The present invention relates to chromatography assay devices and a method of detecting an analyte in a whole blood sample, and more particularly to a device and method employing a red blood cell separating agent to aggregate red blood cells, and a neutralizing agent to neutralize any negative effect the red blood cell separating agent may have on the assay system.
Modern clinical diagnostic methods are routinely carried out on blood samples. Unfortunately, red blood cells interfere with many diagnostic determinations. In assays for an analyte, red blood cells may inhibit binding between specific binding pair members. Likewise, red blood cells have enzyme activity which, depending on the assay employed, may interfere with the signal produced. Further, in a rapid test format using a chromatography assay device, particularly a chromatography immunoassay device, red blood cells may inhibit fluid flow which is necessary for reactions to occur on the device. For these reason and others, many assay methodologies are carried out on plasma or serum which must first be separated from a whole blood sample.
Many known techniques exist 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 clotting) and serum (after clotting) is separated from whole blood. In this procedure, red blood cells settle at the bottom of the test tube, and the serum is separated by decantation or some other method. Stratifying whole blood by centrifugation, however, has many disadvantages. Generally, centrifugation requires a large blood sample to be drawn. Further, the process is time consuming and requires cumbersome laboratory equipment often not maintained in a physician""s office. Finally, the extra handling of the blood increases the exposure to the potential hazards of blood-borne pathogens.
To reduce or eliminate the need for centrifugation, assay devices have been developed which employ gradient membranes or trapping membranes to separate red blood cells from the liquid portion of the blood. Immobilized anti-red blood cell antibodies 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 bibulous element to which is bound at least one specific binding pair member to remove the agglutinated red blood cells; (2) passing whole blood through a glass microfiber filter which may or may not have an agglutinating agent incorporated; (3) employing a barrier or exclusion layer of polysaccharide material to prevent red blood cells from passing through and 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 the separation of red blood cells from plasma are costly, complicated, may result in incomplete separation of red blood cells, and may cause hemolysis. Hemolysis leads to non-specific binding or high backgrounds causing a loss in assay sensitivity. This can be the result of free hemoglobin which can color the detection zone such that the zone can obtain a color that ranges from pink to dark maroon. As a result, the production of a visual chemical signal can be wholly or partly obscured by the presence of the hemoglobin color in the detection zone. Further, the use of a separating agent, such as a polycation, in an assay system tends to interfere with the system, often by aggregating other reagents or binding members in addition to the red blood cells.
Accordingly, need exists for a device and method for detecting an analyte in a blood sample without adversely effecting the assay system. Such device and method should be suitable for whole blood samples of various sizes, including small samples.
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 on the chromatography carrier spaced apart from the application site, a diffusively bound labeled substance located downstream of the application site, a diffusively bound red blood cell separating agent for separating plasma or serum from the blood sample upstream of the detection site, and a diffusively bound neutralizing agent capable of binding with the separating agent downstream of the bound separating agent and upstream of said detection site whereby a positive charge of said separating agent is neutralized. Preferably, the red blood cell separating agent is located at the application site 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 capillary flow, along which are (a) an application site for the blood sample in fluid flow contact with said chromatography carrier, (b) a detection site on the chromatography carrier spaced apart from the application site, (c) a diffusively bound labeled substance located downstream of the application site, (d) a diffusively bound red blood cell separating agent for separating plasma or serum from said blood sample upstream of the detection site, and (e) a diffusively bound neutralizing agent capable of binding with the separating agent located downstream of the bound separating agent and upstream of the detection site whereby a positive charge of the separating agent is neutralized; contacting the application site with the blood sample such that the red blood cell separating agent separates the plasma or serum from the blood sample, and the neutralizing agent neutralizes the positive charge of the separating agent as the sample flows along the flow path; and detecting the presence of analyte in the blood sample.