MXPA00006963A - Neutralization of polycations in a chromatographic device for whole blood use - Google Patents

Abstract

A chromatography assay device and method for use with whole blood samples utilizing a red blood cell separating agent to aggregate red blood cells and permit plasma or serum to flow by capillary action and a neutralizing agent to neutralize any effects the red blood cell separating agent may have on the device and method.

Description

Neutralization of polycations in a chromatographic device for use of whole blood TECHNICAL FIELD OF THE INVENTION The present invention relates to chromatographic assay devices and a method for detecting an analyte in a whole blood sample, and more particularly, to a device and method employing a red blood cell separator agent to add red blood cells, and a neutralizing agent to neutralize any negative effects that the red blood cell separation agent may have on the test system.

BACKGROUND OF THE INVENTION Modern clinical diagnostic methods are routinely performed on blood samples. Unfortunately, red blood cells interfere with many diagnostic determinations. In assays for an analyte, red blood cells can inhibit the binding between specific binding partner members. Similarly, red blood cells have enzymatic activity, which, depending on the assay used, can interfere with the signal produced. Additionally, in a rapid test format using a chromatography assay device, particularly a chromatography immunoassay device, red blood cells can inhibit the flow of fluids, which is necessary for reactions to occur in the device. For these and other reasons, many assay methodologies are performed in plasma or serum, which must first be separated from a whole blood sample. There are many known techniques for separating red blood cells from the plasma in a whole blood sample. Centrifugation is a method well known in the art, whereby plasma (before coagulation) and serum (after coagulation) is separated from whole blood. In this procedure, the red blood cells settle to the bottom of the test tube, and the serum is separated by decanting or some other method. However, stratification of whole blood by centrifugation can have many disadvantages. In general, centrifugation requires the extraction of a large blood sample. In addition, the process is slow and requires cumbersome laboratory equipment that is often not maintained in a doctor's office. Finally, extra blood management increases exposure to potential hazards of pathogens carried in the blood. To reduce or eliminate the need for centrifugation, test devices have been developed that employ gradient membranes or trap membranes to separate the red blood cells from the liquid portion of the blood. Anti-immobilized anti-red blood cells have also been used. Other known techniques for separating red blood cells from plasma or serum include (1) combining a whole blood sample with a red blood cell binding agent, filtering the mixture through a solid spongy element, to which at least one member binds. specific binding pair to remove agglutinated red blood cells; (2) passing whole blood through a glass microfiber filter, which may or may not have a built-in binder; (3) employing a barrier or exclusion layer of polysaccharide material to prevent red blood cells from passing through by interfering with detection or visualization of a signal on a dry test strip; and (4) using a support having a polycationic surface, which binds red blood cells but not plasma. Many of these techniques for separating red blood cells from plasma are expensive and complicated, can result in incomplete separation of red blood cells and can cause hemolysis. Hemolysis leads to a non-specific binding or high background that causes a loss in assay sensitivity. This can be the result of free hemoglobin, which can color the detection zone, so that the area can obtain a color that varies from pink to dark brown. As a result, the production of a visual chemical signal can be partially or completely obscured by the presence of the color of the hemoglobin in the detection zone. Additionally, the use of a separation agent, such as a polycation, in a test system tends to interfere with the system, often by adding other reagents or binding members in addition to the red blood cells. Accordingly, there is a need for a device and method for detecting an analyte in a blood sample without adversely affecting the assay system. Such a device and method should be suitable for whole blood samples of various sizes, including small samples.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a chromatography device comprising a chromatography carrier, which defines a path for fluid flow capable of supporting capillary flow, an application site for said blood sample. in fluid flow contact with the chromatography carrier, a detection site in the chromatography carrier separated from the application site, a diffusely bound labeled substance located downstream from the application site, a red blood cell separation agent attached in a diffuse manner to separate plasma or serum from the blood sample upstream of the detection site, and a diffusely linked neutralizing agent capable of binding with the separation agent downstream of the separation agent bound upstream of said site of separation. detection, whereby a positive charge of said separation agent is neutralized. Preferably, the red blood cell separation agent is located at the site of application, so that the red blood cells will be separated from the serum or plasma before the serum or plasma moves down the chromatography carrier. The present invention is also directed to a method for detecting the presence of an analyte in a sample, preferably a blood sample, which comprises providing a chromatography carrier, which defines a path for fluid flow capable of supporting flow capillary, along which are (a) an application site for the blood sample in fluid flow contact with said chromatography carrier, (b) a detection site in the chromatography carrier separated from the site of application, (c) a diffusely bound labeled substance located downstream from the site of application, (d) a red blood cell separation agent diffusely linked to separate plasma or serum from said blood sample upstream of the detection site. , and (e) a diffusely bound neutralizing agent capable of binding to the separation agent located downstream of the attached separation agent and stream from the detection site, whereby a positive charge of the separation agent is neutralized; contacting the application site with the blood sample, so that the red blood cell separation agent separates the plasma or serum from the blood sample, and the neutralizing agent neutralizes the positive charge of the separation agent as the sample flows to along the flow path; and detecting the presence of analyte in the blood sample.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a preferred embodiment of the chromatography assay device of the present invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the observation that red blood cells in whole blood samples interfere with the determinations of the presence or amount of analyte in a blood sample, which could easily be done otherwise via systems of testing. For example, in an immunoassay, a whole blood sample in contact with an application site is unlikely to move down the strip via capillary action, due to the presence of obstruction or interference of red blood cells. The present invention overcomes this problem without interfering with the sensitivity of the assay system. The following definitions may be useful in understanding the embodiments of the present invention. "Analyte" or "analyte of interest" refers to the compound or composition to be detected or measured, which has at least one epitope or binding site. The analyte can be any substance for which there is a specific binding member-analyte that occurs naturally or for which a specific binding member-analyte can be prepared. Analytes include, but are not limited to, toxins, organic compounds, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, medications (including those administered for therapeutic purposes, as well as those administered for illicit purposes), and metabolites of, or antibodies to, , any of the above substances. The term "analyte" also includes any antigenic substance, hapten, antibody, macromolecule and combinations thereof. "Chromatographic carrier" refers to any suitable porous, absorbent, spongy, isotropic or capillary material, which includes the detection site of the device and through which the analyte or test sample can be transported by capillary or wicking action. One skilled in the art will appreciate that the chromatography carrier can be made of a single material or more than one material (for example, different zones, portions, layers, areas or sites can be made of different materials), as long as the multiple layers they are in fluid flow contact with another, allowing the passage of the test sample between the materials. The fluid flow contact allows the passage of at least some of the components of the sample, ie analyte, between the areas of the porous material and, preferably, is uniform along the contact interface between the different zones . Natural, synthetic or naturally occurring materials that are synthetically modified as the chromatography carrier can be used, and include, but are not limited to: paper (fibrous), or (microporous) membranes of cellulose materials, such as paper; cellulose and cellulose derivatives, such as cellulose acetate and nitrocellulose; fiberglass; fabric, both those that occur naturally (for example, cotton), and synthetic (for example, nylon); porous gels; and similar. "Brand" refers to any substance that is capable of producing a signal that is detectable by visual or instrumental means. Various suitable labels for use in the present invention include labels that produce signals through either chemical or physical means. Examples include enzymes and substrates, chromogens, fluorescent compounds, chemiluminescent compounds, organic polymer latex particles, and liposomes or other vesicles containing directly visible substances. Preferably, radioactive labels, colloidal metal particles or colloidal non-metallic particles are used in the present invention. Preferred brands include latex particles and colloidal gold. "Marked substance" or "conjugate" refers to a substance that comprises a detectable label attached to a specific binding member. The binding may be covalent or non-covalent binding, and may include nucleic acid hybridization. The label allows the labeled substance to produce a detectable signal that is directly or indirectly related to the amount of analyte in a test sample. The specific binding member component of the labeled substance is selected to bind directly or indirectly to the analyte. "Specific binding member" refers to a member of a specific binding pair, ie, two different molecules wherein one of the molecules specifically binds to the second molecule through a chemical or physical medium. If the specific binding member is an immunoreactive, it can be, for example, an antibody, antigen, hapten or complex thereof, and if an antibody is used, it can be a monoclonal or polyclonal antibody, a recombinant protein or antibody, an antibody chimeric, one or a few mixtures or fragments thereof, as well as a mixture of an antibody and other specific binding members. Specific examples of specific binding members include biotin and avidin, an antibody and its corresponding antigen (both having no relation to a sample to be tested), a single-stranded nucleic acid and its complement and the like. "Trap substance" refers to one or more specific binding members that are bound within or on a portion of the chromatographic carrier to form one or more "capture sites", wherein the analyte, labeled reagent, and / or reagent control become immobilized in the chromatography carrier. The binding method is not critical to the present invention. The trap substance facilitates the observation of the detectable signal by substantially separating the analyte and / or the labeled substance from unbound test reagents and the remaining components in the test sample. The cheat substance can be immobilized in the chromatography carrier before or during the performance of the assay by any suitable binding method. In addition, the trap substance can be provided in a single detection site or in multiple sites on or in the chromatography carrier. The trap substance can also be provided in a variety of configurations to produce different measurement or detection formats. For example, the trap substance can be configured as a letter, number, icon or symbol, or any combination thereof. In particular, the present invention provides a chromatography assay device for detecting the presence of an analyte in a sample, preferably a blood sample. The device is preferably in the form of a chromatographic strip having a chromatographic carrier defining a path for fluid flow, and which is capable of supporting capillary flow, an application site for the blood sample, and a detection site. separated from the application site to detect the presence or amount of analyte present in the blood sample. Preferably, the device also includes a labeled substance (or conjugate) diffusely bound to the chromatographic carrier.

In a preferred embodiment, the labeled substance will bind to the analyte or compete with the analyte to bind to the detection site. The device preferably contains two additional agents diffusely bound to the chromatographic carrier; (1) an upstream red blood cell separating agent (hereinafter, the direction of movement of a sample caused by capillary action is called "downstream" and the opposite direction is called "upstream") of the detection site , which is capable of separating plasma or serum from the blood sample, and (2) a neutralizing agent downstream of the red blood cell separation agent and upstream of the detection site to neutralize any effect, particularly an adverse effect, of the agent for separating red blood cells in the chromatography system. In the context of the present invention, the phrase "diffuse bound", as applied to a given reagent, can be defined as any reagent used in the present invention, including but not limited to, a labeled substance, specific binding member , red blood cell separating agent or neutralizing agent, is meant to denote that the reagent (s) are attached in a manner that allows the reagent (s) to flow along the flow path. For purposes of the present invention, any test system may be employed. Immunoassay systems are preferred, including but not limited to, lateral flow systems, vertical flow systems, soaking systems, and immersion sticks. A general description of known test systems is discussed below.

Generally, in a chromatography strip, at least one sample application site and one detection site are arranged in a chromatography carrier. A sample solution, in this case preferably a blood sample, which is suspected to have an analyte of interest, eg, an analyte, is moved through the chromatography carrier by capillary action when added to the sample application site. , and a labeled or conjugated substance, which is contained in a marking medium arranged in a chromatography carrier in advance, accumulates at the detection site in direct or inverse proportion to the presence or amount of the substance to be tested in the sample solution, effected by a binding reaction (such as an immunological reaction), so that the presence or quantity of substance to be assessed in the sample solution can be found by measuring the presence or amount of the conjugate or labeled substance accumulated in this way. Various types of chromatography strips are known, and all of these known chromatography strips, including those that will be described later, can be used in the present invention. The term "chromatography assay device", as used herein, means a chromatography strip which is produced in such a form that it can be used in an assay and is capable of being stored and transported. The following describes a normal example of a chromatography strip. A sample application site may be located in the same place where the marked substance is present, preferably in an upstream position of the labeled substance.

When a sample solution, which is suspected to contain an analyte to be tested, comes into contact with the sample application site, the sample solution is moved through the chromatography carrier in the downstream direction together with the analyte, performed by capillary action. Typically, the analyte is a compound that binds in a specific manner to a trap substance fixed to the detection site, or is a compound that binds in a specific manner to a conjugate that specifically binds to the trap substance in the detection site. For example, the analyte is an antibody when the trap substance is an antigen or the conjugate contains an antigen, and the analyte is an antigen when the trap substance is an antibody or the conjugate contains an antibody. By way of further example, the analyte can be a nucleic acid, which binds to a complementary conjugate and trap substance. When the sample application site is located in an upstream position to a labeled substance, the labeled substance may be arranged adjacent to the sample application site or in a position disconnected from the sample application site. The addition of the labeled substance can be effected by various means, for example, by adding it to a certain position outside the detection site of the chromatographic strip after the addition of the solution of the sample. Because the labeled substance is arranged in such a way that it is moved by the capillary action of the sample solution, the labeled substance moves in the downstream direction when the sample solution is added to the sample addition media . The detection site is generally located in a downstream position of the labeled substance and at a certain distance from the marked substance. At the detection site, a trap substance that binds only to an analyte or a conjugate in a specific manner, or binds specifically to each of the substances to be tested and a labeled substance, is fixed to the chromatography carrier. Accordingly, in one embodiment the analyte (sometimes bound to a labeled substance), moved by capillary action of the sample solution, is attached to the trap substance or a conjugate, which in turn binds to a substance of trap. The labeled substance is bound to the substance thus bound to be tested, thereby effecting the accumulation of the labeled substance in the detection means, in response to the presence or amount of the analyte. Alternatively, the labeled substance and the analyte, moved by capillary action, are competitively bound to the trap substance or a conjugate, which in turn binds to the trap substance, thereby effecting the accumulation of the trap. substance marked in inverse proportion to the amount of substance to be tested. There is a case in which a certain marked substance binds both to a trap substance (or a conjugate which in turn attaches to a trap substance) and to an analyte, but not in a simultaneous way and, in that case , the analyte binds first to the labeled substance and the remaining labeled substance, which did not bind to the substance to be tested, binds to the trap substance. Consequently, the presence or quantity of the analyte can be analyzed by measuring the marked substance accumulated in the detection means. As the occasion demands, several substances are located upstream of the detection site. For example, a conjugate can be placed in this manner in a movable manner. In some cases, one or more additional detection sites can be arranged downstream of the first detection site. In addition, downstream of the detection site there can be a further extension of the chromatography carrier, so that a sample solution can be completely discharged or the carrier can be equipped with a material to be used in the absorption of the sample solution. In this way, the presence or quantity of an analyte of interest in a sample solution can be found by measuring the presence or amount of a marked substance accumulated at the detection site. In one case, this can be achieved visually. The present invention is intended to be used with a blood sample, including serum and plasma, but is preferably used with a blood sample containing red blood cells, for example, whole blood. Before testing the analyte of interest in the blood sample, the red blood cells are preferably removed, if the trial will work with the desired sensitivity. Thus, according to the present invention, a red blood cell separating agent is bound to the chromatography carrier. The red blood cell separation agent can be attached to the chromatography carrier at any location, which will work to separate the red blood cells from the plasma or serum. Preferably, it diffusely binds to the chromatographic carrier upstream of the detection site. Most preferably, the red blood cell separating agent is diffusely bound at the sample application site. This location is preferable because it causes aggregation of red blood cells as soon as they are applied to the chromatography carrier resulting in minimal interference, if any, in the flow of serum or plasma along the carrier by capillary action. The red blood cell separating agent of the present invention can be any substance capable of adding red blood cells. Preferred agents are positively charged materials, such as polycations, including, for example, poly-L-lysine hydrobromide.; poly (dimethyl diallyl ammonium) chloride (Merquat®-100, Merquat® 280, Merquat® 550); poly-L-arginine hydrochloride; poly-L-histidine; poly (4-vinylpidirine), poly (4-vinylpyridine) hydrochloride; quaternary salt of crosslinked poly (4-vinylpyridine) methylchloride; poly (4-vinylpyridine-co-styrene); poly (hydrogen fluoride) of poly (4-vinylpyridinium); poly (4-vinylpyridinium-P-toluene sulfonate); poly (4-vinylpyridinium-tribromide); poly (4-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate); poly vinylpyrrolidone, crosslinked; polyvinylpyrrolidone, poly (melamine-co-formaldehyde); partially methylated hexadimetre bromide; hydrobromide 1: 4 poly (Glu, Lys); hydrobromide 3: 1 poly (Lys, Ala); hydrobromide 2: 1 poly (Lys, Ala); succinylated poly-L-lysine; hydrobromide 1: 1 poly (Lys, Ala); and hydrobromide 1: 4 poly (Lys, Trp). The most preferred polycation is poly (dimethyl diallyl ammonium) chloride (Merquat®-100). The red cell separation agent of the present invention can be used in any suitable amount, which functions to separate the red blood cells from the rest of the sample. Preferably, the red blood cell separating agent may be present in a concentration of from about 0.04% to about 1.3% (weight per volume), with from about 0.13% to about 0.33% (weight per volume) being preferred, and more preferred. approximately 0.20% to approximately 0.33% (weight by volume). A positive charge on the red blood cell separation agent has a tendency to add any negatively charged agent present in the strip. For example, a labeled or conjugated substance bound to the chromatography carrier can also be added by the red blood cell separating agent interfering with the binding of the analyte to the conjugate or, in a competitive assay, the binding of the labeled substance and the analyte of interest to the trap substance at the detection site or a conjugate. Finally, the sensitivity and accuracy of the immunoassay system can be compromised. Accordingly, when the globule separating agent is a positively charged material, the present invention preferably employs a neutralizing agent. The neutralizing agent is capable of neutralizing the positive charge of the red blood cell separation agent, thereby eliminating, or at least minimizing, any interference to the assay system caused by the red blood cell separating agent. Preferably, the neutralization agent is diffusely bound to the chromatographic carrier. The neutralizing agent can be diffusely bound at any location on the chromatographic carrier where it will function to neutralize a red blood cell separating agent, but preferably it is located downstream of the red blood cell separation agent and upstream of the detection site, and more preferably it is located in the same place in the chromatography as the labeled substance diffusely bound. The neutralizing agent can be any polyanion capable of neutralizing the positive charge of the red blood cell separating agent. Preferred polyanions include poly (acrylic acid), poly (Na salt of acrylic acid), poly (methyl methacrylic acid), poly (Na-4-styrene sulfonate), poly (vinyl sulfonic acid), poly-L- acid aspartic and carboxymethyl cellulose, dextran sulfate being the most preferred. The neutralizing agent may be present in any amount that functions to neutralize the positive charge of the red blood cell separating agent. Generally, the concentration of the neutralizing agent is dependent on the concentration of the red blood cell separation agent being used. Preferably, the neutralizing agent is present in a concentration from about 0.33% to about 20% (weight per volume), with about 0.34% to about 10% (weight per volume) being more preferred, and 0.34% to 10 being more preferred. % (weight by volume).

Figure 1 shows one embodiment of an immunochromatography assay device according to the present invention. The device 1 0 comprises a chromatography carrier 20. Located on the chromatography carrier 20 is an application site 30 for the blood sample. In this preferred embodiment, the red blood cell separation agent, ie, Merquat® 1 00, is located at the application site 30. Adjacent to the application site 30 is a conjugate pad 40 containing the conjugate, i.e. Selenium-labeled binding substance and a neutralizing agent, ie, dextran sulfate. In addition, downstream is the detection site 50, which after the test has been run, will display a control bar 60 and if the substance to be tested is present, a test bar 70. In another embodiment of the test present invention, a damper may be contacted with the application site, preferably after the application site has had contact with the sample. The buffer helps maintain an acceptable fluid flow rate along the flow path in the chromatographic carrier. The buffer may be any substance that is capable of flowing by capillary action along the fluid flow path including, but not limited to, phosphate buffer, phosphate buffer saline, Tris-HCl buffer, buffer carbonate, citrate buffer, buffer of HEPES (2-hydroxypiperazine-N'-2-ethanesulfonic acid), buffer of MOPS (3- (N-morpholino) propanesulfonic acid), MES buffer (2- (N-morpholino) ethanesulfonic acid) and the like. Although the concentration and pH can be any concentration and pH working in the desired test device, preferably, the molarity is in a range from about 10 mM to about 100 mM and the pH is from about 5-9 and more preferably from approximately 6-8. Most preferably, the buffer used is 50 mM phosphate buffer, pH 7.4. The volume of fluid employed in the present invention is dependent on the size of the device. Suitably, sufficient volume of fluid is used to allow the flow of fluids through the device to the detection site. Preferably, the volume of fluid is in a range from about 25 μl to about 100 μl, and more preferably from about 40 μl to about 60 μl. Accordingly, the buffer, when necessary, can be added in a volume range from about 10 μl to about 40 μl, and more preferably from about 20 μl to about 30 μl. The present invention is also directed to a method for detecting the presence of an analyte in a blood sample. Preferably, the method employs the one-shot assay device of the present invention. Specifically, the method comprises (1) providing a chromatography carrier, which defines a path for fluid flow capable of supporting capillary flow, along which an application site for the blood sample is located. , which is in fluid contact with the chromatography carrier, a detection site in the chromatography carrier separated from the application site, a diffusely labeled substance (or a conjugate), which binds to or competes with the analyte for joining the detection site, a red blood cell separation agent diffusely linked to separate plasma or serum from said blood sample upstream of the detection site, and a conjugate bound to the chromatography carrier; (2) contacting the application site with the blood sample, so that the red blood cell separation agent separates the red blood cells from the plasma or serum from the blood sample, and the neutralizing agent neutralizes the positive charge of the agent from separation; and (3) detecting the presence of analyte in the blood sample. Preferably, the red blood cell separating agent is a positively charged material and the fluid flow path contains a diffusely bound neutralizing agent, which is capable of binding with said red blood cell separation agent and is located downstream. of said red blood cell separation agent and upstream of said detection site, whereby the positive charge of said separation agent is neutralized. Thus, in the preferred embodiment of Figure 1, a blood sample is applied to the application site 30 and the red blood cell separation agent separates the red cells by adding them and allowing the plasma or serum to move by capillary action downwards. of the chromatographic carrier 20. The neutralizing agent in the conjugate pad 40 neutralizes the effects of the red blood cell separating agent in the device 10 and conjugate, and the analyte binds to the conjugate present in the conjugate pad 40. The analyte bound to the conjugate continues to move downstream to the detection site 50. If the analyte of interest is present, the test bar 70 will appear. To indicate that the test is working properly, the control bar 60 will appear whether or not it is present. analyte of interest. The present invention may preferably include a non-reactive cover or enclosure around the device. Preferably, the cover encloses at least the chromatographic carrier to avoid contact with, and contamination of, the capture sites. The cover may also include a raised area adjacent to the application site to facilitate receiving and / or containing a certain volume of the sample. Additionally, the cover may include a cut area or areas in the form of a letter, number, icon or symbol, or any combination thereof. In this embodiment, the cutting area or areas form the design for one or a few particular detection sites when the strip is completely closed. It is preferred that a sufficient portion of the strip can be enclosed to prevent the applied sample from contacting the detection sites without first passing through a portion of the strip. The device and method of the present invention can be used in any test system in which a blood sample contains an analyte of interest. Examples of preferred systems include, but are not limited to, Hepatitis C virus ("HCV"), hepatitis A virus ("HAV"), human immunodeficiency virus ("H IV"), hepatitis B surface antibody ( "HBsAb"), hepatitis B surface antigen ("HBsAg"), hepatitis B core antibody ("HBcAb"), hepatitis B core antigen ("H BcAg"), carcinogenic antigen ("CEA") , alpha-fetoproten ("AFP"), a marker of pancreatic cancer ("CA1 9-9"), syphilis, tuberculosis, malaria, Leishmania, and Dengue fever. The following examples further illustrate the present invention, but should not be interpreted, in any way, as limiting its scope.

EXAMPLES Example 1 Aggregation of red blood cells vs. aggregation of se-conjugate by polycations For the purpose of the present invention, aggregation of red blood cells (rbc's) in whole blood is desired while aggregation of the selenium conjugate is not desired. Several polycations were tested to see which could cause sufficient aggregation of rbc's while only minimally adding the selenium conjugate. The recombinant protein selenium conjugate was prepared in the following manner: first, selenium colloid was prepared by reacting 32 mM selenium oxide with 91 mM L-ascorbic acid in an aqueous solution for 72 hours at 42 ° C. This selenium colloid was diluted to an optical density of 30 at a wavelength of 550 nm and then reacted with 40 μg / ml of recombinant H IV-1 envelope protein in 30 mM Tris buffer, pH 7.4 for 20 minutes at room temperature. This protein conjugate of H IV-1 labeled with selenium colloid was then diluted to an optical density of 30 at a wavelength of 550 nm in Tris buffer 1 0 mM, pH 7.4 containing 0. 1% of casein and it was incubated for 20 minutes at room temperature. The conjugate solution was centrifuged at 1970 x g for 20 min at 4 ° C, the supernatant was removed and the pellet discarded. A buffer volume of 30 mM Tris, pH 7.4 containing 2% casein, equivalent to one tenth of the volume of the supernatant, was then added. Finally this conjugate solution was diluted to an optical density of 10 at a wavelength of 550 nm in 50 mM Tris buffer, pH 7.4 containing 1% casein, 2% sucrose and 2% lactose. Aqueous solutions of the following polycations were prepared at 0.25% (w / v): poly-L-lysine hydrobromide, molecular weight (mp) 37000; poly-L-arginine hydrochloride, MW 12100, 42400 and 92000; poly-L-histidine, MW 1 8400; hexadimetrin bromide, hydrobromide 3: 1 poly (lysine, alanine), MW 35000; 2: 1 hydrobromide urea poly (lysine, alanine), MW 49300; hydrobromide 1: 1 poly (lysine, alanine), MW 41600, hydrobromide 1: 4 poly (lysine, tryptophan), MW 38000 (all previous polycations were purchased from Sigma, St. Louis, MO); poly (diallyldimethylammonium chloride), MW 05 to 06 (Merquat®-1 00, Calgon, Pittsburgh, PA). The ability of these polycations to add either rbc's in whole blood or the selenium conjugate were observed in separate reactions by adding 350 μl of 0.25% of the various polycation solutions to an equal volume of either whole blood or the selenium conjugate . The solutions were mixed and stored at room temperature for 10 minutes, then the aggregation was visually evaluated. The results are summarized in Table 1. A plus sign (+) indicates a weak aggregation, 2+ indicates moderate aggregation, and 3+ and 4+ indicate strong aggregation.

TABLE 1 A good choice would be a polycation that causes the aggregation of rbc's (2+ or greater) while causing minimal aggregation of selenium conjugate (2+ or less). Those polycations with a 2+ in both categories conform to this criterion. HBr poly-L-lysine and Merquat®-100 were chosen for additional work, with Merquat® being the most cost effective.

Example 2 Prevention of conjugate aggregation with polyanion neutralization A. Prevention of aggregation and conjugate flow using dextran sulfate Using poly-L-lysine as the polycation rbc aggregation reagent, various concentrations of dextran sulphate were tested. polyanion to see if the positive charge of the polycation could be neutralized by the dextran sulfate, thus preventing the aggregation of the selenium conjugate caused by the polycation. Dextran sulfate was added after the polycation had already caused the aggregation of the rbc's, but before the interaction of the polycation with the selenium conjugate. The following experiment evaluated the effect of polycation and dextran sulfate on the aggregation of the selenium conjugate and its subsequent ability to flow along the strip of inm unicochromatog raphy. An immunochromatography strip was assembled, consisting of a Sample Cushion, a Neutralization Cushion, a Conjugate Cushion, and a Detection Strip. The Sample Cushion was prepared by soaking a glass fiber filter 4 mm wide by 20 mm long (Lypore 9524, Lydall, Rochester, NH) in an aqueous solution of 0.33% poly-L-lysine hydrobromide. , pm 37000 (Sigma, St. Louis, MO), then drying under vacuum. The Neutralization Cushions, containing different concentrations of Dextran Sulphate, were prepared by soaking filters 4 mm wide by 13 mm long made of wood pulp and polyester (Sontara 8801, Du Pont, Wilmington, Delaware) in aqueous solutions containing 0%, 1 .1%, 3.3% or 10% Dextran Sulfate, p.m. 5000 (Sigma, St. Louis, MO). After soaking, the cushions were dried under vacuum. The Conjugate Cushion was prepared by soaking a 4 mm wide by 4.3 mm long glass fiber filter (Lypore 9524, Lydall, Rochester, NH) in recombinant protein conjugate of H IV-1 labeled with selenium colloid, prepared and diluted as in Example 1. After soaking, the Conjugate Cushion was dried under vacuum. The Detection Strip was a nitrocellulose membrane filter 4 mm wide by 40 mm long (catalog # H9643G1, Millipore, Bedford, MA). The HIV-1 envelope antigen was added at a concentration of 5 mg / ml in 100 mM Tris buffer, pH 7.4 containing 1% sucrose, to the nitrocellulose membrane in order to form a line across the width of pull it in a position approximately 1 cm from the end of the membrane. The delineated region is supported with Polyester Laminate (Code # 7733, Adhesives Research Inc., Glen Rock, PA). This was allowed to dry sufficiently, in order to fix the antigen to the nitrocellulose. The 4 mm wide nanochromatography strips were assembled using the above components by placing them end-to-end lengthwise with a 1 mm overlap between each section, with the 20 mm long Sample Cushion at one end , near which one of the Neutralization Cushions of 1 3 mm long was placed, followed by a Conjugate Cushion of 4.3 mm in length, and finally a Detection Strip of 40 mm in length. Then the reinforced strip was covered with Polyester Laminate (code # 8648, Adhesives Research Inc., Glen Rock, PA) from the top of the Detection Strip to 1 0 mm from the bottom of the strip, leaving approximately 10 mm of the Cushion of Sample expueesto. Eighty μl of plasma was then applied to the Sample Cushions of each of the immunochromatography strips containing Neutralization Cushions with either 0%, 1.1%, 3.3% or 1.0% Dextran Sulfate. The aggregation of the red selenium conjugate and the ability of the conjugate to flow along the strip were visually observed. The results, shown in Table 2 below, indicated that, without the presence of Dextran Sulfate to neutralize the charge of the polycation solution, the selenium conjugate was added and was not able to flow along the strip. There was an inverse relationship between conjugate aggregation and flow, with Dextran Sulfate concentrations of 3.3% or higher being sufficient to prevent aggregation of conjugate and allow the conjugate to flow along the strip.

TABLE 2 B. Aggregation of rbc in the presence of dextran sulfate In order to assess the effect of dextran sulfate on rbc aggregation, the previous experiment was repeated using whole blood as the sample with 10% dextran sulfate on a cushion. Neutralization of 4.3 mm long by 4 mm wide. After mounting the immunochromatography strip as before, using this Neutralization Cushion, 80 μl of whole blood was applied to the Sample Cushion. Fifteen minutes later, the result of rbc aggregation was visually observed and the ability of the resulting plasma to flow along the strip was measured. The rcbs were added, being retained in the Sample Pad and did not flow over the strip, while the plasma flowed 33 mm along the strip in 15 minutes. This indicated that the polycation in the Sample Cushion was still able to cause the aggregation of the rcbs in the whole blood sample, and that the presence of the polyanion, Dextran Sulfate, in the Neutralization Cushion did not interfere with this aggregation of rbc. .

C Prevention of conjugate aggregation by polyanions Other polyanions were tested to evaluate their ability to prevent aggregation of the selenium conjugate as in Example 2. A. A Sample Pad of 5.5 mm in length and 4 mm in width was soaked. in an aqueous solution containing 0.26% Merquat®-100, and then dried at 55 ° C. The Neutralization Cushions were not used, and instead the selenium conjugate was diluted in buffer of Tris 1 0 mM, pH 7.4 containing 1% of casein, 2% of sucrose, 2% of lactose and 0%, 1 .1 % or 3.3% of the Dextran sulphate polyanion, MW 5000 (Sigma, St. Louis, MO), or 0%, 0.5%, 1% or 2% of one of the following polyanions (all from Aldrich Chemical Co., Milwaukee, Wl): poly (acrylic acid), MW 5000; poly (sodium sulfonate-4-styrene), MW 70,000; poly (sodium salt of vinyl sulfonic acid); poly (methyl methacrylic acid), MW 9500; poly (sodium salt of acrylic acid), mp 21 00. The Conjugate Cushions were soaked in the various selenium conjugate solutions and dried under vacuum. The Detection Strip was prepared as in Example 2. A. and the immunochromatography strips were armed. Fifty μl of plasma was then applied to the Sample Cushions of each of the immunochromatography strips containing Conjugate Cushions with the various polyanions. The aggregation of the red selenium conjugate was observed visually. Table 3 shows the relative amount of aggregation of conjugate seen with the various concentrations of polyanions tested.

TABLE 3 nt = not tested As before, the selenium conjugate was added if there was not a polyanion present to neutralize the positive charge of the Sample Cushion polycation (which is necessary for rbc aggregation when whole blood is tested). All polyanions used in the Conjugation Cushion prevented conjugate aggregation from occurring at least at one of the concentrations tested. This experiment also showed that the polyanion did not have to be applied to a separate cushion, but could be combined with the selenium conjugate in the Conjugation Cushion.

Example 3 Use of dextran sulfate in Cojín de Neutralización vs. Conjugation Pad in an HIV-1 antibody assay Immunochromatography strips were prepared as in Example 2. A. either with or without a 4 mm wide by 4.3 mm glass fiber filter Neutralization Cushion long (Lypore 9524, Lydall, Rochester, NH). When used, the Neutralization Cushion was soaked in an aqueous solution containing 3.3% dextran sulfate, then dried under vacuum. In the strips without a Neutralization Cushion, the selenium conjugate was diluted in 10 mM Tris buffer, pH 7.4 containing 1% casein, 2% sucrose, 2% lactose and 3.3% dextran sulfate, and the Cushion Conjugate was soaked in this solution and then dried under vacuum. The Sample Pad used was as in Example 2. A., except that it was soaked in a 0.2% aqueous solution of Merquat®-100. Human serum containing HIV-1 antibodies was diluted 1: 2048 either in HIV negative human whole blood (based on plasma volume) with a hematocrit value of 50% or in human plasma negative for HIV. Three more serial 1: 2 dilutions were made, again using either whole blood or plasma as the diluent. Eighty μl of negative whole blood or samples from the series of HIV-1 positive whole blood dilutions were added to the Sample Pad of the immunochromatography strips prepared with dextran sulfate on a separate Neutralization Pad or dextran sulfate in the solution of conjugate of selenium in the Conjugate Cushion. Eighty μl of negative plasma or samples from the HIV-1 positive plasma dilution series were tested only on dextran sulfate immunochromatography strips in the Conjugate Cushion. The results were read 15 minutes after the application of the sample (Table 4). A positive result showed a red color on the Detection Strip, where the HIV-1 antibody complex conjugated with red selenium HIV-1 antigen to the HIV-1 antigen was joined in the delineated region of the strip. A negative result showed no color in this region on the Detection Strip.

TABLE 4 nt = not tested The results in Table 4 indicate that the HIV-1 antibodies are detectable from whole blood in an immunochromatography strip assay using the Merquat®-1 00 polycation to add rbc's and allow the sample to flow along the strip, and the dextran sulfate polyanion as a neutralizing agent, preventing the aggregation of the selenium conjugate by the polycation and allowing the conjugate to bind and complex with a positive sample and flow along the strip to the detection area . The polyanion was shown to be effective when used either on a separate Neutralization Cushion or combined with the selenium conjugate on the Conjugate Cushion. In this assay, the sensitivity to detect H IV-1 antibodies showed a 2-fold improvement when the polyanion (dextran sulfate) was used in the Conjugate Pad instead of a separate Neutralization Pad. Additionally, the results in Table 4 indicate that the polycation is effectively adding the rbc's in the whole blood, as shown by the same detection sensitivity of HIV-1 antibodies either in whole blood, where the rcbs should be added so that the sample flows, or plasma, where there are no RBCs to avoid the flow of the sample. This also shows that the presence of the polyanion, either in a separate Neutralization Cushion or in the Conjugate Cushion, does not interfere with the capacity of the polycation to effectively cause the aggregation of rbc's in whole blood.

Example 4 Use of Merguat® and several polyanions in an HBsAg assay. Immunochromatography strips were prepared for the detection of Hepatitis B surface antigen (HBsAg) in whole blood samples. Merquat®-1 00 was used as the polycation for the aggregation of rbc's in the Sample Cushion, and various polyanions in the Conjugate Cushion were evaluated as polycation neutralization reagents to prevent aggregation of the selenium conjugate.

The strips of unmromatochromatography, composed of a Sample Cushion, a Conjugate Cushion, and a Detection Strip. He Sample Pad was prepared by soaking a glass fiber filter 4 mm wide by 15.5 mm long in an aqueous solution of 0.26% Merquat®-1 00, then drying at 55 ° C. The selenium conjugate was prepared using selenium colloid, as in Example 1, and 12 μg / ml of mouse monoclonal antibody to HBsAg (anti-H Bs). This conjugate was then diluted anti-HBs labeled with selenium colloid to an optical density of 2.6 at a wavelength of 550 nm in Tris buffer, containing one of the following polyanions: 0.5% poly (acrylic acid), 2000 ppm (PAA-2000); 0.5% poly (acrylic acid), MW 240,000 (PAA-240,000); 0.5% dextran sulfate, p.m. 5000; 0.8% poly-L-aspartic acid, MW 36, 300; 0.5% carboxymethyl cellulose, MW 90, 000 (CMC). Dextran sulfate and poly-L-aspartic acid were from Sigma, St. Louis, MO, and the remaining polyanions were from Aldrich Chemical Co., Milwaukee, Wl. The Conjugate Cushion was prepared by soaking a 4 mm wide by 4.3 mm long fiberglass filter in anti-HBs conjugate labeled with selenium colloid, prepared and diluted with one of the above polyanions. After soaking, the Conjugate Cushion was dried under vacuum. The Detection Strip was a nitroceulose membrane filter 4 mm wide by 40 mm long, prepared as in Example 2 using mouse monoclonal anti-HBs at a concentration of 3 mg / ml and added to the membrane. nitrocellulose, in order to form a line across the width of the strip at a position approximately 1 cm from the end of the membrane. The delineated region was supported with Polyester Laminate. This was allowed to dry sufficiently in order to bind the antibody to the nitrocellulose. The immunochromatography strips were assembled using the above components when placing them end-to-end lengthwise, with a 1 mm overlap, with the Sample Cushion at one end, after which a Conjugate Cushion was placed, and finally a Detection Strip. Then the strip armed with Polyester Laminate was covered, leaving approximately 10 mm of the Sample Cushion exposed. Recombinant BsAg H was added to human negative whole blood to HBsAg with a hematocrit value of 50% at a concentration of 12.5 ng / ml. Three additional 1: 2 serial dilutions were made in whole blood. Fifty μl of negative whole blood or samples from the positive whole blood dilution series to HBsAg were added to the Sample Pad of the immunochromatography strips prepared with various polyanions in the Conjugate Pad. The results were read 15 minutes after the application of the sample (Table 5). A positive result showed a red color on the Detection Strip, where the anti-H Bs red selenium-HBsAg conjugate complex was bound to the anti-HBs in the delineated region of the strip. A negative result showed no color in this region on the Detection Strip. The aggregation of the red selenium conjugate at the entrance to the Detection Strip was visually observed.

TABLE 5 Although all polyanions allowed the detection of HBsAg to occur, those polyanions that prevented the conjugate aggregation, PAA-2000, dextran sulfate and poly-L-aspartic acid, exhibited a 2- to 4-fold more sensitive detection of HBsAg in samples of whole blood. The above experiment was repeated, using the conjugate labeled with selenium colloid diluted in Tris buffer containing PAA-2000 as the polyanion, except 25 μl of 50 mM phosphate buffer, pH 7.4, was added to the sample pad one minute later of the addition of the whole blood samples of H BsAg. The results obtained using this procedure, with the addition of the buffer after the application of the sample, were identical to the results without this step. In this way, these tests can be done either with or without addition of buffer after application of the sample.

Example 5 Use of Merquat® and dextran sulfate in a tuberculosis assay Immunochromatography strips were prepared for the detection of antibody to Mycobacterium tuberculosis (anti-Mtb) in whole blood samples. Merquat®-1 00 was used as the polycation for the aggregation of rbc's in the Sample Cushion, and several concentrations of the dextran sulphate polyanion were evaluated in the Cushion.

Conjugate as the polycation neutralization reagent to prevent aggregation of the selenium conjugate. The immunochromatography strips were assembled, composed of a Sample Cushion, a Conjugate Cushion, and a Detection Strip. He Sample Pad was prepared by soaking a glass fiber filter 4 mm wide by 1 5.5 mm long in an aqueous solution of 0.26% Merquat®-100, then drying under vacuum. The selenium conjugate was prepared using selenium colloid, as in Example 1, and 3.5 μg / ml recombinant Mtb antigen from E. coli. This conjugate of Mtb labeled with selenium colloid was then diluted to an optical density of 2.5 at a wavelength of 550 nm in Tris buffer containing either 0%, 0.34%, 1.1% or 3.3% dextran sulfate. . The Conjugate Cushion was prepared by soaking a glass fiber filter 4 mm wide by 4.3 mm long in conjugate of Mtb labeled with selenium colloid, prepared and diluted with one of the above concentrations of dextran sulfate. After soaking, the Conjugate Cushion was dried under vacuum. The Detection Strip was a nitrocellulose membrane filter 4 mm wide by 40 mm long, prepared as in Example 2 using a recombinant Mtb antigen at a concentration of 0.1 5 mg / ml and added to the membrane of nitrocellulose, in order to form a line across the width of the strip at a position approximately 1 cm from the end of the membrane. The delineated region was supported with Polyester Laminate. This was allowed to dry sufficiently for the purpose of fixing the antigen to the nitrocellulose. The immunochromatography strips were assembled using the anterior components by placing them longitudinally end-to-end, with a 1 mm overlap, with the Sample Cushion at one end, after which a Conjugate Cushion is placed, and finally a Detection strip. Then the reinforced strip was covered with Polyester Laminate, leaving approximately 10 mm of the Sample Cushion exposed. The anti-Mtb positive serum was diluted 1: 1000 in negative human whole blood with a hematocrit value of 50%. Two additional 1: 2 serial dilutions were made in whole blood. Fifty μl of negative whole blood or samples from the positive whole blood dilution series to anti-Mtb were added to the Sample Pad of immunochromatography strips prepared with various concentrations of dextran sulfate in the Conjugate Cushion. The results were read 15 minutes after the application of the sample (Table 6). A positive result showed a red color on the Detection Strip where the conjugate complex of Mtb selenium red-anti-Mtb bound to Mtb in the delineated region of the strip. A negative result showed no color in this region on the Detection Strip. The aggregation of the red selenium conjugate at the entrance of the Detection Strip was visually observed.

TABLE 6 The data in Table 6 show that the assay does not work without the presence of a polyanion, in this case dextran sulfate, to prevent aggregation of the conjugate. There is an inverse relationship between the aggregation of conjugate and the sensitivity of the assay. At a dextran sulfate concentration of 3.3%, conjugate aggregation does not occur and the assay shows the most sensitive detection of anti-Mtb.

Example 6 Syphilis Test Using Merquat® and Dextran Sulfate Immunochromatography strips were prepared for the detection of Treponema pallidum antibody (anti-TP) in whole blood or plasma samples. By comparing the sensitivity of detection in whole blood to plasma, one could determine if the rcbs in the whole blood are being effectively added by the polycation, in order not to interfere with, and therefore decrease, the sensitivity of detection of the essay. Merquat®-100 is used as the polycation for the aggregation of rbc's in the Sample Cushion, and the dextran sulfate polyanion was used in the Conjugate Cushion as the polycation neutralization reagent to prevent aggregation of the selenium conjugate. The immunochromatography strips were assembled, composed of a Sample Cushion, a Conjugate Cushion, and a Detection Strip. He Sample Cushion was prepared by soaking a glass fiber filter 4 mm wide by 15.5 mm long in a 0.2% aqueous solution of Merquat®-1 00, then drying at 55 ° C. The selenium conjugate was prepared using selenium colloid, as in Example 1, and 7.5 μg / ml of Treponema pallidum (TP) lysate. This conjugate of TP labeled with selenium colloid was then diluted to an optical density of 2.8 at a wavelength of 550 nm of Tris buffer containing 3.3% dextran sulfate. The Conjugate Cushion was prepared by soaking a 4 mm wide by 4.3 mm long fiberglass filter in the PT conjugate labeled with selenium colloid prepared above. After soaking, the Conjugate Cushion was dried under vacuum.

The Detection Strip was a 4-mm-wide by 40-mm long n-cell membrane filter prepared as in Example 2 using Treponema pallidum lysate at a concentration of 44 μg / ml and added to the nitrocellulose membrane. , in order to form a line across the width of the strip to a position approximately 1 cm from the end of the membrane. The delineated region was supported with Polyester Laminate. This was allowed to dry sufficiently in order to fix the TP lysate to the nitrocellulose. The immunochromatography strips were assembled using the above components by placing them longitudinally end-to-end, with a 1 mm overlap, with the Sample Cushion at one end, after which a Conjugate Cushion was placed, and finally a Strip detection. The reinforced strip was then covered with Polyester Laminate, leaving approximately 10 mm of the exposed Sample Cushion. The positive human anti-TP serum was diluted 1: 10.8 either in negative human whole blood with a hematocpto value of 50% or in human negative plasma. Four additional 1 2 serial dilutions were made, again using either whole blood or plasma as the diluent. Sixty μl of whole blood or negative plasma or samples of the anti-TP positive or whole blood dilutions series were added to the Sample Cushion of the immunochromatography strips. The results were read 15 minutes after the application of the sample (Table 7). A positive result showed a red color on the Detection Strip where the TP-red selenium-anti-TP conjugate complex was bound to the TP lysate in the delineated region of the strip. A negative result showed no color in this region on the Detection Strip.

Table 7 shows that the sensitivity for the detection of anti-TP was the same in both whole blood and plasma, indicating that the polycation, Merquat®-100, effectively added the rcbs in the whole blood.

TABLE 7 All publications, patents and patent applications cited herein are incorporated by reference to the same extent as if each individual document was individually and specifically indicated to be incorporated by reference and is set forth in its entirety herein. Although this invention has been described with emphasis on preferred embodiments, it will be obvious to those of ordinary skill in the art that the preferred embodiments may be varied. It is intended that the invention be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as set forth in the specification and accompanying claims.

Claims (18)

1. A chromatography assay device for detecting the presence of an analyte in a blood sample, comprising: a chromatography carrier, which defines a path for fluid flow capable of supporting capillary flow, an application site for said blood sample in fluid flow contact with said chromatography carrier, a detection site in said chromatography carrier separated from said application site, a diffusely linked labeled substance located downstream of said application site; a red blood cell separation agent diffusely linked to separate plasma or serum from said blood sample upstream of said detection site, and a neutralizing agent diffusely linked downstream of said bounding agent and upstream of said blood. said detection site.

2. The chromatography assay device of claim 1, wherein said device is an immunoassay device.

3. The chromatography assay device of claim 1, wherein said red blood cell separating agent is attached to said site for application of said blood sample.

4. The chromatography device of claim 1, wherein said red blood cell separating agent is a positively charged material.

5. The chromatography assay device of claim 4, wherein said positively charged material is a polycation selected from the group consisting of poly-L-lysine hydrobromide, poly-L-arginine hydrochloride, poly-L-histidine, hydrobromide 3: 1 poly (lysine, alanine), hydrobromide 2: 1 poly (lysine, alanine), hydrobromide 1: 1 poly (lysine, alanine), hydrobromide 1: 4 poly (lysine, tryptophan), and poly (diallyldimethylammonium chloride) ).

6. The chromatography assay device of claim 4, wherein said positively charged material is poly (diallyldimethylammonium chloride).

7. The chromatography assay device of claim 1, wherein said neutralizing agent is a negatively charged material.

8. The chromatography assay device of claim 7, wherein said negatively charged material is a polyanion selected from dextran sulfate, poly (acrylic acid), poly (sodium sulfonate-4-styrene), poly (vinyl sulfonic acid), poly (methyl methacrylic acid, poly-L-aspartic acid, and carboxymethyl cellulose) 9.

The chromatography assay device of claim 7, wherein said negatively charged material is dextran sulfate 10.

The chromatography assay device of the claim 1, wherein said neutralizing agent is diffusely bound to the chromatography carrier in the same location as said labeled substance. eleven .

The chromatography assay device of claim 1, wherein said labeled substance is a selenium-labeled substance.

12. A method for detecting the presence of an analyte in a blood sample, comprising the steps of: providing a chromatography carrier, which defines a path for fluid flow capable of supporting capillary flow, along which find (a) an application site for said blood sample in fluid flow contact with said chromatography carrier, (b) a detection site in said chromatography carrier separated from said application site, (c) a labeled substance located downstream of said application site, (d) a red blood cell separation agent diffusely bound to separate plasma or serum from said blood sample, upstream of said detection site, and (e) a bound neutralizing agent diffusely located downstream of said separation agent attached and upstream of said detection site; contacting said application site with said blood sample; and detecting the presence of analyte in said blood sample.

The method of claim 12, wherein said labeled substance comprises a substance marked with selenium.

The method of claim 12, wherein said neutralizing agent is diffusely bound to the chromatography carrier in the same location as said labeled substance.

15. The method of claim 12, wherein said red blood cell separating agent is a positively charged material.

16. The method of claim 15, wherein said positively charged material is a polycation selected from the group consisting of poly-L-lysine hydrobromide, poly-L-arginine hydrochloride, poly-L-histidine, hydrobromide 3: 1 poly (lysine, aianine), hydrobromide 2: 1 poly (lysine, alanine), hydrobromide 1: 1 poly (lysine, alanine), hydrobromide 1: 4 poly (lysine, tryptophan), and poly (diallyldimethylammonium chloride).

17. The method of claim 12, wherein said neutralizing agent is a negatively charged material. The method of claim 17, wherein said negatively charged material is a polyanion selected from dextran sulfate, poly (acrylic acid), poly (sodium sulfonate-4-styrene), poly (vinyl sulfonic acid), poly ( methyl methacrylic acid), poly-L-aspartic acid and carboxymethyl cellulose. SUMMARY A chromatography assay device and method for use with whole blood samples using a red blood cell separation agent to add red blood cells and allow the plasma or serum to flow by capillary action, and a neutralizing agent to neutralize any effect it may have the agent separating red blood cells on the device and the method.