample flows in an upstream to downstream direction. Thus, in some embodiments, a lateral flow device comprises an upstream sample receiving zone, a labeling zone downstream of the sample receiving zone, and a capture zone downstream of the labeling zone, all of which are in the same liquid flow path such that added sample flows from the sample receiving zone, through the labeling zone, to the capture zone.

[0043]

[0046] In some embodiments, the lateral flow devices described herein comprise a sample receiving zone including a sample pad impregnated with an agglutinating agent, such as a lectin or red blood cell specific antibody, such that the red blood cells are chemically and / or physically retained or captured within the pad material, and the agglutinating agent interacts with the red blood cells. The systems, methods, and kits may include devices as described herein, blood capillaries such as capillaries with volume monitoring mechanisms for sample collection. The embodiments may facilitate removal of red blood cells from a whole blood sample by venipuncture or finger prick, where a whole blood sample is added to the sample receiving zone of a lateral flow immunoassay, while the sample travels through the lateral flow device. Thus, plasma and buffer pass through the test strip for analysis, and red blood cells are agglutinated and captured / retained on the sample pad in the sample receiving zone. The agglutinating agent and devices and methods including same may simplify the end user's workflow and may be broadly applicable to assays utilizing a whole blood sample for detecting an analyte of interest, particularly lateral flow immunoassay strip tests for analyzing whole blood for an analyte of interest.

[0044]

[0047] In some embodiments, the lateral flow device comprises a sample receiving zone including a sample pad configured to physically interact with red blood cells such that the red blood cells are retained or trapped within the pad material. In such embodiments, the sample pad comprises a means for physical interaction with red blood cells such that the sample pad maintains, retains, traps, and / or otherwise slows the flow of red blood cells from the sample pad to a downstream portion of the lateral flow strip test device. For example, the sample pad can comprise a means for physical interaction with red blood cells having a structure such as a net, mesh, screen, or lattice having an appropriate mesh size to physically retain and / or trap red blood cells within the sample pad of the lateral flow strip test device.

[0045]

[0048] In another embodiment, the aggregation and capture of red blood cells on the sample pad of the sample receiving zone of the lateral flow device improves the efficiency of plasma separation. Specifically, the capture of red blood cells on the agglutinant-impregnated sample pad requires only the addition of a whole blood sample in a buffer to the sample receiving zone, and the red blood cells are agglutinated, captured, and retained on the sample pad. This prevents the red blood cells from entering the sample labeling or testing zone of the lateral flow immunoassay, and therefore prevents the red blood cells from interfering with the generation or interpretation of the strip test results. The aggregation of red blood cells on the agglutinant-impregnated sample pad allows the red blood cells to be captured and retained on the sample pad without lysing or leaking from the sample pad to downstream components and areas of the lateral flow immunoassay device. This prevents the red blood cells from interfering with downstream agents for labeling and immobilization of the analyte of interest that may also be present in the whole blood sample being tested.

[0046] kit

[0049] According to embodiments, kits using devices containing the agglutinating agents are also provided. In addition to the agglutinating agents, devices, and methods described herein, instructions and additional components, such as sample collection means, are also provided. In some embodiments, the means or device for collecting a whole blood sample may include a capillary tube.

[0047]

[0050] According to embodiments, a kit of parts may include a device as described herein, as well as additional components for sample collection, sample dilution (buffers), and / or use of the device. Instructions for Use ("DFU") may be included in the kit. One or more collection devices may be provided, and such collection devices may be disposable, intended for a single use.

[0048]

[0051] method

[0052] According to other embodiments, methods are provided that employ components of devices or kits as described herein that, in some embodiments, accomplish analysis of a fluid sample, such as analysis of a whole blood sample for the presence or absence of an analyte of interest, without interference from certain components of the whole blood sample, such as red blood cells. Thus, a method of detecting the presence or absence of an analyte of interest in a whole blood sample, in embodiments, includes providing a device as described herein, placing a whole blood sample in the device, such as on or in a sample receiving zone that includes an agglutinant-impregnated sample pad, where red blood cells are retained in the sample receiving zone and the presence or absence of a labeled analyte immobilized in the capture zone is visualized.

[0049]

[0053] The method may employ components of a system or kit as described herein to achieve agglutination of red blood cells present in a whole blood sample. According to some embodiments, a sample such as whole blood is drawn, applied to the device, and agglutinated, such as by agglutination with an agglutinating agent present in a sample pad in a sample receiving zone of a lateral flow immunoassay device, and the agglutinated red blood cells are captured / retained on the sample pad while the remainder of the sample is flowed downstream for analysis for the presence of an analyte of interest by interaction of the sample with components present in the label zone and test zone. EXAMPLES

[0050]

[0054] The following examples are illustrative in nature and are not intended to be limiting in any way.

[0051] Example 1: Human hemagglutination assay using various lectins

[0055] A study was conducted to evaluate the ability of various lectins to agglutinate human red blood cells. The lectins tested were biotinylated forms commercially available from Vector Labs with sugar specificity for mannose, galactose, fucose, N-acetylgalactosamine, and N-acetylglucosamine. Specifically, the lectins tested included biotinylated forms of concanavalin A (Con A), soybean (glycine max) agglutinin (SBA), wheat germ (triticum vulgaris) agglutinin (WGA), Ulex europaeus agglutinin I (UEA I), peanut (arachis hypogaea) agglutinin (PNA), pea (pisum sativum) agglutinin (PSA), lentil (lens culinaris) agglutinin (LCA), and phaseolus vulgaris hemagglutinin (PHA-E).

[0052]

[0056] Agglutination assays were performed in round-bottom 96-well plates using an orbital shaker / incubator for mixing and temperature control. Lectins were analyzed for their ability to agglutinate pooled human red blood cells suspended in 10% PBS solution (Rockland #R407-0050). Agglutination assays for each lectin were performed with and without the addition of streptavidin (SA).

[0053]

[0057] Serially diluted lectin solutions were prepared with and without streptavidin, and 95 μL of the serially diluted lectin solutions were pipetted into wells of a 96-well plate. 5 μL of pooled human red blood cell suspension was then added to the lectin solution. The solution was mixed thoroughly by pipetting and on an orbital shaker. The lectin / human red blood cell mixtures were incubated for 1 hour at room temperature. After 1 hour of incubation, the agglutination results were visualized.

[0054]

[0058] The results of the agglutination assay of several lectins are shown in Figure 1 as follows: Column 1: WGA without SA, Column 2: UEA without SA, Column 3: PNA without SA, Column 4: PSA without SA, Column 5: LCA without SA, Column 6: PHA-E without SA, Column 7: WGA with SA, Column 8: UEA with SA, Column 9: PNA with SA, Column 10: PSA with SA, Column 11: LCA with SA, Column 12: PHA-E with SA. The lectin concentrations in the test wells after serial dilution are: Row A: 100 μg / mL, Row B: 30 μg / mL, Row C: 10 μg / mL, Row D: 3 μg / mL, Row E: 1 μg / mL, Row F: 0 μg / mL.

[0055]

[0059] Based on the results of the agglutination assay, the 50% effective concentrations (EC50) of the tested lectins were calculated, as shown in Table 2.

[0056] [Table 2]

[0057]

[0060] The data demonstrate that lectins with various carbohydrate specificities confer hemagglutination activity on pooled human red blood cells (hRBCs). In addition, some biotinylated lectins, such as WGA, UEA-I, PSA, LCA, and PHA-E, improve hemagglutination of pooled human red blood cells when tested in combination with streptavidin, as demonstrated by low EC50 values.

[0058] Example 2: Human erythrocyte agglutination test using lectin and anti-human erythrocyte antibody

[0061] A study was conducted to evaluate the ability of various lectins and anti-human red blood cell antibodies to agglutinate human red blood cells. The lectins tested were commercially available biotinylated forms from Vector Labs with sugar specificity for mannose and galactose. Specifically, the lectins tested included biotinylated forms of Concanavalin A (Con A) and Glycine max (soybean) agglutinin (SBA). The anti-human red blood cell antibody tested was a biotinylated monoclonal anti-hRBC H101 engineered at Avitag and biotinylated in vitro. Anti-hRBC mAb H101 was recombinantly produced to exhibit specificity for the H antigen of hRBC.

[0059]

[0062] Agglutination assays were performed in round-bottom 96-well plates using an orbital shaker / incubator for mixing and temperature control. Anti-hRBC mAb H101 and lectins were analyzed for their ability to agglutinate pooled human red blood cells suspended in 10% PBS solution (Rockland #R407-0050). Agglutination assays for each agglutinant (Ab and lectin) were performed with and without the addition of streptavidin (SA).

[0060]

[0063] Serially diluted antibody and lectin solutions were prepared with and without streptavidin, and 95 μL of the serially diluted antibody / lectin solutions were pipetted into wells of a 96-well plate. 5 μL of pooled human red blood cell suspension was then added to the lectin solution. The solutions were mixed thoroughly by pipetting and on an orbital shaker. The agglutinant / human red blood cell mixtures were incubated for 1 hour at room temperature. After 1 hour of incubation, the agglutination results were visualized.

[0061]

[0064] The results of the agglutination assay for several lectins are shown in Figure 2 as follows: Column 1: mAb H101 without SA, Column 2: Con A without SA, Column 3: SBA without SA, Column 4: mAb H101 with SA, Column 5: Con A with SA, Column 6: SBA with SA. The concentrations of antibodies and lectins in the test wells after serial dilution are: Row A: 100 μg / mL, Row B: 30 μg / mL, Row C: 10 μg / mL, Row D: 3 μg / mL, Row E: 1 μg / mL, Row F: 0 μg / mL.

[0062]

[0065] Based on the results of the agglutination assay, the median effective concentration (EC50) of the tested antibodies and lectins was calculated, as shown in Table 3.

[0063] [Table 3]

[0064]

[0066] The data demonstrate that mAb H101 produces hemagglutination activity of pooled human red blood cells (hRBCs) with or without streptavidin, as demonstrated by low EC50 values.

[0065] Example 3: Evaluation of a lateral flow immunoassay containing a hemagglutinating agent

[0067] A study was conducted to evaluate the activity of mAb H101 as a hemagglutinating agent when present in a lateral flow strip test. The same mAb H101 antibody as described above in the agglutination assay of Example 2 was added to the sample receiving zone of a lateral flow strip test to analyze whether red blood cells could be captured / retained on the sample pad when a whole blood sample was added to the sample receiving zone of the lateral flow immunoassay (strip test).

[0066]

[0068] The lateral flow strip test consisted of three different treatments applied to the sample pads of the sample receiving zone of the lateral flow test. The sample pad treatments included condition (1) a blank control without any antibody (Figure 3A), condition (2) mAb H101 antibody at 0.74 mg / mL in PBS (Figure 3B), and condition (3) mAb H101 antibody at 0.74 mg / mL and 1 mg / mL streptavidin in PBS (Figure 3C). 1 mL of the prepared treatment solution was added to each bottom of the sample pad and allowed to dry at room temperature for 1 h.

[0067]

[0069] In configurations where the pad treatment included condition (1), a blank control with no antibody, retention of red blood cells on the sample pad is achieved by physical means, such as trapping red blood cells on the reticulated mesh of the sample pad (FIG. 3A). In configurations where the pad treatment included conditions (1) and (2), mAb H101 with or without streptavidin, retention of red blood cells on the sample pad is achieved by agglutination of red blood cells by antibody and / or streptavidin in addition to physical means, such as trapping agglutinated red blood cells on the reticulated mesh of the sample pad (FIGS. 3B and 3C).

[0068]

[0070] Next, various amounts of pooled human red blood cells (Rockland #R407-0050) suspended in 10% PBS solution were pipetted onto the sample pad, followed by a chase volume of 10% PBS to a final volume of 100 μL. Four different red blood cell / PBS volumes were tested: (1) 50 μL RBCs + 50 μL PBS; (2) 25 μL RBCs + 75 μL PBS; (3) 12.5 μL RBCs + 87.5 μL PBS; (4) 6.2 μL RBCs + 93.8 μL PBS.

[0069]

[0071] After adding red blood cells and additional PBS, the lateral flow strip test was performed for 10 minutes at room temperature. After 10 minutes, capture / retention of red blood cells in the sample receiving zone was visualized for conditions 1, 2, and 3, and RBC / PBS volumes 1, 2, 3, and 4, as shown in FIG.

[0070]

[0072] This data demonstrates that the control strip retains red blood cells on the sample pad through physical capture (condition 1, see Figure 3A). Additionally, the inclusion of biotinylated mAb H101 efficiently captured hRBCs at sample receiving zones containing RBC volumes of approximately 12.5 μL (condition 2, see Figure 3B). Furthermore, in the presence of streptavidin, biotinylated mAb H101 was even more effective at capturing / retaining hRBCs at sample receiving zones containing RBC volumes of at least approximately 50 uL (condition 3, see Figure 3C).

[0071]

[0073] The red blood cell retention and agglutination agents, devices, and methods described herein have several advantages. For example, a lateral flow immunoassay device that includes a physical red blood cell capture means and / or agglutination agent in the sample receiving zone can be used to analyze a whole blood sample without optical interference from red blood cells. Also, the described physical capture and / or hemagglutination lateral flow assays have the advantage that they allow separation of red blood cells from a whole blood sample without additional steps, devices, or operations, but instead, this separation occurs directly in the sample receiving zone of the lateral flow immunoassay device. This allows laboratory assistants, or other device users, such as users of over-the-counter home strip tests, to quickly, simply, and accurately use the lateral flow strip test to analyze whole blood, such as blood from a finger prick, without the need for complex and time-consuming extra steps. By way of further example, the described whole blood capture / agglutination lateral flow immunoassay provides a single-use separation and analysis device without the need for additional equipment and without the need for laboratory equipment such as a centrifuge to separate blood fractions.

[0072]

[0074] The foregoing description is presented to enable one skilled in the art to practice the various configurations described herein. While the subject technology has been specifically described with reference to various figures and configurations, it should be understood that these are for illustrative purposes only and should not be construed as limiting the scope of the subject technology.

[0073]

[0075] The use of a phrase such as "embodiment" or "aspect" does not imply that such aspect is essential to the subject technology or that such aspect applies to all configurations of the subject technology. Disclosure related to an aspect may apply to all configurations or one or more configurations. An aspect may provide one or more examples of the disclosure. A phrase such as "aspect" may refer to one or more aspects, and vice versa. A phrase such as "embodiment" does not imply that such an embodiment is essential to the subject technology or that such an embodiment applies to all configurations of the subject technology. Disclosure related to an embodiment may apply to all embodiments or one or more embodiments. An embodiment may provide one or more examples of the disclosure. Such a phrase "embodiment" may refer to one or more embodiments, and vice versa. A phrase such as "configuration" does not imply that such an embodiment is essential to the subject technology or that such an embodiment applies to all configurations of the subject technology. Disclosure related to a configuration may apply to all configurations or one or more configurations. A configuration may provide one or more examples of the disclosure. A phrase such as "a configuration" may refer to one or more configurations, and vice versa.

[0074]

[0076] There may be many other ways to implement the subject technology. The various functions and elements described herein may be partitioned differently than shown without departing from the scope of the subject technology. Various modifications to these configurations will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other configurations. Thus, numerous changes and modifications may be made to the subject technology by those skilled in the art without departing from the scope of the subject technology.

[0075]

[0077] It will be understood that the particular order or hierarchy of steps in the disclosed processes is illustrative of example approaches. Based on design preferences, it will be understood that the particular order or hierarchy of steps in the processes can be rearranged. Some steps may also be performed simultaneously. The accompanying method claims present elements of the various steps in a sample order, and are not intended to be limited to the particular order or hierarchy presented.

[0076]

[0078] As used herein, the phrase "at least one" preceding a series of items with the term "and" or "or" separating any of the items modifies the list as a whole, rather than each member (i.e., each item) of the list. The phrase "at least one" does not require the selection of at least one of each item listed, rather the phrase allows for the meaning of including at least one of any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. By way of example, the phrase "at least one of A, B, and C" or "at least one of A, B, or C" refers, respectively, to only A, only B, or only C, any combination of A, B, and C, and / or at least one of each of A, B, and C.

[0077]

[0079] Terms such as "top," "bottom," "front," "rear," and the like, as used in this disclosure, should be understood to refer to any frame of reference other than the typical gravitational frame. Thus, top, bottom, front, and rear surfaces may extend upward, downward, diagonally, or horizontally in the gravitational frame.

[0078]

[0080] Furthermore, to the extent that terms such as "including" or "having" are used in this specification or the claims, such terms are intended to be encompassed in the same manner as the term "comprising," which when used as a transitional term in the claims is interpreted as "comprising."

[0079]

[0081] The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

[0080]

[0082] Reference to a singular element is not intended to mean "one and only one" unless otherwise specified, but rather "one or more." Masculine pronouns (e.g., his) include feminine and neuter genders (e.g., her and its), and vice versa. The term "several" refers to one or more. Underlined and / or italicized headings and subheadings are used for convenience only, do not limit the subject art, and are not to be referred to in connection with the interpretation of the subject art description. All structural and functional equivalents to the elements of the various configurations described throughout this disclosure that are known or later become known to those skilled in the art are expressly incorporated herein by reference and are intended to be encompassed by the subject art. Furthermore, nothing disclosed herein is intended to be made available to the public, regardless of whether such disclosure is expressly set forth in the above description.

[0081]

[0083] Although specific aspects and embodiments of the subject technology have been described, they have been presented by way of example only and are not intended to limit the scope of the subject technology. Indeed, the methods and systems described herein may be embodied in a variety of other forms without departing from the spirit thereof. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the subject technology.

Claims

1. 1. A device for detecting an analyte of interest in a whole blood sample, comprising: (i) a sample receiving zone containing a non-lytic hemagglutinating agent, the hemagglutinating agent being a biotinylated antibody or a biotinylated lectin that captures red blood cells from a whole blood sample added to the sample receiving zone without lysing the red blood cells; (ii) a labeling zone comprising means for specifically labeling said analyte of interest; and (iii) a capture zone comprising means for specifically binding and immobilizing said labeled analyte of interest; A device wherein the sample receiving zone, the label zone, and the capture zone are disposed in a liquid flow path.

2. The device of claim 1 , wherein the hemagglutinating agent binds to the H antigen of human red blood cells.

3. The device of claim 2 , wherein the antibody is a monoclonal antibody.

4. The device of claim 2 , wherein the antibody is a polyclonal antibody.

5. The device of claim 1 , wherein the hemagglutinating agent is self-immobilized in the sample receiving zone.

6. The device of claim 5 , wherein the sample receiving zone further comprises streptavidin.

7. The device of claim 6 , wherein the streptavidin is on particles immobilized in the sample receiving zone.

8. The device of claim 1 , wherein the hemagglutinating agent is a biotinylated lectin.

9. 2. The device of claim 1, wherein the hemagglutinating agent is wheat germ agglutinin, gorse (Ulex europaeus) agglutinin I, pea (Pisum sativum) agglutinin, lentil (Lens culinaris) agglutinin, or kidney bean (Phaseolus vulgaris) hemagglutinin.

10. The device of claim 8 , wherein the lectin self-immobilizes in the sample receiving zone.

11. The device of claim 8 , wherein the sample receiving zone further comprises streptavidin.

12. 12. The device of claim 11, wherein the streptavidin is on particles immobilized in the sample receiving zone.

13. 10. The device of claim 1, wherein said means for specifically labeling said analyte of interest is a detectable antibody.

14. The device of claim 13 , wherein the detectable antibody is a monoclonal antibody.

15. The device of claim 13 , wherein the detectable antibody is a polyclonal antibody.

16. 10. The device of claim 1, wherein said means for specifically labeling said analyte of interest is a detectable antigen.

17. The device of claim 16 , wherein the antigen is a protein.

18. The device of claim 16 , wherein the antigen is a peptide.

19. The device of claim 16 , wherein the antigen is a macromolecule.

20. The device of claim 16 , wherein the antigen is a small molecule.

21. 10. The device of claim 1, wherein said means for specifically binding and immobilizing said labeled analyte of interest is an immobilized antibody.

22. 22. The device of claim 21, wherein the immobilized antibody is a monoclonal antibody.

23. 22. The device of claim 21, wherein the immobilized antibody is a polyclonal antibody.

24. 10. The device of claim 1, wherein said means for specifically binding and immobilizing said labeled analyte of interest is an immobilized antigen.

25. 25. The device of claim 24, wherein the antigen is a protein.

26. 25. The device of claim 24, wherein the antigen is a peptide.

27. The device of claim 24 , wherein the antigen is a macromolecule.

28. 25. The device of claim 24, wherein the antigen is a small molecule.

29. 1. A method for detecting the presence or absence of an analyte of interest in a whole blood sample, comprising: Providing a device according to any one of claims 1 to 28, placing a whole blood sample into the device wherein red blood cells are retained in the sample receiving zone; and determining the presence or absence of a labeled analyte immobilized in said capture zone.

30. 30. The method of claim 29, further comprising providing a device for collecting the whole blood sample and collecting the whole blood sample in the device.

31. 29. A device according to any one of claims 1 to 28, a device for collecting a whole blood sample; and Kit includes instructions for use.

32. 32. The kit of claim 31, wherein the device is a capillary tube.

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