Abstract:
A system for testing for HDL-C and HDL-P includes a first lateral flow test strip, a second lateral flow test strip, and a dosing area, the dosing area interconnected with the first and second lateral flow test strip. The system further includes a collector for collecting a sample and a first mixer for receiving the sample from the collector, the mixer including buffers for mixing with the sample, the first mixer for dosing the sample pad a first time. The first lateral flow test strip provides for the detection of HDL-C and the second lateral flow test strip provides for the detection of HDL-P.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims the benefit of U.S. Provisional Patent Application No. 62/234,525 filed Sep. 29, 2015, and hereby incorporated by reference to the same extent as though fully disclosed herein. 
     
    
     BACKGROUND 
       [0002]    Heart disease is a leading cause of death in today&#39;s society. The monitoring of blood analytes to assist in the monitoring and prediction of heart disease is frequently conducted by medical personnel. In many scenarios, a blood sample must be taken far in advance of meeting with medical personnel so that laboratory testing may occur. Furthermore, recent developments in research related to heart disease have determined that certain blood analytes may be more relevant to the prediction and monitoring of heart disease than others. Typically, high-density lipoprotein is thought to play a protective role against cardiovascular (or heart) disease by transporting cholesterol away from the vessel wall. Studies exist showing the cholesterol carrying capacity of such molecules. However, it has been determined that HDL cholesterol (HDL-C) is only one property. Additionally, HDL particles (HDL-P) play a part in the carrying of cholesterol away from the vessel wall. In fact, in some scenarios, it has been determined that the measurement of HDL-P is more instructive in determining heart disease prevention than measuring HDL-C. Therefore, it would be desirable to provide a point-of-care test that would test for HDL-C and possibly HDL-P simultaneously, since HDL-C is a traditional measure of HDL related interactions. 
       BRIEF SUMMARY 
       [0003]    In one embodiment, a system for testing for HDL-C and HDL-P includes a first lateral flow test strip, a second lateral flow test strip, and a dosing area, the dosing area interconnected with the first and second lateral flow test strips. The system further includes a collector for collecting a sample and a first mixer for receiving the sample from the collector, the mixer including buffers for mixing with the sample, the first mixer for dosing the sample pad a first time. The first lateral flow test strip provides for the detection of HDL-P, and the second lateral flow test strip provides for the detection of HDL-C. In one alternative, the system further includes a second mixer, the second mixer containing an enzyme formulation, the enzyme formulation for dosing the sample pad. Optionally, the first lateral flow test strip includes an antibody-antigen stripe and an antigen stripe. Alternatively, the antibody-antigen stripe is an anti Apo A-1 Ab-latex conjugate. In one alternative, the antibody-antigen stripe includes blue particles connected to the anti Apo A-1 antibody. Optionally, the antigen stripe is an Apo A antigen stripe, located in a direction of flow distal from the dosing area on the first lateral flow test strip. Alternatively, the second lateral flow test strip includes an anti Apo A-1 antibody stripe. In one configuration, the first and second lateral flow test strips include conjugate nitrocellulose and nylon 120 membranes. 
         [0004]    In one embodiment, a method of testing for HDL-C and HDL-P includes providing a meter, a first lateral flow test strip, a second lateral flow test strip, and a dosing area, the dosing area interconnected with the first and second lateral flow test strips, a collector for collecting a sample, and a first mixer. The method further includes collecting a sample with the collector and mixing the sample with the mixer. The method further includes dosing the sample on the dosing area. The method further includes laterally flowing the sample across the first and second lateral flow test strips and reading the first lateral flow test strip to determine a concentration of HDL-P in the sample. In one alternative, the method further includes providing a sampler, the sampler containing an enzyme formula, dosing the dosing area with the enzyme formula, and reading the second lateral flow test strip with the meter to determine a concentration of HDL-C in the sample. In another alternative, the first lateral flow test strip includes an antibody-antigen stripe and an antigen stripe. Optionally, the antibody-antigen stripe is an anti Apo A-1 Ab-latex Conjugate. Alternatively, the antibody-antigen stripe includes blue particles connected to the anti Apo A-1 antibody. Optionally, the antigen stripe is an Apo A antigen stripe, located in a direction of flow distal from the dosing area on the first lateral flow test strip. In another alternative, the second lateral flow test strip includes an anti Apo A-1 antibody stripe. Alternatively, the first and second lateral flow test strips include conjugate nitrocellulose and nylon 120 membranes. Optionally, the method further includes, after the dosing of the sample, flowing the sample to the antibody-antigen stripe of the first lateral flow test strip; binding the anti Apo A-1 antibody to HDL-P in the sample; flowing the sample to the antigen stripe of the first lateral flow test strip; and capturing unbound portions of the anti Apo A-1 antibody to the antigen stripe. Alternatively, the method further includes, after the dosing of the sample, flowing the sample to the anti Apo A-1 antibody stripe of the second lateral flow test strip; and binding HDL to the anti Apo A-1 antibody stripe of the second lateral flow test strip. Alternatively, the method further includes, after the dosing with the enzyme formula, flowing the enzyme formula to bound HDL, the bound HDL being a result of the binding HDL to the anti Apo A-1 antibody stripe; and reacting the enzyme formula with the bound HDL. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0005]      FIG. 1  shows one embodiment of a lateral flow test strip; 
           [0006]      FIG. 2  shows one embodiment of a first lateral flow test strip and a second lateral flow test strip for use in the detection HDL-C and HDL-P; 
           [0007]      FIG. 3  shows one embodiment of a sample collector for use with the first and second test strips for use in the detection of HDL-C and HDL-P; 
           [0008]      FIG. 4  shows a sample collector (Si) with a mixer containing reagents for use with the first and second test strips for use in the detection of HDL-C and HDL-P; 
           [0009]      FIGS. 5 a  and 5 b    show a second sampler (S 2 ) containing a proprietary enzymatic formulary for use with the first and second test strips for use in the detection HDL-C and HDL-P; 
           [0010]      FIG. 6  shows a typical dose response of Apo A-1 particle concentration to the referenced method; and 
           [0011]      FIG. 7  shows a typical dose response of HDL cholesterol concentration to the referenced method. 
       
    
    
     DETAILED DESCRIPTION 
       [0012]    Certain terminology is used herein for convenience only and is not to be taken as a limitation on the embodiments of the systems and methods for point-of-care determination of HDL-C and HDL-P. In the drawings, the same reference letters are employed for designating the same elements throughout the several figures. 
         [0013]    Currently, there are no point-of-care (POC) methods to determine the HDL particle (HDL-P) concentration of HDL cholesterol (HDL-C) simultaneously in a whole blood sample. Embodiments of systems and methods for point-of-care determination of HDL-C and HDL-P determine the number of HDL particles (HDL-P) and HDL cholesterol (HDL-C) concentrations. Embodiments of systems and methods for point-of-care determination of HDL-C and HDL-P yield an HDL-P (particle) concentration and a direct HDL-C (cholesterol) concentration from a single sample using a single device. This point-of-care device test (POCT) can give both particle and cholesterol concentrations. In recent literature, it has been recognized that HDL-P concentration is of greater diagnostic value. In addition, it also is important to provide actual HDL-C concentrations in the lipoprotein fractions. Embodiments of systems and methods for point-of-care determination of HDL-C and HDL-P include a POCT for determining both the HDL-P (particle) and HDL-C (cholesterol) concentrations in one simple test. 
         [0014]    Embodiments of systems and methods for point-of-care determination of HDL-C and HDL-P include a POCT that employs a lateral flow methodology.  FIG. 1  shows one embodiment of a lateral flow test strip that may be used in conjunction with the systems and methods described herein. In lateral flow, the conjugate membrane  105 , nitrocellulose  110 , and nylon  120  membranes are layered in such a way to obtain easy plasma/fluid flow which enables the analytes to be captured on the membranes in different zones as shown in  FIG. 1 . The flow is towards end pad  130 , where the concentration of analytes is read. Additionally, the lateral flow test strip includes a sprocket hole  140  for mounting and positioning the strip in a cassette or other holder. This lateral flow based device has two parts: the left arm of the lateral flow which will quantify the concentration of Apo A-1 concentration, and the right arm of the device will quantify the cholesterol concentration present in the HDL&#39;s Apo A fractions. The resulting device will give physicians a dual value to make effective diagnoses or treatment options for patients with a hypercholesterolemic condition. 
       Description of Quantification of HDL-P 
       [0015]    As shown in  FIG. 2 , embodiments of a device for quantification of HDL-P and HDL-C include two arms  201 ,  202 . Each arm  201 ,  202  is a lateral flow test strip with a common sample dosing pad  210 . In this embodiment, the HDL-P arm  201 , a lateral flow method using an antibody-antigen interaction to quantify the levels of HDL&#39;s Apo A protein in the sample is utilized. An antibody-antigen stripe  230  (anti Apo A-1 Ab-latex conjugate stripe) is included in the lateral flow strip. Blue particles (or alternatively other detectable particle colors) coated with anti Apo A-1 antibody will be striped as shown in  FIG. 2  at stripe  230 . The Apo A antigen will be striped in zone one at stripe  240 . 
       Description of Quantification of HDL-C 
       [0016]    In the second arm, arm  202 , a lateral flow method using an enzymatic reaction of the HDL&#39;s fraction in the sample will be quantified. Here, only the anti Apo A antibody will be striped in zone one at stripe  250 . No blue dye particles will be used. As can be seen in the figures, the fluid flows from the dosing pad  210  toward the other end of the lateral flow strip in the flow direction toward stripe  250 . 
         [0017]    As shown in  FIG. 2 , the architecture of the device includes arms  201 ,  202  where the HDL-P concentration and HDL-C concentration can be determined. The left arm  201  of the device yields an HDL-P concentration based on the lipoprotein Apo A concentration, while the right arm  202  of the device yields an HDL-C concentration based on enzymatic reaction of the cholesterol present in the HDL Apo lipoprotein.  FIG. 2  shows the locations of the striped components and the zones for each lipoprotein. 
         [0018]    Dosing and quantification occur according to a number of steps.  FIG. 3  shows a sample collector with whole blood and the method for collecting capillary whole blood from a finger stick.  FIG. 4  shows a sample collector (S 1 ) with a mixer containing reagents.  FIGS. 5 a  and 5 b    show a second sampler (S 2 ) containing proprietary enzymatic formulary.  FIG. 5 b    shows that the second sampler includes a one-stop snap cap  510  with a perforator for perforating the chamber  520  containing water which may later be mixed with the chamber  530  containing enzymes and then dosed by removing the cap  540 . 
         [0019]    Step  1 : A venous or capillary sample will be collected by the collector as shown in  FIG. 3 , mixed with proprietary buffers by sample mixers (S 1 ) (as shown in  FIG. 4 ), and dosed on the sample pad. After dosing, the solution will wick on both arms evenly. The following physical phenomenon will ensue. 
         [0020]    The blue particles in stripe  230  in the left arm  201  of the device will interact with the sample and migrate along the length of the lateral flow strip. The blue particle coated with anti Apo A-1 antibody will be captured in the test zone one at stripe  240  (which is striped with Apo A-1 antigen-protein conjugate) when a very small amount of HDL-P is present in the sample. If a large amount of HDL-P is present in the sample, the HDL will stick to the blue particles resulting in proportionally lower capture of the blue particles in zone one at stripe  240 . In this particular immunochemistry method, a direct relationship exists between the analyte concentrations to the light reflected from the “capture” in zone one at stripe  240  (for both arms of the device) as shown in the dose response,  FIG. 6 . A lower reflectance reading will yield a lower value, while a high reflectance reading will yield a higher analyte concentration. An HDL-P can be calibrated ( FIG. 6 ) against the Liposcience NMR® method or via the Roche&#39;s “Tina-quant Apo lipoprotein A-1 ver.2®” assay, thus enabling a POC device to now report the particle count for the HDL lipoproteins. 
         [0021]    In the same amount of time, the HDL&#39;s Apo A-1 fractions will migrate on the right arm and will be captured on the zone one at stripe  250  where the anti-Apo A-1 will be striped, letting all other lipoproteins flow to the end pad and be ready for step  2 . 
         [0022]    Step  2 : When the HDL-P concentration has been determined (from the left arm strip) by the meter, the meter will prompt the user to dose a second sampler S 2  (as shown in  FIG. 5 ) containing a proprietary enzyme formulary. In this embodiment, the enzymes will wick to capture Apo A fractions in zone one of the left arm and trigger a series of reactions where the cholesterol concentration for HDL will be determined (HDL-C). The enzymatic formulary contains surfactants to de-coat the Apo A lipoprotein followed by a series of chemical reactions of the cholesterol esters by cholesterol esterase, cholesterol oxidase. The resulting hydrogen peroxide then will be reacted with 4-amino antipyrine (4-AAP) and N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline, sodium salt, monohydrate (MAOS) with the help of horse radish peroxidase (HRP) enzyme to yield a blue colored quinoneimine dye which will be measured by the meter in reflectance mode. This reaction scheme is merely an example of a reaction scheme that may be utilized in the development of a color change, and other reaction schemes may be apparent to those of ordinary skill in the art. In certain embodiments, an HDL-C is calibrated ( FIG. 7 ) against reference methods like via the Roche&#39;s “HDL-Cholesterol plus assay®” or by Sekisui&#39;s “Sekisui HDL Ultra Cholesterol Test Kit®”, thus enabling a POC device to now report the HDL cholesterol concentration. 
         [0023]    In one of the embodiments of step  2 , the enzymes can be lyophilized and placed in a hand held mixer that contains two compartments. Compartment one  530  is for lyophilized enzymes, while compartment two  520  is for holding the right amount of water (see  FIG. 5 b   ) for mixing the enzyme prior to dosing it. The water and the enzymes will be mixed by simply puncturing the barrier between them.  FIG. 6  shows a typical dose response of Apo A-1 particle concentration to the reference method.  FIG. 7  shows a typical dose response of HDL cholesterol concentration to reference method. 
         [0024]    While specific embodiments have been described in detail in the foregoing detailed description and illustrated in the accompanying drawings, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure and the broad inventive concepts thereof. It is understood, therefore, that the scope of this disclosure is not limited to the particular examples and implementations disclosed herein but is intended to cover modifications within the spirit and scope thereof as defined by the appended claims and any and all equivalents thereof. Note that, although particular embodiments are shown, features of each may be interchanged between embodiments.