Assay and method for determining antibody fragments in a sample

Abstract

The present disclosure provides a method for determining the presence or quantity of antibody fragments in a sample by capturing said fragments on a line or zone comprising immobilized antibodies or binders specific to the antibody fragments The method may further comprise detecting whole antibody on a line comprising immobilized binders specific to the whole antibody. The immobilized binders specific to the antibody fragments may be immobilized on a test line of a test strip, and the immobilized binders specific to the whole antibodies may be immobilized on a control line of the test strip. The capturing of the antibody fragments results in generation of a first result, and the binding of the whole antibody results in generation of a second result. The method may determine total antibody concentration by aggregating measurements of antibody fragments and intact antibodies.

Description

[0001] ASSAY AND METHOD FOR DETERMINING ANTIBODY FRAGMENTS IN A SAMPLE

[0002] FIELD OF INVENTION

[0003] The present disclosure relates to diagnostic assays for biological samples, and more particularly to an assay and method for quantifying antibody fragments in biological samples using lateral flow technology.

[0004] BACKGROUND

[0005] Point-of-care diagnostic testing has become increasingly important in healthcare and home-based applications due to its convenience, rapid results, and cost-effectiveness. Among various diagnostic platforms, lateral flow assays have emerged as a dominant technology, widely used for pregnancy testing, infectious disease detection, and molecular diagnostics. These assays offer advantages such as ease of use, portability, and minimal equipment requirements, making them suitable for diverse clinical and consumer settings.

[0006] Lateral flow assays operate on the principle of immunochromatography, enabling detection of biomolecules including proteins, hormones, and antibodies. While effective for full-length antibodies, these assays face challenges in detecting smaller molecular entities such as antibody fragments. Antibody fragments differ structurally from intact antibodies, possessing fewer binding sites and reduced stability, which affects their interaction with capture reagents and compromises assay sensitivity and specificity.

[0007] Thus, there is a need for an assay for quantifying the antibody fragments that overcomes the aforementioned limitations.

[0008] SUMMARY

[0009] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0010] In an aspect of the present disclosure, a method for determining the presence or quantity of antibody fragments in a sample includes detecting the presence of antibody fragments by capturing said fragments on a line or zone including immobilized antibodies or binders specific to the antibody fragments.

[0011] In some aspects, the immobilized antibodies, or binders specific to the antibody fragments are immobilized on the test line of a test strip.

[0012] In some aspects, capturing the antibody fragments by the immobilized antibodies or binders specific to the antibody fragments results in generation of a first result.

[0013] In some aspects, the method further includes detecting the presence of whole or intact antibody on a line or zone including immobilized antibodies or binders specific to the whole or intact antibody.

[0014] In some aspects, the immobilized antibodies, or binders specific to the whole or intact antibodies are immobilized on the control line or zone of the test strip.

[0015] In some aspects, binding of the whole or intact antibody with the immobilized antibodies or binders specific to the whole or intact antibody results in generation of a second result.

[0016] In some aspects, the method further includes determining the total antibody concentration in the biological sample by aggregating measurements of antibody fragments and measurements of intact antibodies.

[0017] In some aspects, the first and second result are visually distinct signals.

[0018] In some aspects, the antibody or binder is selected from the group including antiidiotype antibodies, anti -Fab antibodies, anti-Fc antibodies, Protein A, Protein G, Protein L, engineered protein scaffolds including Affibody molecules and Designed Ankyrin Repeat Proteins (DARPins), nanobodies, aptamers, Fc-binding peptides, and combinations thereof. In some aspects, the biological sample is selected from a group including urine, semen, saliva, tears, blood, sweat, or pre-ejaculate.

[0019] In another aspect of the present disclosure, a method for determining an antibody in a biological sample by an assay includes detecting antibody fragments by capturing said fragments on a test line including immobilized antibodies or binders specific to the antibody fragments and generating a first result; detecting whole or intact antibodies by capturing said antibodies on a control line including immobilized antibodies or binders specific to the whole or intact antibodies and generating a second result; and analyzing the first result and the second result to determine the concentration of the antibody present in the biological sample.

[0020] In some aspects, the assay further includes a test strip including a sample receiving pad configured for receiving biological sample from a subject; a conjugate pad having one or more detector reagents configured to bind with the received biological sample; a test line immobilized with antibodies or binders specific to the antibody fragments and configured to capture antibody fragments; and a control line immobilized with antibodies or binders specific to the whole or intact antibody and configured to capture said antibody.

[0021] In some aspects, analyzing the total antibody concentration includes computing the concentration based on a combined analysis of the first result and the second result.

[0022] In some aspects, the first and second result are visually distinct signals.

[0023] In some aspects, the assay is a lateral flow assay.

[0024] In some aspects, the one or more detector reagents are selected from a group including an enzyme label, fluorescent labels, quantum dots, or luminescent nanoparticles.

[0025] In some aspects, the biological sample is selected from a group including urine, semen, saliva, tears, blood, sweat, or pre-ejaculate.

[0026] In some aspects, the antibody or binder is selected from the group including antiidiotype antibodies, anti -Fab antibodies, anti-Fc antibodies, Protein A, Protein G, Protein L, engineered protein scaffolds including Affibody molecules and Designed Ankyrin Repeat Proteins (DARPins), nanobodies, aptamers, Fc-binding peptides, and combinations thereof.

[0027] In some aspects, the analysis is performed using a handheld imaging sensor configured to detect colorimetric, fluorescent, or chemiluminescent signals.

[0028] In yet another aspect of the present disclosure, an assay for determining the presence or quantity of antibody fragments in a sample includes capturing said fragments on a line or zone including immobilized antibodies or binders specific to the antibody fragments.

[0029] In some aspects, the assay further includes a test strip including a sample receiving pad configured for receiving biological sample from a subject; a conjugate pad having one or more detector reagents configured to bind with the received biological sample; a test line immobilized with antibodies or binders specific to the antibody fragments and configured to capture antibody fragments; and a control line immobilized with antibodies or binders specific to the whole or intact antibody and configured to capture said antibody.

[0030] In some aspects, the assay is a lateral flow assay.

[0031] In some aspects, the one or more detector reagents are selected from a group including an enzyme label, fluorescent labels, quantum dots, or luminescent nanoparticles.

[0032] In some aspects, the biological sample is selected from a group including urine, semen, saliva, tears, blood, sweat, or pre-ejaculate.

[0033] In some aspects, the sample flows from a first end to a second end of the test strip by capillary action.

[0034] In some aspects, the antibody or binder is selected from the group including antiidiotype antibodies, anti -Fab antibodies, anti-Fc antibodies, Protein A, Protein G, Protein L, engineered protein scaffolds including Affibody molecules and Designed Ankyrin Repeat Proteins (DARPins), nanobodies, aptamers, Fc-binding peptides, and combinations thereof.

[0035] In yet another aspect of the present disclosure, an assay for determining quantity of antibody in a biological sample includes a test strip configured for receiving biological sample from a subject; a conjugate pad including one or more detector reagents configured to bind with a target molecule in the biological sample and release antibody fragments or antibody nanoparticles; a test line immobilized with antibodies or binders specific to the antibody fragments and configured to capture said antibody fragments; and a control line immobilized with antibodies or binders specific to the whole or intact antibody and configured to capture said antibody; wherein analyzing the total antibody concentration includes computing the concentration based on a combined analysis of the first result and the second result.

[0036] The foregoing general description of the illustrative embodiments and the following detailed description thereof are merely exemplary aspects of the teachings of this disclosure and are not restrictive.

[0037] BRIEF DESCRIPTION OF FIGURES

[0038] Non-limiting and non-exhaustive examples are described with reference to the following figures.

[0039] FIG. 1 illustrates a method for determining total antibody concentration in a biological sample, according to aspects of the present disclosure.

[0040] FIG. 2 illustrates an assay for determining antibody fragments in a sample, according to aspects of the present disclosure.

[0041] DETAILED DESCRIPTION

[0042] The following description sets forth exemplary aspects of the present disclosure. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure. Rather, the description also encompasses combinations and modifications to those exemplary aspects described herein. This section is intended to provide an explanation and description of various possible aspects of the present disclosure. The aspects used herein, and the various features and advantageous details thereof are explained more fully with reference to non-limiting aspects illustrated in the accompanying drawing / s and detailed in the following description. The examples used herein are intended only to facilitate an understanding of ways in which the aspects may be practiced and to enable the person skilled in the art to practice the aspects used herein. Also, the examples / aspects described herein should not be construed as limiting the scope of the aspects herein. Various aspects of the present disclosure provide an assay for quantifying antibody fragments in a sample. The following description provides specific details of certain aspects of the disclosure illustrated in the drawings to provide a thorough understanding of those aspects. It should be recognized, however, that the present disclosure can be reflected in additional aspects and the disclosure may be practiced without some of the details in the following description. The various aspects including the example aspects are now described more fully with reference to the accompanying drawings, in which the various aspects of the disclosure are shown. The disclosure may, however, be embodied in different forms and should not be construed as limited to the aspects set forth herein.

[0043] Rather, these aspects are provided so that this disclosure is thorough and complete, and fully conveys the scope of the disclosure to those skilled in the art. In the drawings, the sizes of components may be exaggerated for clarity. The subject matter of example aspects, as disclosed herein, is described with specificity to meet statutory requirements. However, the description itself is not intended to limit the scope of this patent. Rather, the inventor / inventors have contemplated that the claimed subject matter might also be embodied in other ways, to include different features or combinations of features similar to the ones described in this document, in conjunction with other technologies. Generally, the various aspects including the example aspects relate to the assay for quantifying antibody fragments present in the biological sample. The terms "test strip" and "strip" may refer to similar meaning / interpretation and may be interchangeably used throughout the specification. Such test strips may be immunoassay strips or immunochromatographic strips. The test strip may refer to a capillary, an atomizer, medium or base, or any paper-based device on which a biochemical reagent and other antibodies are integrated / affixed / immobilized to react with the one or more analytes present in the biological sample associated with the user. The terms 'user,' 'patient,' 'body,' and 'subject' may refer to similar meaning / interpretation and may be interchangeably used throughout the specification. The term "assay" may refer to a "lateral flow assay" or a "lateral flow assay cartridge" and are interchangeably used across the context.

[0044] The terms "bodily fluid," "biological sample" and "sample" may be interchangeably used throughout the specification. Examples of the biological samples may include, but are not limited to, urine, semen, pre-ejaculate, saliva, blood, sweat, tears, and the like. Aspects of the present disclosure are intended to include or otherwise cover any type of biological sample, without deviating from the scope of the present disclosure.

[0045] As used herein, the term "whole antibody" or "intact antibody" refers to a complete immunoglobulin molecule comprising two heavy chains and two light chains arranged in a Y-shaped structure, including both antigen-binding regions (Fab) and the constant region (Fc). Intact antibodies retain full structural integrity and functional capability for antigen recognition and effector activity.

[0046] As used herein, the term "antibody fragments" refers to portions of an antibody molecule that include at least one antigen-binding site but lack the full structure of an intact antibody. Examples include Fab, F(ab’) 2, scFv, and other engineered or naturally occurring fragments generated by enzymatic cleavage or recombinant methods. These fragments maintain antigen-binding specificity but may differ in size, valency, and functional properties compared to whole antibodies.

[0047] As used herein, the term "antibody nanoparticles" refers to nanoscale particulate structures comprising antibody molecules or antibody fragments, either aggregated or engineered into nanoparticle constructs, that preserve antigen-binding functionality. Such nanoparticles may occur naturally in biological samples or be formed during assay processing and are detectable through interaction with detector reagents configured to bind, release, or signal their presence.

[0048] As used herein, the term "target molecule" refers to a specific component within a biological sample that is intended to interact with one or more detector reagents in the assay. The target molecule may include an antigen, epitope, or other binding site associated with antibody fragments or antibody nanoparticles, and is configured to facilitate binding, release, or detection of said fragments or nanoparticles during the assay process.

[0049] Aspects of the present disclosure are intended to include or otherwise cover any configuration of capture regions, whether referred to as test lines, control lines, zones, or equivalent structures, without deviating from the scope of the present disclosure. References to “test line” and “control line” in this disclosure are intended for illustrative purposes only. These terms should not be construed as limiting the invention to specific line-based configurations. The described features may be implemented as zones, regions, or any equivalent structures capable of performing the stated functions within the assay format.

[0050] Antibodies in biological samples frequently undergo fragmentation due to mechanisms such as enzymatic cleavage, oxidative stress, mechanical shear forces, pH-induced degradation, and thermal instability. These processes often target regions like the hinge, generating Fab, Fc, and smaller peptide fragments. Conformational changes induced by antigen binding can further destabilize antibody structure, accelerating fragmentation. Current antibody quantification assays primarily detect intact antibodies and fail to account for fragmented forms, resulting in incomplete or inaccurate measurements of total antibody concentration. Since total antibody concentration comprises both intact antibodies and fragments, there remains a critical need for a reliable and practical solution that enables simultaneous detection and quantification of all antibody populations present in a sample.

[0051] The present disclosure provides an assay and method for comprehensive quantification of antibody and antibody fragments in a biological sample. The assay includes a test strip configured with multiple sets of capture antibodies immobilized at distinct regions, each designed to selectively bind different components present in the sample. These components include intact antibodies, antibody fragments such as Fab and Fc regions, and target molecules or antigen-antibody complexes. The test strip may operate in a lateral flow format, enabling sample migration by capillary action and facilitating simultaneous capture of all relevant populations. Detection can be achieved through labeled secondary antibodies or signal-generating reagents, allowing visual or instrument-based readout. By incorporating multiple capture zones and specific binding interactions, the invention overcomes the limitations of conventional assays that fail to account for fragmented antibodies, thereby providing accurate and complete measurement of total antibody concentration. The dual detection system architecture employs two distinct capture lines, one designed to bind antibody fragments and the other to bind whole antibodies. This configuration enables comprehensive quantification by accounting for fragmented molecules that would otherwise remain undetected, ensuring that all antibody species present in the sample contribute to the total concentration measurement.

[0052] FIG. 1 illustrates a method 200 for determining antibody concentration in a biological sample. The method 200 may include a sequential multi-step process designed to comprehensively analyze both fragmented and intact antibody populations within a single assay format. The method 200 may provide enhanced accuracy in total antibody quantification by separately detecting and measuring different antibody forms present in biological samples.

[0053] The method 200 may begin with step 202, which involves introducing a biological sample to a test strip 102 via a sample receiving pad 108. The biological sample may be selected from a group including, but not limited to, a urine, semen, saliva, tears, blood, sweat or pre- ejaculate. Aspects of the present disclosure are intended to include or otherwise cover any type of biological sample, without deviating from the scope of the present disclosure. The introduction of the biological sample may be accomplished through various application methods including dropper application for precise volume control, dipping the test strip 102 into the sample, or direct pouring of the sample onto the sample receiving pad 108. Aspects of the present disclosure are intended to include or otherwise cover any type of sample introduction method, without deviating from the scope of the present disclosure. The sample receiving pad 108 may be configured to accommodate different sample volumes ranging from microliters to milliliters and viscosities while ensuring uniform distribution across the width of the test strip 102.

[0054] In some aspects, the biological sample may be pre-treated or conditioned before application to the test strip 102. Pre-treatment may include dilution with appropriate buffers to optimize pH and ionic strength, filtration to remove particulate matter that could interfere with flow dynamics, or addition of stabilizing agents to preserve antibody integrity during processing. The sample receiving pad 108 may incorporate pre-treatment reagents or buffers that automatically condition the biological sample upon contact, eliminating the need for separate sample preparation steps.

[0055] The method 200 may be implemented using various assay formats including lateral flow assay, ELISA, immunoassay, Enzyme immunoassays (EIAs) and chemiluminescent immunoassays (CLIAs), or immune-chromatographic assay. Aspects of the present disclosure are intended to include or otherwise cover any type of assay, without deviating from the scope of the present disclosure. In some aspects, the method 200 may utilize a lateral flow assay format that enables rapid point-of-care testing without requiring sophisticated laboratory equipment or extensive user training. The lateral flow format may provide results within 5-30 minutes of sample application, making it suitable for clinical, field, or home-based testing applications. The method 200 may proceed to step 204, which involves binding a target molecule in the sample with one or more detector reagents on a conjugate pad 110. The conjugate pad 110 may be positioned downstream of the sample receiving pad 108 to receive the biological sample after initial uptake and conditioning. The one or more detector reagents may be selected from a group including, but not limited to, enzyme labels such as horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, or 0- galactosidase that catalyze colorimetric or chemiluminescent reactions, fluorescent labels including fluorescein isothiocyanate (FITC), rhodamine derivatives, Alexa Fluor dyes, or cyanine dyes for optical detection, quantum dots providing enhanced photostability and narrow emission spectra, or luminescent nanoparticles such as europium chelates or terbium complexes for time-resolved fluorescence detection. Aspects of the present disclosure are intended to include or otherwise cover any type of detector reagents, without deviating from the scope of the present disclosure.

[0056] In some aspects, the binding of the target molecule with the one or more detector reagents may result in conformational changes that may cause the controlled release of antibody fragments or antibody nanoparticles from the detector reagents. These conformational changes may be triggered by specific molecular recognition events between the target molecules and the one or more detector reagents, leading to structural rearrangements that may weaken intramolecular bonds, expose previously buried cleavage sites, or cause dissociation of multi-subunit detector reagent complexes.

[0057] In some embodiments, the one or more detector reagents may be engineered to undergo structural changes upon target binding, resulting in fragmentation or activation of a signal-generating component. For example, certain biosensor designs employ split enzyme systems where binding of a target molecule induces conformational rearrangement that separates inhibitory domains and restores enzymatic activity, producing a detectable signal. Such mechanisms demonstrate how binding-induced structural changes can be utilized to trigger fragmentation or functional switching of engineered proteins, enabling controlled signal generation within the assay 100.

[0058] In some aspects, the conformational changes may occur through various mechanisms including allosteric effects where target binding at one site induces structural changes at distant sites, competitive displacement where target binding displaces stabilizing cofactors or inhibitory molecules, or cooperative binding effects where multiple target molecules binding simultaneously amplify structural changes. The magnitude and kinetics of these conformational changes may be controlled through protein engineering approaches such as introduction of flexible linker regions, incorporation of protease-sensitive sequences, or modification of binding domain orientations.

[0059] The method 200 may continue with step 206, which may involve detecting antibody fragments by capturing said fragments on a test line 112 and generating a first result. The test line 112 may include immobilized antibodies or binders specific to the antibody fragments that have been released from the conjugate pad 110. The test line 112 may be positioned downstream of the conjugate pad 110 at a distance that allows sufficient time for conformational changes and fragment release to occur while maintaining optimal flow dynamics.

[0060] The capturing of the antibody fragments by the immobilized antibodies or binders specific to the antibody fragments may result in generation of the first result. The immobilized antibodies or binders may be selected from the group including, but not limited to, anti-idiotype antibodies that recognize variable regions of antibody fragments and provide specificity for particular antibody clones or families, anti-Fab antibodies that specifically bind to antigen-binding fragments and can distinguish between different Fab orientations or conformations, anti-Fc antibodies that recognize crystallizable fragment regions and may be specific for different immunoglobulin classes or subclasses, Protein A which binds to the Fc region of IgG antibodies from various species with high affinity, Protein G providing broader species reactivity and stronger binding to certain IgG subclasses compared to Protein A, Protein L which binds to the light chain variable regions and can capture antibody fragments lacking Fc regions, engineered protein scaffolds including Affibody molecules that provide customizable binding specificities through directed evolution approaches and Designed Ankyrin Repeat Proteins (DARPins) offering high stability and specific target recognition, nanobodies providing single-domain binding capabilities with enhanced stability and tissue penetration, aptamers providing nucleic acid-based recognition with high specificity and affinity that can be selected against virtually any target, Fc-binding peptides for synthetic binding interactions that can be chemically synthesized and modified, and combinations thereof. Aspects of the present disclosure are intended to include or otherwise cover any type of antibodies or binders, without deviating from the scope of the present disclosure.

[0061] In some aspects, the selection of specific antibodies or binders for the test line 112 may depend on the type of antibody fragments expected to be generated, the species origin of the target antibodies, and the desired specificity and sensitivity of the assay. For example, when detecting human antibody fragments, anti-human Fab antibodies or human-specific Protein A variants may be preferred. When broad-spectrum detection is desired, combinations of different binding molecules may be employed to capture various fragment types simultaneously.

[0062] The first result may be represented by a detectable signal that correlates with the concentration of antibody fragments present in the biological sample. The signal intensity may correlate directly with the amount of captured antibody fragments, enabling quantitative assessment of fragment concentrations through optical measurement techniques. The relationship between signal intensity and fragment concentration may follow linear, logarithmic, or sigmoidal curves depending on the binding kinetics, detection method, and concentration range being measured.

[0063] In some aspects, the signal generation mechanism may vary depending on the labeling strategy employed. For colorimetric detection, enzyme-conjugated secondary antibodies may catalyze substrate conversion reactions that produce colored products at the test line 112. For fluorescent detection, fluorophore-labeled antibodies may accumulate at the test line 112 and produce measurable fluorescence upon excitation. For chemiluminescent detection, enzyme labels may catalyze light-producing reactions that can be detected using photosensitive devices. Aspects of the present disclosure are intended to include or otherwise cover any type of signal, without deviating from the scope of the present disclosure.

[0064] The method 200 may proceed to step 208, which involves detecting whole or intact antibodies by capturing said antibodies on a control line 114 and generating a second result. The control line 114 may be positioned downstream of the test line 112 to capture intact antibodies that have not undergone fragmentation during processing at the conjugate pad 110. The control line 114 may include immobilized antibodies or binders specific to the whole or intact antibodies. Aspects of the present disclosure are intended to include or otherwise cover any type of antibodies or binders, without deviating from the scope of the present disclosure.

[0065] The binding of the whole or intact antibody with the immobilized antibodies or binders specific to the whole or intact antibody may result in generation of the second result. The antibodies or binders on the control line 114 may be selected from the same group as those used on the test line 112, but may be specifically chosen for their affinity toward intact antibody structures rather than fragmented forms. For example, antibodies that recognize epitopes spanning multiple domains or conformational epitopes that are only present in intact antibodies may be preferred for the control line 114.

[0066] In some aspects, the control line 114 may serve multiple functions including validation of assay performance by confirming the presence of antibodies in the sample, normalization of results by providing a reference signal for calculating relative fragment concentrations, and quality control by indicating proper sample flow and reagent function. The control line 114 may also enable calculation of fragmentation ratios by comparing the signal intensities between the test line 112 and control line 114. The first and second results may be visually distinct signals that enable differentiation between antibody fragments and whole antibodies. The visual distinction may be achieved through different approaches including the use of different chromogenic substrates that produce distinct colors when activated by different enzyme labels, different signal intensities based on the relative concentrations of fragments versus intact antibodies and the binding affinities of the capture reagents, different fluorescence wavelengths for multiplexed detection using spectrally distinct fluorophores, or different temporal development characteristics where signals develop at different rates or reach maximum intensity at different time points due to differences in binding kinetics or enzymatic activity.

[0067] In some aspects, the visual distinction may be enhanced through the use of different nanoparticle labels such as gold nanoparticles producing red signals, silver nanoparticles producing brown signals, or colored latex beads producing various colors depending on the incorporated dyes. The size and optical properties of these nanoparticles may be optimized to produce distinct visual signals that can be easily differentiated by users or optical detection systems. Aspects of the present disclosure are intended to include or otherwise cover any type of nanoparticles, without deviating from the scope of the present disclosure.

[0068] The method 200 may conclude with step 210, which involves analyzing the first result and the second result to determine the concentration of the antibody present in the biological sample. The analysis may include computing the total antibody concentration based on a combined analysis of the first result and the second result. This computational approach may involve direct summation of signal intensities from both detection zones, weighted averaging those accounts for differences in binding affinities or detection efficiencies between the test line 112 and control line 114, or mathematical modeling using predetermined calibration curves that relate signal intensities to antibody concentrations. The total antibody concentration may be determined by aggregating measurements of antibody fragments and measurements of intact antibodies using the mathematical relationship:

[0069] Total Antibody Concentration = Fragment Concentration + Intact Antibody Concentration.

[0070] This aggregation process may provide a comprehensive assessment of total antibody content that accounts for all antibody forms present in the biological sample, including both fragmented and intact populations that may exist simultaneously due to various degradation mechanisms or sample processing conditions.

[0071] In some aspects, the analysis may incorporate correction factors to account for differences in binding efficiencies, signal generation capabilities, or detection sensitivities between the fragment and intact antibody detection systems. These correction factors may be determined through calibration studies using known concentrations of purified antibody fragments and intact antibodies, and may be specific to particular antibody types, sample matrices, or assay conditions.

[0072] In some aspects, the analysis may be performed using a handheld imaging sensor configured to detect colorimetric, fluorescent, or chemiluminescent signals from both the test line 112 and control line 114. The handheld imaging sensor may include optical components such as light-emitting diodes (LEDs) or laser diodes for excitation, optical filters for wavelength selection and background rejection, photodetectors such as photodiodes, photomultiplier tubes, or charge-coupled device (CCD) sensors for signal capture, and microprocessors with embedded algorithms for signal analysis, background subtraction, and concentration calculation. Aspects of the present disclosure are intended to include or otherwise cover any type of imaging sensor, without deviating from the scope of the present disclosure.

[0073] The handheld imaging sensor may provide various output formats including numerical concentration values displayed on integrated screens, graphical representations showing signal intensities over time, data logging capabilities for storing multiple measurements, and wireless communication features for transmitting results to external devices or databases. The sensor may also include quality control features such as automatic calibration using internal standards, detection of assay failures or invalid results, and user guidance for proper sample application and result interpretation.

[0074] FIG. 2 discloses the assay 100 for quantifying concentration of antibody fragments in a sample through a comprehensive detection approach that captures both fragmented and intact antibody populations within a single integrated platform.

[0075] The assay 100 may include a test strip 102 designed to facilitate sequential sample processing through multiple functional zones. The test strip 102 may be configured as an elongated substrate that supports capillary-driven flow of biological samples through various detection regions while maintaining optimal flow rates and reagent interactions.

[0076] The test strip 102 may be fabricated from various membrane materials selected based on their flow characteristics, binding properties, and compatibility with different detection methods. Suitable materials may include, but not limited to, a nitrocellulose membranes providing high protein binding capacity and consistent flow rates, cellulose acetate membranes offering low background binding and good optical clarity, polycarbonate membranes with precise pore size control and chemical resistance, nylon membranes providing high mechanical strength and protein binding capacity, polytetrafluoroethylene (PTFE) membranes offering chemical inertness and hydrophobic properties, or composite membranes combining multiple materials to optimize specific performance characteristics. Aspects of the present disclosure are intended to include or otherwise cover any type of materials, without deviating from the scope of the present disclosure.

[0077] The test strip 102 may include a first end 104 and a second end 106 that define the directional flow path of the biological sample. The first end 104 may be positioned to receive the biological sample through direct application, dipping into a sample container, or connection to sample delivery systems such as pipettes or automated dispensers. The first end 104 may be designed with features such as sample wells, absorption pads, or flow restrictors to control sample introduction and initial flow dynamics. The second end 106 may serve as an overflow region where excess sample may be collected, absorbed, or vented to prevent backflow or sample pooling that could interfere with assay performance. The second end 106 may include absorbent materials such as cellulose pads, synthetic wicking materials, or porous polymers that can accommodate varying sample volumes while maintaining consistent flow characteristics.

[0078] The test strip 102 may include a sample receiving pad 108 positioned near the first end 104. The sample receiving pad 108 may serve as the initial contact point between the biological sample and the test strip 102, providing controlled sample uptake and conditioning before downstream processing. The sample receiving pad 108 may be constructed from absorbent materials such as glass fiber, cellulose, polyester, or synthetic polymer matrices that facilitate rapid sample uptake while filtering out particulate matter or cellular debris that could interfere with assay performance.

[0079] The sample receiving pad 108 may be treated with various reagents to optimize sample processing including pH buffers to maintain optimal conditions for antibody stability and binding interactions, detergents or surfactants to improve sample wetting and flow characteristics, stabilizing agents such as trehalose or bovine serum albumin to preserve antibody integrity during processing, or antimicrobial agents to prevent bacterial growth during extended storage or processing times.

[0080] The sample may flow from the first end 104 to the second end 106 of the test strip 102 by capillary action. The capillary action may be facilitated by the porous structure of the membrane materials, the surface tension properties of the biological sample, and the hydrophilic nature of the membrane surfaces. The capillary flow mechanism may provide consistent and reproducible sample flow rates without requiring external pumping mechanisms, electrical power, or user intervention beyond initial sample application. The flow rate through the test strip 102 may be controlled through various design parameters including membrane pore size and distribution, membrane thickness and porosity, surface chemistry and hydrophilicity, and the presence of flow control elements such as barriers, channels, or restrictors. Typical flow rates may range from 0.1 to 10 millimeters per minute, with optimal rates selected to provide sufficient contact time for binding interactions while maintaining reasonable assay completion times. Aspects of the present disclosure are intended to include or otherwise cover any type of parameters, without deviating from the scope of the present disclosure.

[0081] The test strip 102 may include a conjugate pad 110 located downstream of the sample receiving pad 108. The conjugate pad 110 may contain one or more detector reagents configured to bind with target molecules in the received biological sample and undergo conformational changes that result in the release of antibody fragments or antibody nanoparticles.

[0082] The one or more detector reagents may be stored in the conjugate pad 110 in a dried, stabilized form and may be reconstituted when the biological sample flows through the pad. The drying and stabilization process may involve lyophilization, spray drying, or air drying in the presence of stabilizing agents such as sugars, polymers, or proteins that preserve reagent activity during storage and enable rapid reconstitution upon sample contact. Aspects of the present disclosure are intended to include or otherwise cover any type of drying and stabilization process, without deviating from the scope of the present disclosure.

[0083] The one or more detector reagents may be selected from a group including, but not limited to, an enzyme labels, fluorescent labels, quantum dots, or luminescent nanoparticles that provide different detection modalities based on assay requirements, sensitivity needs, and available detection equipment. Enzyme labels such as horseradish peroxidase, alkaline phosphatase, or glucose oxidase may be preferred for colorimetric detection systems that can be read visually or with simple optical devices. Fluorescent labels such as fluorescein, rhodamine, or Alexa Fluor dyes may be preferred for high-sensitivity applications requiring fluorescence detection equipment. Quantum dots may be preferred for multiplexed detection applications requiring multiple distinct signals, while luminescent nanoparticles may be preferred for time- resolved detection systems that eliminate background fluorescence. Aspects of the present disclosure are intended to include or otherwise cover any type of detector reagents, without deviating from the scope of the present disclosure.

[0084] The conjugate pad 110 may include one or more detector reagents configured to bind with a target molecule in the biological sample and release antibody fragments or antibody nanoparticles through various mechanisms. The binding interaction may cause conformational changes in the detector reagents, leading to controlled fragmentation through weakening of intramolecular bonds, exposure of protease cleavage sites, dissociation of multi-subunit complexes, or activation of self-cleaving peptide sequences.

[0085] In some embodiments, the one or more detector reagents may be engineered fusion proteins including a target-binding domain linked to an antibody fragment or nanoparticle through a cleavable linker sequence. Upon target binding, conformational changes may expose the linker sequence to endogenous proteases present in the biological sample, resulting in controlled release of the antibody fragment or nanoparticle. The linker sequence may be designed to be specific for particular proteases or to have defined cleavage kinetics that control the rate and extent of fragment release.

[0086] The test strip 102 may include a test line 112 positioned further along the strip downstream of the conjugate pad 110. The test line 112 may be immobilized with antibodies or binders specific to the antibody fragments and configured to capture antibody fragments released from the conjugate pad 110. The test line 112 may generate a first result proportional to the concentration of antibody fragments present in the biological sample. In some aspects, the antibodies or binders on the test line 112 may be selected from the group including, but not limited to, anti-idiotype antibodies that recognize the variable regions of antibody fragments and provide specificity for particular antibody families or clones, anti-Fab antibodies that specifically bind to the antigen-binding fragments and can distinguish between different Fab conformations or orientations, anti-Fc antibodies that recognize the crystallizable fragment regions and may be specific for different immunoglobulin classes such as IgG, IgA, IgM, or IgE, Protein A which binds to the Fc region of IgG antibodies from various species including human, mouse, and rabbit with high affinity and specificity, Protein G providing broader species reactivity and stronger binding to certain IgG subclasses compared to Protein A, particularly IgG3 and mouse IgGl, Protein L which binds to the kappa light chain variable regions and can capture antibody fragments lacking Fc regions such as Fab, scFv, or singlechain antibodies, engineered protein scaffolds including Affibody molecules that provide customizable binding specificities through directed evolution approaches and can be selected against virtually any target protein and Designed Ankyrin Repeat Proteins (DARPins) offering high stability, specific target recognition, and resistance to proteolysis, nanobodies derived from camelid antibodies providing single-domain binding capabilities with enhanced stability, small size, and ability to recognize cryptic epitopes, aptamers providing nucleic acid-based recognition with high specificity and affinity that can be selected against virtually any target through SELEX procedures, Fc-binding peptides for synthetic binding interactions that can be chemically synthesized, modified with various functional groups, and optimized for specific binding characteristics, and combinations thereof that may provide synergistic binding effects or broader capture capabilities. Aspects of the present disclosure are intended to include or otherwise cover any type of antibodies or binders, without deviating from the scope of the present disclosure.

[0087] In some aspects, the immobilization of antibodies or binders on the test line 112 may be achieved through various methods including physical adsorption where proteins are allowed to bind to the membrane surface through hydrophobic interactions, electrostatic interactions, or van der Waals forces, covalent coupling using crosslinking agents such as glutaraldehyde, carbodiimides, or NHS esters that form stable bonds between protein amino groups and membrane functional groups, biotin-streptavidin interactions where biotinylated antibodies are captured by streptavidin immobilized on the membrane surface, or specialized membrane chemistries that provide reactive groups for specific protein attachment. Aspects of the present disclosure are intended to include or otherwise cover any immobilization method, without deviating from the scope of the present disclosure.

[0088] The test strip 102 may further include a control line 114 situated near the second end 106 of the strip, downstream of the test line 112. The control line 114 may be immobilized with antibodies or binders specific to the whole or intact antibody and configured to capture intact antibodies that have not undergone fragmentation during processing at the conjugate pad 110. The control line 114 may generate a second result proportional to the concentration of whole antibodies present in the biological sample. The control line 114 may serve multiple functions within the assay 100 including validation of assay performance by confirming the presence of antibodies in the sample and proper flow through the test strip 102, normalization of results by providing a reference signal that can be used to calculate relative fragment concentrations and account for variations in sample volume or antibody concentration, quality control by indicating proper sample flow, reagent reconstitution, and binding function, and calculation of fragmentation ratios by comparing signal intensities between the test line 112 and control line 114 to determine the extent of antibody fragmentation in the sample.

[0089] The antibodies or binders on the control line 114 may be selected to preferentially bind intact antibodies over fragments, which may be achieved through the use of antibodies that recognize conformational epitopes spanning multiple domains, antibodies specific for intact Fc regions that may be disrupted in fragmented antibodies, or binding molecules that require multivalent interactions only possible with intact antibody structures.

[0090] The first and second results may be visually distinct signals that enable simultaneous or sequential detection and differentiation between antibody fragments and whole antibodies. The visual distinction may be achieved through different labeling strategies where the test line 112 and control line 114 use different enzyme labels that produce different colored products, different fluorescent labels with distinct emission wavelengths, or different nanoparticle labels with distinct optical properties.

[0091] The signal intensities at both lines may be quantified using various detection methods including visual comparison with reference color charts or intensity standards, densitometric analysis using flatbed scanners or digital cameras, fluorescence measurement using portable fluorometers or smartphone-based detection systems, or specialized lateral flow readers that provide automated signal quantification and data analysis.

[0092] The total antibody concentration in the biological sample may be determined by aggregating measurements of antibody fragments from the test line 112 and measurements of intact antibodies from the control line 114. The analysis may include computing the concentration based on a combined analysis of the first result and the second result, providing comprehensive quantification of total antibody content that accounts for all antibody populations present in the sample.

[0093] In some aspects, the mathematical relationship for total antibody concentration may be expressed as:

[0094] Total Antibody Concentration = (Fragment Signal x Fragment Calibration Factor) + (Intact Signal x Intact Calibration Factor), where the calibration factors account for differences in binding efficiencies, signal generation capabilities, and detection sensitivities between the two detection systems. In another embodiment, the assay (100) may be configured for quantitative detection of vitamin D, such as 25 -hydroxy vitamin D, in a biological sample including whole blood or serum. The assay (100) may include the test strip (102) including the sample receiving pad (108), conjugate pad (110), test line (112), and control line (114) arranged sequentially along the flow path. The sample receiving pad (108) may be adapted to accept a small volume of whole blood and may include a separation membrane to filter plasma from cellular components. The conjugate pad (110) may include one or more engineered detector reagents, such as antibodies or binding proteins, which undergo a conformational change or fragmentation upon binding to vitamin D. In some aspects, the detector reagents may be conjugated with labeling moieties including fluorescent tags, enzyme labels, or nanoparticle conjugates to enable signal generation.

[0095] Upon application of the sample, the conjugate pad (110) may release the engineered detector reagents, which interact with vitamin D present in the sample. Target binding may induce structural changes in the detector reagents, exposing a previously hidden epitope or releasing a fragment. The test line (112) may include capture reagents configured to selectively bind the altered form of the detector reagent, thereby generating a first visual or optical signal proportional to the vitamin D concentration. The control line (114) may include capture reagents that bind the detector reagent irrespective of target binding, providing a second signal to validate assay performance and normalize measurements.

[0096] Detection may be performed visually or using an external reader such as a handheld imaging sensor or smartphone-based system. In some aspects, the external reader may quantify fluorescence intensity or colorimetric changes at the test line (112) and control line (114) and compute vitamin D concentration using calibration curves. This embodiment demonstrates the application of binding-induced structural changes for signal generation in lateral flow formats, enabling quantitative point-of-care testing for vitamin D. In another embodiment, a system for measuring concentration of antibody fragments may include the test strip 102 and an external device configured to analyze the test strip 102 and quantify antibody fragment concentrations. The external device may be configured to interface with the test strip 102 to perform optical measurements, signal analysis, and quantitative calculations that convert optical signals into concentration measurements for antibody fragments and whole antibodies present in biological samples.

[0097] The external device may be implemented in various embodiments including portable readers, handheld analyzers, smartphone-based systems, and dedicated optical readers. Aspects of the present disclosure are intended to include or otherwise cover any type of reader, without deviating from the scope of the present disclosure.

[0098] In some aspect, the external device may be configured as a portable reader that includes optical components, processing electronics, and user interface elements integrated into a compact, battery-powered device suitable for field use or point-of-care applications. The portable reader may be designed to accommodate the test strip 102 dimensions and provide consistent positioning for reliable optical measurements.

[0099] In some aspects, the external device may be configured as a handheld analyzer that includes advanced optical detection capabilities and processing functions for quantitative analysis of the test strip 102. In some aspect, the handheld analyzer may include multiple detection modes, calibration capabilities, and data storage functions that enable comprehensive analysis of antibody fragment concentrations. The handheld analyzer may be configured to perform automated scanning sequences and provide digital readouts of measurement results.

[0100] In some aspects, the external device may be configured as a smartphone-based system that utilizes the optical and processing capabilities of smartphones to analyze the test strip 102. In some aspect, the smartphone-based system may include a mechanical attachment or adapter that positions the test strip 102 relative to the smartphone camera and provides controlled lighting conditions for optical measurements. The smartphone- based system may utilize software applications that control image capture, perform signal analysis, and calculate antibody fragment concentrations using the smartphone's processing capabilities.

[0101] In some aspects, the external device may be configured as a dedicated optical reader specifically designed for analysis of the test strip 102 format. In some aspect, the dedicated optical reader may include specialized optical components, detection systems, and processing algorithms optimized for the specific signal types and measurement requirements of the antibody fragment assay. The dedicated optical reader may provide enhanced sensitivity, accuracy, and reproducibility compared to general-purpose optical devices.

[0102] In some aspect, the connection between the test strip 102 and the external device may be established through various mechanical coupling, alignment mechanisms, and positioning systems that ensure consistent and reproducible optical measurements. In some aspect, the external device may include a slot or holder configured to receive and position the test strip 102 at a predetermined location relative to the optical detection components. The slot or holder may be designed to accommodate the dimensions of the test strip 102 and provide secure retention during the measurement process.

[0103] In some aspects, the external device may include optical readers configured to perform both visual imaging and fluorescence imaging of the test strip 102. The optical readers may be configured to detect various types of optical signals generated at the test line 112 and control line 114, including colorimetric changes, fluorescent emissions, and light scattering patterns produced by accumulated antibody fragments and whole antibodies. Aspects of the present disclosure are intended to include or otherwise cover any type of optical signals, without deviating from the scope of the present disclosure. The optical reader may include light sources for visual spectrum illumination configured to provide broad-spectrum illumination for colorimetric detection and visual imaging of the test strip 102. In some aspect, the light sources for visual spectrum illumination may include white light LEDs, halogen lamps, or broad-spectrum light sources that enable detection of color changes, reflectance variations, or absorbance measurements at the test line 112 and control line 114. The visual spectrum illumination may be configured to provide uniform lighting conditions across the test strip 102 surface.

[0104] The optical reader may include excitation light sources for fluorescence detection configured to provide specific wavelengths of light for exciting fluorescent labels or fluorescent detector reagents present on the test strip 102. In some aspect, the excitation light sources may include narrow-band LEDs, laser diodes, or filtered light sources that provide excitation wavelengths matched to the absorption characteristics of fluorescent labels used in the assay. The excitation light sources may be positioned to provide uniform excitation across the test line 112 and control line 114 regions. Aspects of the present disclosure are intended to include or otherwise cover any type of light source, without deviating from the scope of the present disclosure.

[0105] The optical reader may include detection systems such as cameras, photodetectors, or imaging sensors configured to capture and measure optical signals generated by the test strip 102. These systems may include CCD or CMOS cameras, photodiode arrays, or linear and area imaging sensors, equipped with optical filters and lenses to optimize signal collection and minimize background interference. Cameras may capture high- resolution images of the test strip under controlled lighting for quantitative analysis of signal intensities at the test line 112 and control line 114. Photodetectors, such as photodiodes or photomultiplier tubes, may measure light intensities at specific wavelengths for colorimetric or fluorescent signals, while imaging sensors provide spatially resolved detection across the strip. Aspects of the present disclosure are intended to include or otherwise cover any type of light source, without deviating from the scope of the present disclosure. The external device may process these signals using algorithms to perform background correction, apply calibration factors, and convert optical measurements into concentration values for antibody fragments and whole antibodies. The external device may include display and communication interfaces for presenting results and transmitting data. Display interfaces, such as LCD or LED screens, may provide real-time feedback and present final results in numerical or graphical formats. Aspects of the present disclosure are intended to include or otherwise cover any type of display interfaces, without deviating from the scope of the present disclosure. Communication interfaces may support wired or wireless protocols, including Bluetooth, Wi-Fi, or USB, enabling data transfer to external devices, networks, or management systems. These interfaces may allow integration with electronic health records, laboratory information systems, or mobile applications for data storage and analysis. Aspects of the present disclosure are intended to include or otherwise cover any type of communication interface, without deviating from the scope of the present disclosure.

[0106] In another embodiment, the assay 100 may be configured as a fertility kit for quantifying fertility-related hormones in biological samples. The kit may enable comprehensive analysis of hormones that exist in both intact and fragmented forms, addressing limitations of conventional methods. In some aspects, the assay 100 may detect and quantify multiple fertility-related hormones, including human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and progesterone-binding antibodies. The fertility kit may utilize the test strip 102 components — sample receiving pad 108, conjugate pad 110, test line 112, and control line 114 — to perform simultaneous detection of whole hormones and fragments. Quantitative measurements may combine fragment and intact hormone concentrations to determine total hormone levels, enabling more accurate fertility status assessment.

[0107] In some embodiments, the assay 100 may be configured to determine the concentration of human chorionic gonadotropin (alpha and / or beta) in biological samples. The conjugate pad 110 may include detector reagents, such as anti -HCG or anti-beta-HCG antibodies, that may bind to target hormones and facilitate migration toward the test line 112 and control line 114. The test line 112 may capture hormone fragments to generate a first result, while the control line 114 may capture intact hormones to generate a second result. Total HCG concentration may be calculated by combining the first and second results, providing a comprehensive measurement of all hormone species. In some aspects, the assay 100 may deliver clinical sensitivity ranges suitable for pregnancy testing and fertility monitoring.

[0108] In some embodiments, the assay 100 may enable simultaneous multi-hormone detection using a test strip 102 that includes multiple test lines along the flow path, each configured to capture fragments of different fertility hormones such as HCG, LH, FSH, and estrogen-binding antibody fragments. Corresponding control lines positioned downstream may capture intact hormones to provide reference measurements for total hormone concentrations. This configuration allows comprehensive fertility status assessment from a single biological sample by quantifying multiple hormone species and their fragments, offering insights into hormone balance, reproductive cycle phase, fertility potential, and related health conditions, thereby enabling more accurate fertility predictions compared to single-hormone measurements.

[0109] ADVANTAGES OF THE INVENTION

[0110] • Comprehensive Total Antibody Quantification: The assay enables accurate total antibody concentration measurement by detecting and quantifying both whole antibodies and antibody fragments separately through the dual detection system, then combining these measurements to determine true total concentration, which conventional assays cannot accomplish because they detect only intact antibodies and miss fragmented molecules present in biological samples.

[0111] • Dual Detection System Architecture: The test line 112 captures antibody fragments while the control line 114 captures whole antibodies, providing a novel mechanism that enables comprehensive quantification accounting for fragmented molecules that would otherwise remain undetected, ensuring that all antibody species present in the sample contribute to the total concentration measurement. • Engineered Fragment Generation Mechanism: The assay 100 utilizes engineered fragment generation mechanisms through detector reagents that undergo conformational changes upon target binding to release antibody fragments, providing a controlled mechanism for fragment generation and detection not available in prior art methods. This engineered approach enables predictable and reproducible fragment release that can be quantitatively measured.

[0112] • Enhanced Clinical Accuracy: The assay 100 improves clinical accuracy and diagnostic reliability by measuring the true total concentration through the mathematical relationship where total amount equals whole antibody plus antibody fragments, rather than underestimating concentrations due to undetected fragments that conventional methods cannot measure.

[0113] • Broad Diagnostic Platform Versatility: The assay 100 demonstrates broad diagnostic platform versatility by being applicable to multiple diagnostic applications including fertility hormone testing for HCG, LH, and FSH detection, vitamin D quantification, and general antibody measurement across various biological sample types including urine, semen, saliva, tears, blood, sweat, and pre-ejaculate.

[0114] • Point-of-Care Accessibility: The assay 100 provides point-of-care accessibility by combining simple lateral flow technology with quantitative dual-line detection that can be analyzed by external devices, making accurate fragment analysis accessible in point- of-care settings without requiring complex laboratory instrumentation or specialized technical expertise.

[0115] • Recognition of Biological Sample Complexity: The assay 100 addresses a fundamental limitation in immunoassay technology by recognizing that biological samples contain both intact and fragmented forms of target molecules due to natural degradation processes, enzymatic cleavage, and structural instability, and providing a comprehensive solution to measure both populations.

[0116] • Improved Sensitivity Through Fragment Recovery: The assay 100 provides improved sensitivity through fragment recovery by recovering antibody fragments that would be lost in conventional detection methods, effectively increasing the total measurable antibody content, and improving overall assay sensitivity by accounting for all antibody species present in the sample.

[0117] • Quantitative External Device Integration: The assay 100 enables quantitative external device integration through integration with external devices including handheld readers, smartphone-based systems, and optical analyzers that can convert optical signals from both the test line 112 and control line 114 into precise concentration values.

[0118] • Multiple Sample Type Compatibility: The assay 100 provides multiple sample type compatibility by accommodating various biological sample types through the sample receiving pad 108 configuration that can be positioned at different locations on the test strip 102 to optimize sample introduction and flow characteristics for different sample viscosities and compositions.

[0119] • Standardized Mathematical Framework: The assay 100 provides a standardized mathematical framework for total antibody concentration calculation by establishing the relationship that total concentration equals fragment concentration plus intact antibody concentration, enabling consistent and reproducible quantitative measurements across different applications and sample types.

[0120] A number of implementations have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other implementations are within the scope of the following claims.

[0121] The foregoing discussion of the present disclosure has been presented for purposes of illustration and description. It is not intended to limit the present disclosure to the form or forms disclosed herein. In the foregoing Detailed Description, for example, various features of the present disclosure are grouped together in one or more aspects, configurations, or aspects for the purpose of streamlining the disclosure. The features of the aspects, configurations, or aspects may be combined in alternate aspects, configurations, or aspects other than those discussed above. This method of disclosure is not to be interpreted as reflecting an intention, the present disclosure requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed aspects, configuration, or aspect. Thus, the following claims are hereby incorporated into this Detailed Description, with each claim standing on its own as a separate aspect of the present disclosure.

[0122] Moreover, though the description of the present disclosure has included description of one or more aspects, configurations, or aspects and certain variations and modifications, other variations, combinations, and modifications are within the scope of the present disclosure, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative aspects, configurations, or aspects to the extent permitted, including alternate, interchangeable and / or equivalent structures, functions, ranges, or steps to those claimed, whether or not such alternate, interchangeable and / or equivalent structures, functions, ranges, or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.

Claims

WE CLAIM1. A method (200) for determining the presence or quantity of antibody fragments in a sample comprising: detecting the presence of antibody fragments by capturing said fragments on a or zone comprising immobilized antibodies or binders specific to the antibody fragments.

2. The method (200) as claimed in claim 1, wherein the immobilized antibodies or binders specific to the antibody fragments are immobilized on the test line (110) or zone of the test strip (102).

3. The method (200) as claimed in claim 1, wherein the capturing of the antibody fragments by the immobilized antibodies or binders specific to the antibody fragments result in generation of a first result.

4. The method (200) as claimed in claim 1, further comprising detecting the presence of whole or intact antibody on a line / zone comprising immobilized antibodies or binders specific to the whole / intact antibody.

5. The method (200) as claimed in claim 1, wherein the immobilized antibodies or binders specific to the whole / intact antibodies are immobilized on the control line (114) or zone of the test strip.

6. The method (200) as claimed in claim 4, wherein the binding of the whole / intact antibody with the immobilized antibodies or binders specific to the whole or intact antibody result in generation of a second result.

7. The method (200) as claimed in 1 , further comprising determining the total antibody concentration in the biological sample by aggregating measurements of antibody fragments and measurements of intact antibodies8. The method (200) as claimed in claim 1, wherein the first and second result are visually distinct signals.

9. The method (200) as claimed in claim 1, wherein the antibody or binder is selected from the group comprising anti-idiotype antibodies, anti-Fab antibodies, anti-Fc antibodies, Protein A, Protein G, Protein L, engineered protein scaffolds including Affibody molecules and Designed Ankyrin Repeat Proteins (DARPins), nanobodies, aptamers, Fc-binding peptides, and combinations thereof.

10. The method (200) as claimed in claim 1, wherein the biological sample is selected from a group comprising a urine, semen, saliva, tears, blood, sweat or pre-ejaculate.

11. A method (200) for determining an antibody in a biological sample by an assay (100), comprising: detecting antibody fragments by capturing said fragments on a test line (110) comprising immobilized antibodies or binders specific to the antibody fragments, and generating a first result; detecting whole or intact antibodies by capturing said antibodies on a control line (112) comprising immobilized antibodies or binders specific to the whole or intact antibodies, and generating a second result; and analyzing the first result and the second result to determine the concentration of the antibody present in the biological sample.

12. The method (200) as claimed in claim 11, wherein the assay (100) further comprises: a test strip (102) comprising: a sample receiving pad (108) configured for receiving biological sample from a subject;a conjugate pad (110), having one or more detector reagents, configured to bind with the received biological sample; a test line (112), immobilized with antibodies or binders specific to the antibody fragments and configured to capture antibody fragments; and a control line (114), immobilized with antibodies or binders specific to the whole or intact antibody and configured to capture said antibody.

13. The method (200) as claimed in claim 11, wherein analysing the total antibody concentration comprises computing the concentration based on a combined analysis of the first result and the second result.

14. The method (200) as claimed in claim 11, wherein the first and second result are visually distinct signals.

15. The method (200) as claimed in claim 11, wherein the assay is a lateral flow assay.

16. The method (200) as claimed in claim 11, wherein the one or more detector reagents are selected from a group comprising an enzyme label, fluorescent labels, quantum dots, or luminescent nanoparticles.

17. The method (200) as claimed in claim 11 , wherein the biological sample is selected from a group comprising a urine, semen, saliva, tears, blood, sweat or pre-ejaculate.

18. The method (200) as claimed in claim 11, wherein the antibody or binder is selected from the group comprising anti-idiotype antibodies, anti-Fab antibodies, anti- Fc antibodies, Protein A, Protein G, Protein L, engineered protein scaffolds including Affibody molecules and Designed Ankyrin Repeat Proteins (DARPins), nanobodies, aptamers, Fc-binding peptides, and combinations thereof.

19. The method (200) as claimed in claim 11, wherein the analysis is performed using a handheld imaging sensor configured to detect colorimetric, fluorescent, or chemiluminescent signals.

20. An assay (100) for determining the presence or quantity of antibody fragments in a sample by capturing said fragments on a line or zone comprising immobilized antibodies or binders specific to the antibody fragments.

21. The assay (100) as claimed in claim 20, wherein the assay (100) further comprises: a test strip (102) comprising: a sample receiving pad (108) configured for receiving biological sample from a subject; a conjugate pad (110), having one or more detector reagents, configured to bind with the received biological sample; a test line (112), immobilized with antibodies or binders specific to the antibody fragments and configured to capture antibody fragments; and a control line (114), immobilized with antibodies or binders specific to the whole or intact antibody and configured to capture said antibody.

22. The assay (100) as claimed in claim 20, wherein the assay (100) is a lateral flow assay.

23. The assay (100) as claimed in claim 20, wherein the one or more detector reagents are selected from a group comprising an enzyme label, fluorescent labels, quantum dots, or luminescent nanoparticles.

24. The assay (100) as claimed in claim 20, wherein the biological sample is selected from a group comprising a urine, semen, saliva, tears, blood, sweat or pre-ejaculate.

25. The assay (100) as claimed in claim 20, wherein the sample flows from a first end (104) to a second end (106) of the test strip (102) by capillary action.

26. The assay (100) as claimed in claim 20, wherein the antibody or binder is selected from the group comprising anti-idiotype antibodies, anti-Fab antibodies, anti-Fc antibodies, Protein A, Protein G, Protein L, engineered protein scaffolds including Affibody molecules and Designed Ankyrin Repeat Proteins (DARPins), nanobodies, aptamers, Fc-binding peptides, and combinations thereof.

27. An assay (100) for determining quantity of antibody in a biological sample, comprising: test strip (102) configured for receiving biological sample from a subject; a conjugate pad (110) comprises one or more detector reagents configured to bind with a target molecule in the biological sample and release antibody fragments or antibody nanoparticles; a test line (112), immobilized with antibodies or binders specific to the antibody fragments and configured to capture said antibody fragments; and a control line (14), immobilized with antibodies or binders specific to the whole or intact antibody and configured to capture said antibody; wherein analysing the total antibody concentration comprises computing the concentration based on a combined analysis of the first result and the second result.

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