HK1035027B - A chromatorgraphic device for neutrlization of polycations with polyanions and method thereof - Google Patents

Description

Chromatographic device and method using polyanion neutralization polycation action

Technical Field

The present invention relates to a chromatographic assay device and method for detecting an analyte in a whole blood sample, and more particularly, to a device and method for aggregating red blood cells using a red blood cell separating agent and eliminating the possible adverse effects of the red blood cell separating agent on the assay system using a neutralizing agent.

Background

Modern clinical diagnostic methods are usually performed on blood samples. Unfortunately, red blood cells interfere with many diagnostic assays. In the detection of analytes, red blood cells may inhibit the binding between specifically bound partner components. Furthermore, erythrocytes have an enzymatic activity which, depending on the assay method used, may interfere with the signal produced. On the other hand, in a rapid assay format using a chromatographic assay device, particularly a chromatographic immunoassay device, red blood cells may impede the flow of fluid necessary for reactions to occur on such a device. For this and other reasons, many assays are performed using plasma or serum, which must first be separated from whole blood.

There are many known techniques for separating red blood cells from plasma in a whole blood sample. Centrifugation is a well known method in the art by which plasma (before coagulation) and serum (after coagulation) can be separated from whole blood. During this process, the red blood cells settle to the bottom of the tube and the serum can be separated by slow pouring. However, the stratification of whole blood by centrifugation has a number of disadvantages. Centrifugation typically requires the withdrawal of a large sample of blood. In addition, this method is time consuming, requires cumbersome laboratory equipment, and is often not installed in the doctor's office. Finally, additional manipulation of the blood increases the potential risk of exposure to blood-borne pathogens.

To reduce or eliminate the need for centrifugation, assay devices have been developed that employ gradient membranes or capture membranes to separate red blood cells from the liquid portion of blood. Immobilized anti-erythrocyte antibodies were also 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 and then filtering the mixture through a solid, water-absorbent component to which at least one of its specifically bound partner binds, thereby removing agglutinated red blood cells; (2) passing whole blood through a glass microfiber filter, which may or may not have agglutinating agent bound thereto; (3) applying a barrier or spacer layer of polysaccharide material to prevent red blood cells from passing therethrough, thereby preventing it from interfering with the detection or visual observation of the signal on the dry test strip; and (4) applying a support having a polycationic surface which binds red blood cells and not plasma.

Many of these techniques for separating red blood cells from plasma are expensive, complex, may result in incomplete separation of red blood cells, and may cause hemolysis. Hemolysis can cause non-specific binding or high background interference, resulting in loss of detection sensitivity. This may be due to free hemoglobin, which stains the detection zone so that it can display a color in the range of pink to dark brown red. As a result, the visible chemical signal generated may be completely or partially masked due to the color of the hemoglobin present in the detection zone. In addition, the use of separating agents such as polycations in the detection system may interfere with the system, often by aggregating some other reagent or binding component in addition to the red blood cells.

Thus, there is a need for a device and method for detecting an analyte in a blood sample without adversely affecting the detection system. Such devices and methods should be suitable for a variety of volumes of whole blood samples, including small amounts.

Summary of The Invention

The present invention relates to a chromatographic device comprising a chromatographic carrier capable of supporting capillary flow and defining a flow path, a blood sample application site in fluid contact with the chromatographic carrier, a detection site spaced from the application site and on top of the chromatographic carrier, a diffusively bindable labeled substance located downstream of the application site, a diffusible bindable red blood cell separating agent upstream of the detection site for separating plasma or serum from the blood sample, and a diffusible bindable neutralizing agent capable of binding to the separating agent downstream of the binding to the separating agent and upstream of the detection site, whereby the positive charge of the separating agent can be neutralized. Preferably, the red blood cell separating agent is located at the sample application site so that red blood cells can be separated from the serum or plasma before the serum or plasma moves along the chromatographic carrier.

The invention also relates to a method for detecting the presence of an analyte in a sample, preferably a blood sample, which method comprises providing a chromatographic carrier capable of supporting capillary flow and defining a flow path along which there is (a) a sample application site for the blood sample in fluid contact with the chromatographic carrier, (b) a detection site on the chromatographic carrier spaced from the application site, (c) a diffusively bound labelled species located downstream of the application site, (d) a diffusibly bound red blood cell separating agent upstream of the detection site for separating plasma or serum from the blood sample, and (e) a diffusively bound neutralising agent capable of binding the separating agent downstream of the bound separating agent and upstream of the detection site, whereby the positive charge of the separating agent is neutralised; the method further includes contacting the sample application site with the blood sample such that the red blood cell separating agent can separate plasma or serum from the blood sample, and such that the neutralizing agent neutralizes the positive charge of the separating agent as the sample flows along the flow path; and further comprising detecting the presence or absence of the analyte in the blood sample.

Brief Description of Drawings

FIG. 1 shows a preferred embodiment of the chromatographic assay device of the present invention.

Detailed Description

The present invention is based on the observation that red blood cells in a whole blood sample interfere with the determination of the presence or amount of an analyte in the blood sample, and that in the absence of such interference, such detection can be conveniently accomplished by a variety of assay systems. For example, in an immunoassay, a whole blood sample in contact with a sample application site is unlikely to move along the test strip by capillary action due to the presence of red blood cell obstructions or interferences. The present invention overcomes this problem without affecting the sensitivity of the assay system.

Some of the following definitions may be applied in understanding embodiments of the present invention.

An "analyte" or "analyte to be studied" refers to a compound or composition to be detected or assayed having at least one antigenic epitope or binding site. Such analytes may be of the nature of the analyte-specific binding member or any material from which an analyte-specific binding member can be prepared, including but not limited to the following: toxins, organic compounds, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, drugs (including drugs administered for therapeutic purposes as well as drugs administered for illicit purposes), and metabolites of, or antibodies against, any of the foregoing. The term "analyte" also includes any antigenic substance, hapten, antibody, macromolecule and combinations thereof.

"chromatography carrier" refers to any suitable porous, absorbent, bibulous, isotropic material, or capillary material. It comprises the detection site of the device by means of which the analyte or sample to be tested can be transferred by means of capillary action or weak forces. It will be understood by those skilled in the art that the chromatographic carrier may be composed of a single material or may be composed of more than one material (e.g., different zones, portions, layers, regions or regions may be composed of different materials), so long as the multilayer structure is in fluid contact with each other so that the sample to be tested can pass between the materials. The fluid stream contact allows at least some of the components of the sample, i.e. the analyte, to pass between the zones of porous material, preferably uniformly along the contact interface between the different zones. Natural or synthetic materials, or synthetically modified naturally occurring materials, can be used as chromatographic supports, including but not limited to the following: paper (fibrous), or a film of cellulosic material (microporous) such as filter paper; cellulose and cellulose derivatives such as cellulose acetate and cellulose nitrate; glass fibers; a fabric; including natural (e.g., cotton) and synthetic (e.g., nylon) fabrics; and porous gels, and the like.

"label" refers to any substance capable of producing a visual or instrumentally detectable signal. Various labels suitable for use in the present invention include labels that generate a signal by chemical or physical means. Examples include enzymes and substrates, chromogens, fluorescent compounds, chemically reflective compounds, colored or colorable organic polymer latex particles, and liposomes or other microbubbles containing direct visualization materials. Radiolabels, colloidal metal particles or colloidal non-metal particles may be preferably used in the present invention. Preferred labels include colloidal gold and latex microparticles.

"labeled" or "conjugate" refers to a substance that contains a detectable label attached to a specific binding member. Such attachment may be covalent or non-covalent, and may also include nucleic acid hybridization. The label is capable of producing a detectable signal from the labeled substance, which signal is directly or indirectly related to the amount of the analyte in the sample to be tested. The specific binding member of the labeled substance may be selected to bind, directly or indirectly, to the analyte.

"specific binding member" refers to one member of a specific binding pair, i.e., two different molecules, wherein one molecule specifically binds to the second molecule either chemically or physically. If such a specific binding member is an immunoreaction, it may be, for example, an antibody, antigen, hapten, or a complex thereof, and if an antibody is used, it may be a monoclonal or polyclonal antibody, a recombinant protein or antibody, a chimeric antibody, mixtures or fragments thereof, and mixtures of antibodies and other specific binding members. Specific examples of specific binding members include biotin and avidin, antibodies and antigens corresponding thereto (both of which are independent of the sample to be assayed), and single-stranded nucleic acids and complementary strands thereof, and the like.

"capture" refers to one or more specific binding members that are attached to a portion of the interior or surface of a chromatographic carrier to form one or more "capture sites" to which analyte, labeled reagent and/or control reagent will be immobilized. The method of attachment is not critical to the invention. The capture material facilitates the observation of the detectable signal by substantially separating the analyte and/or labeled substance from unbound detection reagent and remaining components of the sample to be tested. The capture material may be immobilised on the chromatographic carrier by any suitable attachment means either before or during the detection. Further, a single detection site or a plurality of detection sites may be provided on the surface of or in the chromatographic carrier. The capture material may also be configured in a variety of configurations to produce different detection or assay modes. For example, the catchment may be formed as letters, numbers, icons or symbols, or in any combination thereof.

In particular, the present invention provides a chromatographic test 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 test strip having a chromatographic carrier defining a fluid flow path and capable of supporting capillary flow, a blood sample application site, and a detection site spaced from the application site for detecting the presence or amount of an analyte in the blood sample. Preferably, the device further comprises a labelled species (or conjugate) which is diffusibly bound to the chromatographic carrier. In a preferred embodiment, the labeled substance will bind to the analyte or compete for binding with the analyte at the detection site. The device preferably further comprises two further reagents diffusibly bound to the chromatographic carrier: (1) a red blood cell separating agent upstream of the detection site (hereinafter, the direction of movement of the sample by capillary action is referred to as "downstream", and the opposite direction is referred to as "upstream") which is capable of separating plasma or serum from the blood sample, and (2) a neutralizing agent downstream of the red blood cell separating agent, upstream of the detection site, for eliminating any influence, in particular adverse effect, of the red blood cell separating agent on the chromatography system.

For the purposes of the present invention, the term "diffusibly bound" as applied to a given reagent may be defined as any reagent used in the present invention, including but not limited to a labeled substance, a specific binding member, a red blood cell separating agent or a neutralizing agent, which term means that the reagent is bound in a manner that allows the bound reagent to flow along the flow path.

Any detection system may be used for the purposes of the present invention. Preferred are immunoassay systems including, but not limited to, horizontal flow systems, vertical flow systems, immersion systems, and dipsticks. Some known detection systems are outlined below.

Generally, for chromatographic test strips, at least one sample application site and one detection site are arranged on a chromatographic carrier. When a sample liquid suspected of containing the analyte of interest, i.e., the analyte, is applied to the sample application site, it will move along the chromatographic carrier by means of capillary action and accumulate the labeled substance or conjugate contained in the labeling means prearranged on the chromatographic carrier at the detection site by the action of a binding reaction (e.g., an immunological reaction) in proportion or inverse proportion to the presence or amount of the substance to be detected in the sample liquid, so that the presence or amount of the substance to be detected in the sample liquid can be detected by determining the presence or amount of the labeled substance or conjugate thus accumulated. Various types of lateral flow test strips are known, and all of these known lateral flow test strips, including those described below, can be used with the present invention. The term "chromatographic detection device" as used herein means a chromatographic test strip that is prepared in such a manner that it can be used for detection and can be stored and transported.

A typical example of the chromatographic test strip is described below. The sample addition site can be located at the same position as the labeled substance, preferably at a position upstream of the labeled substance. When a sample liquid suspected of containing the analyte to be detected is brought into contact with the sample application site, the sample liquid moves in the downstream direction along the chromatographic carrier together with the analyte by means of capillary action. Typically the analyte is a compound which binds in a specific manner to a capture substance immobilised at the detection site, or a compound which binds in a specific manner to a conjugate which has bound specifically to the capture substance at the detection site. For example, when the capture material is an antigen or the conjugate contains an antigen, the analyte is an antibody, and when the capture material is an antibody or the conjugate contains an antibody, the analyte is an antigen. As another example, the analyte may also be a nucleic acid that can bind to a complementary conjugate and a trap.

When the sample addition part is located at a position upstream of the labeled substance, the labeled substance may be arranged near the sample addition part or at a position separated from the sample addition part.

The addition of the labeled substance can be accomplished in a variety of ways, such as by adding the sample fluid to a location outside the detection site of the chromatographic assay strip after it has been added.

Since the labeled substance is arranged in such a manner that it can move by the capillary action of the sample liquid, the labeled substance can move in the downstream direction when the sample liquid is added to the sample addition device.

The detection site is usually located at a position downstream of the labeled substance and spaced apart from the labeled substance. At the detection site, a capture substance is immobilized on the chromatographic carrier, which capture substance binds in a specific manner only to the analyte or the conjugate, or specifically to each substance to be detected and to the labeled substance. Thus, in one embodiment, the analyte (sometimes linked to a labelled species) which is moved by capillary action of the sample fluid is bound to an capture species, or is bound to a conjugate which itself is bound to the capture species. The labeled substance binds to the bound analyte, thereby causing the labeled substance to accumulate in the detection device in response to the presence or amount of the analyte. Alternatively, the labeled substance and the analyte are moved by capillary action, competitively bound to the capture substance, or bound to a conjugate which itself is bound to the capture substance, resulting in accumulation of the labeled substance in inverse proportion to the amount of the analyte to be detected.

In one case, one of the labelled species binds both the capture species (or a conjugate which itself binds to the capture species) and the analyte, but not both, in which case the analyte first binds to the labelled species and the remainder of the labelled species which are not bound to the analyte bind to the capture species. Thus, by measuring the accumulation of labeled species in the detection device, the presence and amount of analyte can be analyzed.

Where occasionally desired, a different substance may be positioned upstream of the detection site. For example, the conjugate may be so positioned in a mobile manner.

In some cases, one or several supplementary detection sites may be arranged downstream of the first detection site. Downstream of the detection site, the chromatographic carrier may also be extended further, so that the sample liquid can be released completely, or the carrier may be equipped with a material for absorbing the sample liquid.

Thus, by determining the presence or amount of the labeled substance accumulated at the detection site, the presence or amount of the analyte of interest in the sample liquid can be detected. In one case, this can be done by visual inspection.

The present invention is intended for use with any blood sample, including serum and plasma, but is preferably used with blood samples containing red blood cells, such as whole blood samples.

If detection with the desired sensitivity is desired, it is preferred that the red blood cells be removed prior to detection of the analyte of interest in the blood sample. Thus, according to the present invention, an erythrocyte separating agent is bound to a chromatographic carrier. It is preferred that the red blood cell separating agent is diffusibly bound to the chromatographic carrier. The red blood cell separating agent may be bound to the chromatographic carrier at any position that will function to separate red blood cells from plasma or serum. It is preferable to make it diffusibly bound to the chromatographic carrier upstream of the detection site. Most preferably, the red blood cell separating agent is diffusibly bound to the sample application site. This position is preferred because it results in red blood cell aggregation immediately after their use in a chromatographic carrier, which causes minimal, if any, interference with the flow of serum or plasma along the carrier by capillary action.

The erythrocyte separating agent of the present invention may be any substance capable of aggregating erythrocytes. Preferred agents are positively charged species such as polycations including, for example, poly-hydrobromic acid L-lysine; poly (dimethyldiallyl) sAmmonium chloride (Merquat)^-100，Merquat^-280，Merquat^-550); poly-L-arginine hydrochloride; poly-L-histidine; poly (4-vinylpyridine), poly (4-ethylpyridine) hydrochloride; crosslinked poly (4-vinylpyridine), methyl chloride quaternary salt; (4-vinylpyridine-styrene) copolymer; poly (4-vinylpyridine poly (hydrogen fluoride)); poly (4-vinylpyridine-p-toluenesulfonate); poly (4-vinylpyridine-tribromide); poly (4-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate); crosslinked polyvinylpyrrolidone; polyvinylpyrrolidone; poly (melamine-co-formaldehyde); partially methylated; hexadimethrine bromide; poly (glutamic acid, lysine) 1: 4 hydrobromide; poly (lysine, alanine) 3: 1 hydrobromide; poly (lysine, alanine) 2: 1 hydrobromic acid; polysuccinylated L-lysine, poly (lysine, alanine) 1: 1 hydrobromide; and poly (lysine, tryptophan) 1: 4 hydrobromide. The most preferred polycation is poly (dimethyldiallylammonium) chloride (Merquat)^-100))。

The red blood cell separating agent of the present invention can be used in any suitable amount that can effectively separate red blood cells from the rest of the sample. Preferably, the red blood cell separating agent is present at a concentration of about 0.04% to about 1.3% (weight per unit volume), more preferably about 0.13% to about 0.33% (weight per unit volume), and most preferably about 0.20% to about 0.33% (weight per unit volume).

The positive charge on the red blood cell separating agent has a tendency to aggregate any negatively charged reagents present on the test strip. For example, a labeled substance or conjugate bound to a chromatographic carrier may also be aggregated by a red blood cell separating agent to interfere with the binding of the analyte to the conjugate, or in a competitive assay, to interfere with the binding of the labeled substance and analyte to a capture substance at the detection site or to the conjugate. Eventually, the sensitivity and accuracy of the immunoassay system may be compromised.

Therefore, when the erythrocyte separating agent is a positively charged substance, a neutralizing agent is preferably used in the present invention. The neutralizing agent neutralizes the positive charge of the red blood cell separating agent, thereby eliminating or at least minimizing any interference caused by the red blood cell separating agent with the detection system. Preferably, the neutralizing agent is diffusibly bound to the chromatographic carrier. The neutralizing agent may be diffusively bound to the chromatographic carrier at any position effective to neutralize the red blood cell separating agent, but is preferably located downstream of the red blood cell separating agent, upstream of the detection site, and more preferably located at the same position on the chromatographic carrier as the diffusively bound labeled substance.

The neutralizing agent may be any polyanion capable of neutralizing the positive charge of the red blood cell separating agent. Preferred polyanions include poly (acrylic acid), poly (acrylic acid, Na salts), poly (methylmethacrylate), poly (Na-4-styrenesulfonate), poly (vinylsulfonic acid), poly-L-aspartic acid, and carboxymethylcellulose, with dextran sulfate being most preferred.

The neutralizing agent can be present in any amount effective to neutralize the positive charge of the red blood cell separating agent. Generally, the concentration of neutralizing agent will depend on the concentration of the red blood cell separating agent used. Preferably, the neutralizing agent is present in a concentration of about 0.33% to 20% (weight per volume), more preferably about 0.34% to 10% (weight per volume), and most preferably 0.34% to 10% (weight per volume).

FIG. 1 depicts one embodiment of an immunochromatographic detection device of the present invention. The device 10 comprises a chromatographic carrier 20. The blood sample application part 30 is located on the chromatographic carrier 20. In this preferred embodiment, the red blood cell separating agent is Merquat^100 is located at the sample application site 30. Adjacent to the sample application site 30 is a conjugate pad 40 containing a conjugate, i.e., a selenium labeled conjugate, and a neutralizing agent, i.e., dextran sulfate. Further downstream is the detection site 50 which will show a control band 60 after the detection has taken place and a detection band 70 if the analyte is present.

In another embodiment of the invention, a buffer solution can be contacted with the sample application site, preferably after the sample application site is contacted with the sample. The buffer helps to maintain an acceptable flow velocity along the flow path on the chromatographic carrier. This buffer may be any substance capable of flowing along the flow path by capillary action, including but not limited to phosphate buffer, phosphate buffered saline, Tris-HCl buffer, carbonate buffer, citrate buffer, HEPES (2-hydroxypiperazine-N' -2-ethanesulfonic acid) buffer, MOPS (3- (N-morpholino) propanesulfonic acid) buffer, MES (2- (N-morpholino) ethanesulfonic acid) buffer, and the like. Although the concentration and pH can be any concentration and pH that can function in the desired assay device, the preferred molarity is within the range of about 10mM to 100mM, and the pH is about 5 to 9, more preferably about 6 to 8. The buffer used is most preferably 50mM phosphate buffer, pH 7.4.

The volume of liquid used in the present invention depends on the size of the device. Ideally, a sufficient volume of liquid is used to allow flow through the device to the detection site. The liquid volume is preferably about 25. mu.l to 100. mu.l, more preferably about 40. mu.l to 60. mu.l. Thus, the buffer may be added in a volume range of about 10. mu.l to 40. mu.l, and more preferably about 20. mu.l to 30. mu.l, as needed.

The invention also relates to a method for detecting the presence of an analyte in a blood sample. This method preferably employs the chromatographic immunoassay device of the present invention. Specifically, the method comprises (1) providing a chromatographic carrier capable of supporting capillary flow and defining a flow path, a blood sample application site along the carrier in fluid contact with the chromatographic carrier, a detection site on the chromatographic carrier spaced from the application site, a diffusible labeled substance (or conjugate) bound to or competing for binding to the analyte at the detection site, a diffusible conjugated red blood cell separating agent upstream of the detection site for separating plasma or serum from the blood sample, and a conjugate bound to the chromatographic carrier; (2) contacting the sample application site with the blood sample such that the red blood cell separating agent is capable of separating red blood cells from plasma or serum in the blood sample and such that the neutralizing agent is capable of neutralizing the positive charge of the separating agent, and (3) detecting the presence of the analyte in the blood sample.

Preferably, the red blood cell separating agent is a positively charged substance and the fluid flow path contains a diffusibly bound neutralizing agent, preferably capable of binding to the red blood cell separating agent and located downstream of the red blood cell separating agent and upstream of the detection site, thereby neutralizing the positive charge of the separating agent.

Thus, in the preferred embodiment of FIG. 1, a blood sample is applied to the sample application site 30, and the red blood cell separating agent separates the red blood cells by aggregating them and allowing the plasma or serum to move along the chromatographic carrier 20 by capillary action. The neutralizing agent in the conjugate pad 40 neutralizes the effects of the red blood cell separating agent on the device 10 and the conjugate to which the analyte is bound that is present in the conjugate pad 40. The analyte bound to this conjugate continues to move downstream to the detection site 50. If the analyte of interest is present, a test band 70 will be displayed. To indicate that the test is working properly, a control color bar 60 will be displayed indicating whether the analyte of interest is present.

The present invention may preferably include a non-reactive cover or sheath surrounding the detection device. Preferably this cover layer encapsulates at least the chromatography carrier, avoiding contact and contamination of the trapping sites. The cover layer may also include a projection adjacent the sample application site that aids in receiving and/or containing a volume of sample. In addition, the cover may include one or more cut-out regions, forming letters, numbers, icons or symbols, or any combination thereof. In this embodiment, the cut-out region constitutes a specific detection site pattern when the test strip is fully packaged. It is preferred that a sufficient portion of the test strip be encapsulated so as to prevent the applied sample from contacting the detection site without first passing through the test strip portion.

The devices and methods of the present invention may be used in any detection system in which a blood sample contains a desired analyte. Examples of preferred detection systems include, but are not limited to, hepatitis C virus ("HCV"), hepatitis A virus ("HAV"), human immunodeficiency virus ("HIV"), hepatitis B surface antibody ("HBsAb"), hepatitis B surface antigen ("HBsAg"), hepatitis B core antibody ("HBcAb"), hepatitis B core antigen ("HBcAg"), carcinoembryonic antigen ("CEA"), alpha-fetoprotein ("AFP"), pancreatic cancer marker ("CA 19-9"), syphilis, tuberculosis, malaria, leishmaniasis, and dengue fever.

The following examples further illustrate the invention but should not be construed as in any way limiting its scope.

Examples

Example 1

The aggregation of erythrocytes by polycations

Comparison of aggregation of selenium conjugates

For the purpose of the present invention, it is required that red blood cells (rbc's) in whole blood aggregate and selenium conjugate aggregation is not desired. A number of polycations were tested and observed to cause sufficient aggregation of rbc's while at the same time only minimally aggregating the selenium-conjugate.

Selenium conjugates of HIV-1 recombinant proteins were prepared as follows: selenium colloid was first prepared by reacting 32mM selenium oxide with 91ml L-ascorbic acid in water solution at 42 ℃ for 72 hours. This selenium colloid was diluted to an optical density of 30 at a wavelength of 550nm, and then reacted with 40. mu.g/ml of recombinant HIV-1 outer membrane protein in 30mM Tris buffer at pH7.4 at room temperature for 20 minutes. This selenium colloid-labeled HIV-1 protein conjugate was again diluted to an optical density of 30 at a wavelength of 550nm in pH7.410mM Tris buffer containing 0.1% casein, and incubated at room temperature for 20 minutes. The conjugate solution was then centrifuged at 1970 Xg for 20 minutes at 4 ℃ and the supernatant removed and the pellet discarded. To this supernatant was added 30mM Tris buffer, pH7.4, containing 2% casein, in a volume equivalent to one tenth of the volume of the supernatant. Finally, this conjugate solution was diluted to an optical density of 10 at a wavelength of 550nm in 50mM Tris buffer, pH7.4, containing 1% casein, 2% sucrose and 2% lactose.

A0.25% (w/v) aqueous solution of the following polycation was prepared: poly-L-lysine hydrobromide, molecular weight (mw) 37000; poly-L-arginine hydrochloride; mw 12100, 42400 and 92000; poly-L-histidine, mw 18400; hexadimethrine bromide, poly (lysine, alanine) 3: 1 hydrobromide, mw 35000; poly (lysine, alanine) 2: 1 hydrobromide, mw 49300; poly (lysine, alanine) 1: 1 hydrobromide, mw 41600; poly (lysine, tryptophan) 1: 4 hydrobromide, mw 38000 (all above polycations purchased from Sigma, st. louis, MO); poly (diallyldimethylammonium chloride), mw 10⁵-10⁶(Merquat^-100，Calgon，Pittsburgh，PA)。

The ability of these polycations to aggregate rbc's or selenium conjugates in whole blood in separate reactions was observed by adding 350 μ l of 0.25% solutions of different polycations to an equal volume of whole blood or selenium conjugate. The two solutions were mixed and left at room temperature for 10 minutes, and then the aggregation was evaluated visually. The results are summarized in table 1.1 plus (+) sign indicates weak aggregation, 2+ indicates moderate aggregation, and 3+ and 4+ indicate strong aggregation.

TABLE 1

		Aggregation
				Polycation	Molecular weight	Red blood cell	Selenium conjugates
Poly-L-lysine HBr	37000	2+	2+
				Merquat-100	105-106	2+	2+
Poly-L-arginine HCl	12100	2+	2+
				Poly-L-arginine HCl	42400	2+	2+
Poly-L-arginine HCl	92000	2+	2+
				poly-L-histidine	18400	+	4+
Hexadimethrin bromide		+	+
				Poly (lysine, alanine) 3: 1 HBr	35000	2+	2+
Poly (lysine, alanine) 2: 1 HBr	49300	+	2+
				Poly (lysine, alanine) 1: 1 HBr	41600	+	2+
Poly (lysine, tryptophan) 1: 4 HBr	38000	3+	3+

Polycations that cause aggregation of rbc's (2+ or more) while at the same time cause minimal aggregation of selenium conjugates (2+ or less) are good choices. Those polycations having a 2+ aggregation effect on both meet this criterion. Para poly-L-lysine HBr and Merquat^100 for further selection tests, Merquat^100 has the best cost effect.

Example 2

Prevention of conjugate aggregation by neutralization with polyanions

A. Use of dextran sulfate to prevent conjugate flow and aggregation

Different concentrations of dextran sulfate polyanion were tested using poly-L-lysine as the polycationic rbc aggregating agent to see if dextran sulfate could neutralize the positive charge of the polycation and thereby prevent aggregation of the selenium conjugate by the polycation. Dextran sulfate is added after the polycation has caused the rbc's to aggregate, but before the polycation interacts with the selenium conjugate. The aggregation of the selenium conjugate by this polycation and dextran sulfate, and the consequent effect on its ability to flow along the immunochromatographic test strip, was evaluated in the following experiments.

An immunochromatographic test strip is assembled, which consists of a sample pad, a neutralization pad, a conjugate pad and a detection pad. Preparation of sample pad: a4 mm wide, 20mm long glass fiber filter paper (Lypore9524, Lydall, Rochester, NH) was soaked in 0.33% poly-L-lysine hydrobromide, mw37000(Sigma, St. Louis, Mo.) aqueous solution and then dried under vacuum.

Preparation of neutralizing pads containing different concentrations of dextran sulfate: filters made from wood pulp and polyester (Sontara 8801, Du pont, Wilmington, Delaware) 4mm wide and 13mm long were soaked in an aqueous solution containing 0%, 1.1%, 3.3% or 10% dextran sulphate, mw 5000(Sigma, st. After soaking, the pad was vacuum dried.

Preparation of conjugate pad: a4 mm wide, 4.3mm long glass fiber filter paper (Lypore9524, Lydall, Rochester, NH) was soaked in the selenium colloid labeled HIV-1 recombinant protein conjugate formulated and diluted as in example 1. After soaking, the conjugate pad was vacuum dried.

The test piece was a 4mm wide, 40mm long nitrocellulose filter (Cat # H9643G1, Millipore, Bedford, Mass.). HIV-1 envelope antigen prepared at a concentration of 5mg/ml in a buffer solution of pH 7.4100 mM Tris containing 1% sucrose was applied to the nitrocellulose filter so as to form a line spanning the width of the test piece at a position about 1cm from the end of the filter. The scribe area substrate was backed with a polyester sheet (code #7733), adhesive research corporation, Glen Rock, Pa.). The test piece was dried sufficiently so that the antigen was immobilized on the nitrocellulose filter.

The above components were assembled into 4mm wide immunochromatographic test strips, arranged end to end longitudinally with a 1mm long sample pad overlapping each other, 20mm long sample pad at one end, followed by a 13mm long neutralization pad, followed by a 4.3mm long conjugate pad, and finally a 40mm long test pad. The test strip was then covered with a polyester sheet (code #8648, adhesive research corporation, Glen Rock, Pa.) from the top of the test piece to 10mm from the bottom of the assembled test strip, leaving a sample pad exposed approximately 10 mm. 80 μ l of plasma was then applied to the sample pad of each immunochromatographic test strip containing a neutralization pad with 0%, 1.1%, 3.3% or 10% dextran sulfate. The aggregation of the red selenium conjugate and the ability of this conjugate to flow along the test strip was visually observed.

The results are shown in table 2 below, which indicates that the selenium conjugate aggregates and is unable to flow along the test strip in the absence of dextran sulfate to neutralize the charge from the polycationic solution. There is an inverse relationship between aggregation and flow of the conjugate, with a dextran sulfate concentration of 3.3% or higher being sufficient to prevent aggregation of the conjugate and allow the conjugate to flow along the test strip.

TABLE 2

Concentration of dextran sulfate	Aggregation of conjugates	Flow of conjugate
			0％	++	-
1.1％	+	+/-
			3.3％	-	+
10％	-	+

B. Aggregation of rbc in the Presence of dextran sulfate

To determine the effect of dextran sulfate on rbc aggregation, the above experiment was repeated with 10% dextran sulfate on a 4.3mm long by 4mm wide neutralization pad using whole blood as the sample. After assembling the immunochromatographic test strip as above with this neutralization pad, 80. mu.l of whole blood was applied to the sample pad. After 15 minutes, the results of rbc aggregation were visually observed and the ability of the formed plasma to flow along the test strip was measured. rbc's are collected, retained on the sample pad, and do not flow up the test strip, while plasma flows 33mm along the test strip within 15 minutes. This indicates that polycations in the sample pad can still cause the aggregation of rbc's in whole blood samples, and that the presence of polyanionic dextran sulfate in the neutralization pad does not interfere with the aggregation of such rbcs.

C. Prevention of conjugate aggregation with polyanions

Several other polyanions were tested as in example 2.A in order to evaluate their ability to prevent aggregation of the selenium conjugate. A15.5 mm long by 4mm wide pad of the sample was soaked in a solution containing 0.26% Merquat^-100 in aqueous solution and then dried at 55 ℃. Instead of using a neutralisation pad, the selenium conjugate was diluted in a ph7.4, 10mM Tris buffer containing 1% casein, 2% sucrose, 2% lactose, and mw 5000(Sigma, st. louis, MO) a polyanionic dextran sulfate containing 0%, 1.1% or 3.3%, or 0%, 0.5%, 1% or 2% of a polyanion of the following (all available from Aldrich chemical company, Milwaukee, WI): poly (acrylic acid), mw 5000; poly (sodium-4-styrenesulfonate), mw 70000; poly (vinylsulfonic acid, sodium salt); poly (methyl methacrylate), mw 9500; poly (acrylic acid, sodium salt), mw 2100. The conjugate pad was soaked in different selenium conjugate solutions and dried under vacuum. Test strips were prepared as in example 2.A and assembled into immunochromatographic test strips. 50 μ l of plasma was then applied to the sample pad of each immunochromatographic test strip having conjugate pads containing different polyanions. Aggregation of the red selenium conjugate was visually observed. Table 3 shows the relative amount of conjugate aggregation observed at the different concentrations of polyanion tested.

TABLE 3

Aggregation of selenium conjugates
							Concentration of polyanion
Polyanions	0％	0.5％	1-1.1％	2％	3.3％
						Dextran sulfate	++	nt	+	nt	-
Poly (acrylic acid)	++	-	-	-	nt
						Poly (Na-4-styrene sulfonate)	++	++	++	+	nt
Poly (vinylsulfonic acid)	++	+/-	-	-	nt
						Poly (methyl methacrylate)	++	-	-	-	nt
Poly (acrylic acid, sodium salt)	++	+	+/-	-	nt

not tested

As described above, the selenium conjugate is aggregated if no polyanion is present to neutralize the positive charge of the polycation from the sample pad (which is essential for the aggregation of rbc when whole blood is tested). All polyanions used in the conjugate pad, at least one of the concentrations tested, prevented aggregation of the conjugate. This experiment also shows that the polyanion need not be applied to a separate pad, but can be applied in combination with the selenium conjugate to a conjugate pad.

Example 3

Use of dextran sulfate in HIV-1 antibody detection assays

Comparison of neutralization and conjugation pads

Pressing and compactingExample 2.A immunochromatographic test strips were prepared with or without a 4mm wide by 4.3mm long glass fiber filter paper (Lypore9524, Lydall, Rochester, NH) neutralization pad. When a neutralization pad is used, the pad is soaked in an aqueous solution containing 3.3% dextran sulfate and then vacuum dried. For test strips without a neutralization pad, the selenium conjugate was diluted with 10mM Tris buffer, ph7.4, containing 1% casein, 2% sucrose, 2% lactose and 3.3% dextran sulfate, and the conjugate pad was soaked in this solution and then dried under vacuum. The sample pad used was the same as in example 2.A, except that it was soaked in 0.2% Merquat^-100 aqueous solution.

Human serum containing HIV-1 antibodies was diluted 1: 2048 in HIV-negative human whole blood (in terms of plasma volume) or in HIV-negative human plasma at a hematocrit of 50%. Whole blood or plasma was again used as diluent and further diluted 1: 2 serially 3 times. Mu.l of a sample of either negative whole blood or serial dilutions of whole blood from HIV-1 positive was applied to the sample pad of an immunochromatographic test strip prepared to contain dextran sulfate in a separate neutralization pad, or in a conjugate pad, dextran sulfate in selenium conjugate solution. 80 μ l negative plasma or serial dilutions of plasma from HIV-1 positive samples were tested only on immunochromatographic test strips with dextran sulfate on conjugate pads. The results were observed 15 minutes after loading (Table 4). A positive result shows a red color on the test strip where the red selenium labeled HIV-1 antigen conjugate-HIV-1 antibody complex is bound to the HIV-1 antigen on the scored area of the test strip. Negative results this area on the test strip was not colored.

TABLE 4

	The dextran sulfate is neutralizedInside the gasket	Dextran sulfate within the conjugate pad
				Dilution of sample	Whole blood	Whole blood	Blood plasma
1∶2048	+	+	+
				1∶4096	+	+	+
1∶8192	-	+	+
				1∶16384	nt	-	-
Negative control	-	-	-

not tested

The results in Table 4 demonstrate that HIV-1 antibodies in whole blood can be detected with an immunochromatographic test strip using a polycation Merquat^-100 aggregating rbc's to allow sample flow along the test strip, and using a polyanionic dextran sulfate as a neutralizing agent to prevent the polycation from aggregating the selenium conjugate, allowing the conjugate to bind to the positive sample, form a complex and flow along the test strip to a detection zone. This polyanion has been shown to be effective whether used separately in a neutralization pad or in combination with a selenium conjugate on a conjugate pad. In this assay, a polyanion (dextran sulfate) was used in the conjugate pad, rather than in the neutralization pad alone, and showed a two-fold increase in sensitivity for detection of HIV-1 antibodies.

Furthermore, the results in Table 4 also indicate that the polycation was effective in aggregating rbc's in whole blood, in which rbc's must be aggregated for sample flow, and in plasma in which rbc's do not interfere with sample flow, and showed the same detection sensitivity for HIV-1 antibody, either in whole blood or plasma. The results also indicate that the presence of the polyanion, either alone in the neutralization pad or in the conjugate pad, does not interfere with the ability of the polycation to efficiently cause the aggregation of rbc's in whole blood. Example 4

Use of Merquat in HBsAg detection^And different polyanions

An immunochromatographic test strip for detecting hepatitis B surface antigen (HBsAg) in a whole blood sample was prepared. With Merquat^100 as polycations to aggregate rbc's in the sample pad, the effect of different polyanions as polycation neutralizers in the conjugate pad to prevent aggregation of selenium conjugates was evaluated.

An immunochromatographic test strip composed of a sample pad, a conjugate pad and a detection pad is assembled. Tong (Chinese character of 'tong')0.26% Merquat^A4 mm wide by 15.5mm long glass fiber filter paper was soaked in-100 aqueous solution and then dried at 55 ℃ to prepare a sample pad.

A selenium conjugate was prepared as in example 1 using selenium colloid and 12 μ g/ml of anti-HBsAg mouse monoclonal antibody (anti-HBs), and this selenium colloid-labeled anti-HBs conjugate was diluted to an optical density of 2.6 at a wavelength of 550nm in Tris buffer containing one of the following polyanions: 0.5% poly (acrylic acid), mw 2000 (PAA-2000); 0.5% poly (acrylic acid), mw 240000(PAA-240000), 0.5% dextran sulfate, mw 5000; 0.8% poly-L-aspartic acid, mw 36, 300; 0.5% carboxymethyl cellulose, mw 90000 (CMC). Dextran sulfate and poly-L-aspartic acid were purchased from Sigma, St.Louis, MO, and the remaining polyanions were purchased from Aldrich chemical company, Milwaukee, Wis.

Preparation of conjugate pad: a4 mm wide by 4.3mm long glass fiber filter paper was soaked in the selenium colloid labeled anti-HBs conjugate prepared as described above and diluted with one of the above polyanions. After soaking, the conjugate pad was vacuum dried.

The test piece was a nitrocellulose filter 4mm wide by 40mm long prepared as in example 2, which was applied to the nitrocellulose filter using anti-HBs mouse monoclonal antibody at a concentration of 3mg/ml so as to form a line crossing the width of the test piece at a position about 1cm from the end of the filter. The area substrate was scribed with a polyester foil. The test piece was dried sufficiently so that the antibody was immobilized on the nitrocellulose filter.

The immunochromatographic test strip was assembled with the above-mentioned components, and they were arranged end-to-end, longitudinally with 1mm overlap, with a sample pad at one end, and next to it was a conjugate pad, and finally a test piece. The assembled test strip was then covered with a thin layer of polyester, leaving the approximately 10mm sample pad exposed.

Recombinant HBsAg was added to HBsAg negative human whole blood at a 50% hematocrit value to a concentration of 12.5 ng/ml. Further 3 1: 2 serial dilutions were made in whole blood. A series of dilutions of 50 μ l of negative whole blood or whole blood from HBsAg positive were applied to the sample pads of the immunochromatographic test strips prepared to contain different polyanions in their conjugate pads. The results were observed 15 minutes after application of the sample (table 5). A positive result shows a red color on the test strip where the red selenium labeled anti-HBs conjugate-HBsAg complex is bound to anti-HBs on the scored area of the test strip. Negative results show no color in this region of the test strip. Aggregation of the red selenium conjugate can be visually observed at the entrance to the test strip.

TABLE 5

	Concentration of HBsAg (ng/ml)
							Polyanions	12.5	6.25	3.13	1.56	0	Aggregation of conjugates
PAA-2000	+	+	+	-	-	-
							PAA-240,000	+	+	-	-	-	+
Dextran sulfate	+	+	+	-	-	-
							poly-L-aspartic acid	+	+	+	-	-	-
CMC	+	-	-	-	-	+

Although all polyanions can be used to detect HBsAg, those polyanions that prevent aggregation of the conjugate, PAA 2000, dextran sulfate and poly-L-aspartic acid, exhibit a 2-4 fold higher detection sensitivity for HBsAg in whole blood samples.

The above experiment was repeated using selenium colloid-labeled conjugates diluted in Tris buffer containing PAA-2000 as polyanion, except that 25. mu.l of 50mM phosphate buffer, pH7.4, was added to the sample pad 1 minute after the HBsAg whole blood sample was added. The same procedure was used to obtain the same results as without the addition of buffer after the sample was applied. Thus, for these assays, the sample may or may not be buffered after application.

Example 5

Use of Merquat in the detection of tuberculosis^And dextran sulfate

An immunochromatographic test strip was prepared for detecting an anti-Mycobacterium tuberculosis antibody (anti-Mtb) in a whole blood sample. With Merquat^100 as polycation for aggregation of rbc's in the sample pad, and the effect of different concentrations of polyanionic dextran sulfate as polycationic neutralizer in the conjugate pad to prevent aggregation of selenium conjugates was evaluated.

An immunochromatographic test strip composed of a sample pad, a conjugate pad and a detection pad is assembled. Preparation of sample pad: at 0.26% Merquat^-100 soaking glass fiber filter paper 4mm wide by 15.5mm long in aqueous solution, followed by vacuum drying.

Selenium conjugates were prepared as in example 1 using selenium colloid and 3.5 μ g/ml recombinant Mtb antigen from e.coli. This selenium colloid labeled Mtb conjugate was then diluted to an optical density of 2.5 at wavelength 550nm in Tris buffer containing 0%, 0.34%, 1.1% or 3.3% dextran sulfate.

Preparation of conjugate pad: glass fiber filter paper 4mm wide by 4.3mm long was soaked in the selenium colloid labeled Mtb conjugate prepared as described above and diluted with one dextran sulfate concentration. After soaking, the conjugate pad was vacuum dried.

The test strip was a nitrocellulose filter 4mm wide by 40mm long, which was applied to a nitrocellulose filter as in example 2 with a recombinant Mtb antigen at a concentration of 0.15mg/ml, to form a line spanning the width of the test strip at a location approximately 1cm from the end of the filter. The back of the scribe area was backed with a polyester sheet. The test piece is dried sufficiently so that the antigen can be immobilized on the nitrocellulose.

The immunochromatographic test strip was assembled with the above components, joined end to end, arranged in a longitudinal array overlapping by 1mm, with the sample pad at one end, the conjugate pad adjoining it, and finally the test piece. The assembled test strips were then covered with a polyester sheet leaving approximately 10mm sample pads exposed.

anti-Mtb positive sera were diluted 1: 100 in negative human whole blood with a hematocrit value of 50%. Further two 1: 2 serial dilutions were made in whole blood. 50 μ l of negative whole blood or a sample from a serial dilution of anti-Mtb positive whole blood was applied to the sample pad of an immunochromatographic test strip prepared to contain dextran sulfate at various concentrations on its conjugate pad. The results were observed 15 minutes after application of the sample (table 6). A positive result shows a red color on the test strip where the red selenium labeled Mtb conjugate-anti-Mtb complex binds to Mtb at the scored area of the test strip. Negative results show no color in this area on the test strip. The aggregation of the red selenium conjugate can be visually observed at the entrance to the test strip.

TABLE 6

Dextran sulfate	anti-Mtb dilution
						(％)	1∶100	1∶2000	1∶4000	Negative control	Aggregation of conjugates
0	-	-	-	-	++
						0.34	-	-	-	-	++
1.1	+	-	-	-	+
						3.3	+	+	-	-	-

The data in table 6 show that detection cannot be performed in the absence of the polyanion, which is dextran sulfate, to prevent aggregation of the conjugate. There is an inverse proportionality between conjugate aggregation and detection sensitivity. At a dextran sulfate concentration of 3.3%, no conjugate aggregation occurred, showing the highest sensitivity of detection against-Mtb.

Example 6

Use of Merquat in syphilis detection^And dextran sulfate

Immunochromatographic test strips were prepared for the detection of anti-Treponema pallidum antibodies (anti-TP) in whole blood or plasma samples. By comparing the sensitivity of detection in whole blood to plasma, it can be determined whether rbc's in whole blood are efficiently collected by polycations so as not to interfere with detection, and thus the detection sensitivity of the assay can be improved. With Merquat^100 as polycation for aggregation of rbc's in the sample pad and polyanionic dextran sulfate as polycationic neutralizer in the conjugate pad to prevent aggregation of selenium conjugate.

An immunochromatographic test strip composed of a sample pad, a conjugate pad and a detection pad is assembled. Preparation of sample pad: at 0.2%Merquat^-100A glass fiber filter paper 4mm wide by 15.5mm long soaked in an aqueous solution and then dried at 55 ℃.

Selenium conjugates were prepared as in example 1 using selenium colloid and 7.5 μ g/ml of treponema pallidum lysate (TP). This selenium colloid-labeled TP conjugate was then diluted to an optical density of 2.8 at a wavelength of 550nm in Tris buffer containing 3.3% dextran sulfate.

Preparation of conjugate pad: a4 mm wide by 4.3mm long glass fiber filter was soaked in the selenium colloid-labeled TP conjugate prepared above. After soaking, the conjugate pad was vacuum dried.

The test piece was a 4mm wide by 40mm long nitrocellulose filter prepared as in example 2 with 44. mu.g/ml concentration of Treponema pallidum lysate, which was applied to the nitrocellulose filter so as to form a line that spanned the width of the test piece at a position of approximately 1cm from the end of the filter. The back of the scribe area was backed with a polyester sheet. The test piece was dried sufficiently to immobilize the TP lysate on the nitrocellulose.

The immunochromatographic test strip was assembled with the above components, joined end to end, arranged longitudinally overlapping by 1mm, with a sample pad at one end, adjacent to which was a conjugate pad, and finally a test pad. The assembled test strip was then covered with a polyester sheet leaving approximately 10mm of the sample pad exposed.

anti-TP positive human serum was diluted 1: 10.8 in negative human whole blood or negative human plasma at a hematocrit of 50%. Whole blood or plasma was again used as diluent and further diluted 4 times in a 1: 2 series. 60 μ l of negative whole blood or plasma, or a sample from serial dilutions of anti-TP positive whole blood or plasma, was added to the sample pad of the immunochromatographic test strip. The results were observed 15 minutes after application of the sample (table 7). A positive result appears in red on the test strip, where the red selenium labeled TP conjugate-anti-TP complex binds to the TP lysate on the scored area of the test strip. Negative results showed no color in this area of the test strip.

Table 7 shows that the detection sensitivity for anti-TP was the same in whole blood and plasma, indicating that the polycation Merquat^100 efficiently accumulated rbc's in whole blood.

TABLE 7

Dilution of anti-TP samples	Whole blood	Blood plasma
			1∶10.8	+	+
1∶21.6	+	+
			1∶43.2	+	+
1∶86.4	+	+
			1∶172.8	-	-
Negative control	-	-

All publications, patents and patent applications cited herein are hereby incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference and was set forth in its entirety herein.

While the invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that many of these preferred embodiments may be varied. It is possible to practice the invention in a different manner than specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as set forth in the detailed description and the appended claims.

Claims (14)

1. A chromatographic assay device for detecting the presence of an analyte in a blood sample, comprising:

a chromatography carrier defining a liquid flow path and supporting capillary flow,

a blood sample application part which is in fluid contact with the chromatographic carrier,

a detection site on the chromatographic carrier spaced apart from the sample application site, to which a non-diffusion-bound capture substance is bound,

a diffusively bindable labeled substance located downstream of the sample application site,

a diffusively bindable polycation for separating plasma or serum from a blood sample upstream of a detection site, and

a diffusively bindable polyanion downstream of the bound polycation and upstream of the detection site for neutralizing the polycation.

2. The chromatographic assay device of claim 1, wherein the device is an immunoassay device.

3. The chromatographic assay device of claim 1 wherein the polycation is bound at the site for application of the blood sample.

4. The chromatographic assay device of claim 1, wherein the polycation is selected from the group consisting of: poly-L-lysine hydrobromide, poly-L-arginine hydrochloride, poly-L-histidine, lysine alanine 3: 1 copolymer hydrobromide, lysine alanine 2: 1 copolymer hydrobromide, lysine alanine 1: 1 copolymer hydrobromide, lysine tryptophan 1: 4 copolymer hydrobromide, and polydimethyldiallylammonium chloride.

5. The chromatographic assay device of claim 1, wherein the polycation is polydimethyldiallylammonium chloride.

6. The chromatographic assay device of claim 1, wherein the polyanion is selected from the group consisting of: dextran sulfate, polyacrylic acid, sodium-4-styrenesulfonate, polyvinylsulfonic acid, polymethacrylic acid, poly-L-aspartic acid, and carboxymethylcellulose.

7. The chromatographic assay device of claim 1, wherein the polyanion is dextran sulfate.

8. The chromatographic assay device according to claim 1, wherein the polyanion is diffusibly bound to the chromatographic carrier at the same site as the labeled substance.

9. The chromatographic assay device of claim 1 wherein the labeled substance is a selenium labeled substance.

10. A method for detecting the presence of an analyte in a blood sample, comprising the steps of:

providing a chromatographic carrier defining a flow path and supporting capillary flow, along which there is (a) a blood sample application site in fluid contact with the chromatographic carrier, (b) a detection site on the chromatographic carrier spaced from the application site, to which a non-diffusively bound capture material is bound; (c) a labeled substance located downstream of the sample application site, (d) a diffusively bindable polycation located upstream of the detection site for separating plasma or serum from a blood sample, and (e) a diffusively bindable polyanion located downstream of the bound polycation and upstream of the detection site for neutralizing the polycation;

contacting the sample application site with the blood sample; and

the blood sample is tested for the presence of the analyte.

11. The method of claim 10 wherein the labeled substance is a selenium labeled substance.

12. The method of claim 10, wherein said polyanion is diffusibly bound to the chromatographic carrier at the same position as the labeled species.

13. The method of claim 10, wherein the polycation is selected from the group consisting of: poly-L-lysine hydrobromide, poly-L-arginine hydrochloride, poly-L-histidine, lysine alanine 3: 1 copolymer hydrobromide, lysine alanine 2: 1 copolymer hydrobromide, lysine alanine 1: 1 copolymer hydrobromide, lysine tryptophan 1: 4 copolymer hydrobromide, and polydimethyldiallylammonium chloride.

14. The method of claim 10, wherein the polyanion is selected from the group consisting of: dextran sulfate, polyacrylic acid, sodium-4-styrenesulfonate, polyvinylsulfonic acid, polymethacrylic acid, poly-L-aspartic acid, and carboxymethylcellulose.