Patent Publication Number: US-2011076697-A1

Title: Lateral-flow immuno-chromatographic assay devices

Description:
BACKGROUND 
     1. Field of the Invention 
     This invention generally relates to lateral-flow immuno-chromatographic assay devices. 
     2. Related Art 
     Various types of lateral-flow immuno-chromatographic assay devices have been developed for carrying out diagnostic assays on blood samples. Lateral-flow immuno-chromatographic assay devices typically include an arrangement of layers of materials in a housing having openings for sample introduction and for reading of assay results. 
     There is a continuing need for lateral-flow immuno-chromatographic assay devices having structures and modes of utilization facilitating improved assay performance capabilities. 
     SUMMARY 
     In an example of an implementation, a device is provided that includes a migration membrane, a conjugate pad on the migration membrane, a plasma separation membrane on the conjugate pad, and a pre-filter on the plasma separation membrane. The migration membrane has a test line configured for loading onto the test line of one or a plurality of capture antibodies having specific binding affinity for an assay target. The migration membrane is configured for allowing lateral flow of blood plasma or serum across the migration membrane to the test line. The conjugate pad is configured for loading onto the conjugate pad of one or a plurality of detection antibodies having specific binding affinity for an assay target. The plasma separation membrane is configured for allowing passage of blood plasma or serum through the plasma separation membrane and for trapping erythrocytes. The pre-filter is configured for loading of an assay sample including erythrocytes and either or both of blood plasma and blood serum onto the pre-filter. Further, the pre-filter is configured for allowing passage of blood plasma or serum through the pre-filter, and configured for causing lateral flow of blood plasma or serum within the pre-filter. 
     As another example of an implementation, a method is provided. The method includes providing a lateral-flow immuno-chromatographic assay device including a migration membrane, a conjugate pad being on the migration membrane, a plasma separation membrane being on the conjugate pad, and a pre-filter being on the plasma separation membrane. In the lateral-flow immuno-chromatographic assay device, the conjugate pad is loaded with one or a plurality of detection antibodies having specific binding affinity for an assay target; and the migration membrane has a test line loaded with one or a plurality of capture antibodies having specific binding affinity for the assay target. The method further includes carrying out a diagnostic assay cycle. In the diagnostic assay cycle, an assay sample including erythrocytes and either or both of blood plasma and blood serum is loaded onto the pre-filter. Also in the cycle, blood plasma or serum is caused to laterally flow within the pre-filter and allowed to pass through the pre-filter to the plasma separation membrane. The diagnostic assay cycle further includes causing erythrocytes to be trapped in the plasma separation membrane; and allowing blood plasma or serum to pass through the plasma separation membrane to the conjugate pad. Additionally, the cycle includes allowing blood plasma or serum to pass through the conjugate pad onto the migration membrane and allowing blood plasma or serum to laterally flow across the migration membrane to the test line. 
     Other devices, methods, features and advantages of the invention will be or will become apparent to one with skill in the art upon examination of the following figures and detailed description. It is intended that all such additional devices, methods, features and advantages be included within this description, be within the scope of the invention, and be protected by the accompanying claims. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       The invention can be better understood with reference to the following figures. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. Moreover, in the figures, like reference numerals designate corresponding parts throughout the different views. 
         FIG. 1  is an exploded perspective view illustrating an example of an implementation of a lateral-flow immuno-chromatographic assay device. 
         FIG. 2  is a cross-sectional view, taken along line A-A, of the lateral-flow immuno-chromatographic assay device shown in  FIG. 1 . 
         FIG. 3  is a flow diagram illustrating an example of an implementation of a method. 
         FIG. 4  is a photograph showing the lateral-flow immuno-chromatographic assay devices utilized to carry out Examples A, B and C. 
         FIG. 5  is a photograph showing the lateral-flow immuno-chromatographic assay devices utilized to carry out Examples D, E, F and G. 
         FIG. 6  is a photograph showing the lateral-flow immuno-chromatographic assay devices utilized to carry out Examples H, I, J and K. 
         FIG. 7  is a photograph showing the lateral-flow immuno-chromatographic assay devices utilized to carry out Examples L, M, N and O. 
         FIG. 8  is a photograph showing the lateral-flow immuno-chromatographic assay devices utilized to carry out Examples P, Q, R and S. 
         FIG. 9  is a photograph showing the lateral-flow immuno-chromatographic assay devices utilized to carry out Examples T, U, V and W. 
         FIG. 10  is a photograph showing the lateral-flow immuno-chromatographic assay devices utilized to carry out Examples X, Y, Z and AA. 
         FIG. 11  is a photograph showing the lateral-flow immuno-chromatographic assay devices utilized to carry out Examples AB, AC, AD and AE. 
         FIG. 12  is a photograph showing the lateral-flow immuno-chromatographic assay devices utilized to carry out Examples AF, AG, AH and AI. 
         FIG. 13  is a photograph showing the lateral-flow immuno-chromatographic assay devices utilized to carry out Examples AJ and AK. 
         FIG. 14  is a photograph showing the lateral-flow immuno-chromatographic assay devices utilized to carry out Examples AL and AM. 
         FIG. 15  is a photograph showing the lateral-flow immuno-chromatographic assay devices utilized to carry out Examples AN and AO. 
         FIG. 16  is a photograph showing the lateral-flow immuno-chromatographic assay device utilized to carry out Example AP. 
         FIG. 17  is a photograph showing the lateral-flow immuno-chromatographic assay devices utilized to carry out Examples AQ and AR. 
     
    
    
     DETAILED DESCRIPTION 
     A lateral-flow immuno-chromatographic assay device may function, for example, by carrying a diagnostic assay sample to a conjugate pad loaded with one or a plurality of detection antibodies having specific binding affinity for an assay target. The diagnostic assay sample may then be laterally carried across a migration membrane to a test line loaded with one or a plurality of capture antibodies also having specific binding affinity for the assay target. If a sufficient concentration of the assay target was present in the diagnostic assay sample, then a detectable quantity of the detection antibodies may specifically bind with the assay target at the conjugate pad and may then laterally flow along with the assay sample to the test line. The capture antibodies at the test line may then also specifically bind there with the assay target, generating a visible mark constituting a positive test result of the diagnostic assay. For example, the detection antibodies may be tagged with a colored marker such as colloidal gold. Where the assay sample carries a sufficient quantity of the detection antibodies to the test line and then a sufficient quantity of the capture antibodies bind the assay target carrying the bound detection antibodies at the test line, a visibly colored mark may be formed. Where the detection antibody marker is colloidal gold, for example, a mark having a reddish, pinkish, or brownish hue may be formed. 
     The diagnostic assay sample may include red blood cells, also being referred to herein as “erythrocytes.” For example, the diagnostic assay sample may include whole blood, or may otherwise include erythrocytes and either or both of blood plasma and blood serum. Blood plasma or blood serum may, for example, be separated from erythrocytes before carrying out a lateral-flow immuno-chromatographic diagnostic assay to reduce or substantially eliminate the reddish color caused by hemoglobin. However, if erythrocytes in an assay sample become ruptured before or during performance of a lateral-flow immuno-chromatographic assay, hemoglobin released from the ruptured erythrocytes may stain the blood plasma or serum. If blood plasma or serum stained by hemoglobin reaches the test line in a lateral-flow immuno-chromatographic assay, then a visibly colored mark may be formed by the hemoglobin at the test line, generating a false positive assay result. These false positive results may be avoided by removing erythrocytes from an assay sample before carrying out a lateral-flow immuno-chromatographic diagnostic assay. For example, whole blood may be centrifuged to remove erythrocytes, so that the assay sample to be tested is in the form of blood plasma. Further, blood coagulation factors may also be removed, so that the assay sample tested is blood serum. However, in some cases, utilization of an assay sample including erythrocytes as well as either or both blood plasma and blood serum may be needed. For example, a medical professional may need to carry out a lateral-flow immuno-chromatographic diagnostic assay where equipment such as a centrifuge for removal of erythrocytes from whole blood is not available, or where rapid test performance is needed, such that waiting for preparation of a blood plasma or serum sample becomes an unacceptable delay. For example, a medical professional may need to carry out a lateral-flow immuno-chromatographic diagnostic assay at a location away from clinical facilities, or where a patient suffers from a critical, life-threatening condition. 
     As another example, a layman lacking both the skill and equipment needed to prepare a blood plasma or serum sample may need to himself carry out a lateral-flow immuno-chromatographic assay using whole blood. Further, such a layman may also lack both the equipment and skill needed to collect venous blood from a patient. Moreover, a layman may be reluctant to collect more than a single drop of the patient&#39;s capillary blood by a fingertip lance, especially if the layman needs to collect and then perform a diagnostic assay on his own blood. Additionally, such a layman may need to self-administer a lateral-flow immuno-chromatographic assay while suffering from a life-threatening condition at a location distant from professional medical personnel. 
     Hence, a lateral-flow immuno-chromatographic assay device capable of utilization with a small assay sample including erythrocytes and either or both of blood plasma and blood serum, such as a minimal sample of whole blood, may be useful in a variety of circumstances. Such a lateral-flow immuno-chromatographic assay device may, for example, need to effectively separate erythrocytes from blood plasma or serum so that false positive results due to ruptured erythrocytes are avoided. Further, for example, such a lateral-flow immuno-chromatographic assay device may need to effectively deliver as much of a small sample of blood plasma or serum as possible to the test line, so that a qualitative assay test result may be generated utilizing a small assay sample, such as a single drop of whole blood. 
     A lateral-flow immuno-chromatographic assay device may include a plasma separation membrane configured for allowing passage of blood plasma or serum through the plasma separation membrane and for trapping erythrocytes. In so trapping erythrocytes, the plasma separation membrane may become partially blocked and then impede the flow of blood plasma or serum through the plasma separation membrane toward the test line, detracting from a capability of the lateral-flow immuno-chromatographic assay device for utilization with an assay sample having a minimal volume, such as a single drop of blood. Hence, lateral-flow immuno-chromatographic assay devices are needed that may be capable of utilization with an assay sample of minimal volume, such as a single drop, including erythrocytes and either or both of blood plasma and blood serum. Such lateral-flow immuno-chromatographic assay devices, and methods for carrying out lateral-flow immuno-chromatographic assays, are provided herein. 
     A lateral-flow immuno-chromatographic assay device is provided herein that includes a migration membrane, a conjugate pad being on the migration membrane, a plasma separation membrane being on the conjugate pad, and a pre-filter being on the plasma separation membrane. The migration membrane has a test line configured for loading onto the test line of one or a plurality of capture antibodies having specific binding affinity for an assay target. The migration membrane is configured for allowing lateral flow of blood plasma or serum across the migration membrane to the test line. The conjugate pad is configured for loading onto the conjugate pad of one or a plurality of detection antibodies having specific binding affinity for an assay target. The plasma separation membrane is configured for allowing passage of blood plasma or serum through the plasma separation membrane and for trapping erythrocytes. The pre-filter is configured for loading of an assay sample including erythrocytes and either or both of blood plasma and blood serum onto the pre-filter. The pre-filter is also configured for allowing passage of blood plasma or serum through the pre-filter. Further, the pre-filter is configured for causing lateral flow of blood plasma or serum within the pre-filter. The pre-filter may additionally be configured for causing selective passage of blood plasma or serum through the pre-filter and for trapping erythrocytes. The device may be configured for being capable of utilizing a single drop of whole blood, such as hanging drop for example, as the assay sample. 
     In examples, the one or plurality of detection antibodies and the one or plurality of capture antibodies may have specific binding affinity for a cardiac Troponin-I epitope. The plurality of detection antibodies may include cardiac Troponin-I antibody clone 19C7 together with either or both of cardiac Troponin-I antibody clones 4C2 and M155. The plurality of capture antibodies may include both cardiac Troponin-I antibody clones MF4 and 16A11. As another example, the lateral-flow immuno-chromatographic assay device may include first and second detection antibodies and third and fourth capture antibodies, each of the first, second, third and fourth antibodies having specific binding affinity for substantially different cardiac Troponin-I epitopes. Throughout this specification, the term “substantially different” as applied to two epitopes of an assay target means that the two epitopes are sufficiently different to allow two antibodies to bind the target simultaneously through binding to the two epitopes. 
     The following conventions apply regarding terminology utilized throughout this specification. A “layer” of a material is any component of a lateral-flow immuno-chromatographic assay device that is bonded or attached to, formed or deposited on, or otherwise provided on any other layer or on or in the housing of the lateral-flow immuno-chromatographic assay device. A layer may include, as examples, a mat, surface, film, foil, region, body, or substrate. When one layer or material is referred to as being “on”, “over”, or “loaded onto” another layer or housing, then all or a portion of the layer or material may be directly on and in contact with all or a portion of the other layer or housing, or alternatively, intervening layers may also be present such that all or portions of the one layer and of another layer that is “on” or “over” the one layer or housing are not mutually in direct contact. When a layer is stated as being “directly on” another layer or the housing, then no intervening layer is present unless otherwise indicated. When a layer is stated as being “between” two other layers, then one or more additional intervening layers may also be present between the two other layers. When one layer is referred to as being “on” (or “over”) another layer, then the one layer may cover the entire surface of the other layer, or may cover only a portion of the other layer. When a material is stated as being “loaded onto” a layer or a surface of a layer, the material may remain on a surface of the layer, or may also penetrate through the surface into the layer, or may penetrate into and pass through the layer. Terms such as “formed on”, “disposed on”, “loaded onto” or “deposited on” are not intended to introduce any limitations relating to specific methods for fabricating a layer except as otherwise designated. 
       FIG. 1  is an exploded perspective view illustrating an example of an implementation of a lateral-flow immuno-chromatographic assay device  100 .  FIG. 2  is a cross-sectional view, taken along line A-A, of the lateral-flow immuno-chromatographic assay device  100  shown in  FIG. 1 . The lateral-flow immuno-chromatographic assay device  100  includes a migration membrane  102 , a conjugate pad  104  being on the migration membrane  102 , a plasma separation membrane  106  being on the conjugate pad  104 , and a pre-filter  108  being on the plasma separation membrane  106 . These components of the lateral-flow immuno-chromatographic assay device  100  are exploded in  FIG. 1  in the directions of an arrow  109 . The migration membrane  102  has a test line  110  configured for loading onto the test line  110  of one or a plurality of capture antibodies (not shown) having specific binding affinity for an assay target (not shown). So configuring the test line  110  may include forming the migration membrane  102  with a surface  111  selected as suitable for loading and binding the one or plurality of capture antibodies onto the surface  111  at the test line  110 . The migration membrane  102  is also configured for allowing lateral flow of blood plasma or serum (not shown) across the migration membrane  102  to the test line  110 . So configuring the migration membrane  102  may include arranging the conjugate pad  104 , the migration membrane  102 , and the test line  110  to form a pathway in the direction of an arrow  113  suitable to allow lateral flow of blood plasma or serum across the migration membrane  102  to the test line  110 . 
     Throughout this specification, the term “blood plasma” means the components of whole blood from which the solid cellular components, including erythrocytes, leukocytes and thrombocytes, have been removed. Throughout this specification, the term “blood serum” means the components of whole blood from which the coagulants and the solid cellular components have been removed. Throughout this specification, all references to “blood plasma” are deemed to designate, except where expressly stated or clear from the context otherwise: “blood plasma” and “blood serum” together in a mixture, as well as either “blood plasma” alone or “blood serum” alone. Throughout this specification, all references to “blood plasma or serum” are deemed to collectively designate and include, except where expressly stated otherwise: “blood plasma”, “blood serum”, and “blood plasma and blood serum”. 
     Throughout this specification, the term “lateral flow” as applied to flow of blood plasma or serum across the migration membrane  102  means that the lateral-flow immuno-chromatographic assay device  100  is configured to allow flow of blood plasma or serum from the conjugate pad  104  to the test line  110 . However, the orientation of the lateral-flow immuno-chromatographic assay device  100  shown in  FIGS. 1-2  is for purposes of illustration and does not indicate a horizontal positioning or any other specific positioning of the lateral-flow immuno-chromatographic assay device  100  relative to gravity during utilization or otherwise. 
     The conjugate pad  104  is on the migration membrane  102 , being configured for loading onto the conjugate pad  104  of one or a plurality of detection antibodies (not shown) having specific binding affinity for an assay target. So configuring the conjugate pad  104  may include forming the conjugate pad  104  with a surface  117  selected as suitable for loading the one or plurality of detection antibodies onto the surface  117 . The one or plurality of detection antibodies may, for example, penetrate into the surface  117  and soak into the conjugate pad  104 . The detection antibodies may include a visibly colored tagging agent, such as colloidal gold particles or blue latex microspheres, as examples. Colloidal gold particles having an average diameter of about 40 nanometers (nm) that may be utilized, and services of tagging antibodies with such particles, are commercially available from Arista Biologicals, having a business address at 1101 Hamilton Street, Allentown, Pa. 18101 USA. The plasma separation membrane  106  is configured for allowing passage of blood plasma or serum through the plasma separation membrane  106  and for trapping erythrocytes (not shown). The plasma separation membrane  106  may further be configured for causing selective passage of blood plasma or serum through the plasma separation membrane  106 . 
     The pre-filter  108  is configured for loading of an assay sample (not shown) including erythrocytes and either or both of blood plasma and blood serum onto the pre-filter  108 . As an example, the assay sample may include whole blood. In further examples, the assay sample may include whole blood together with either or both of blood plasma and blood serum. In additional examples, the assay sample may include either or both of blood plasma and blood serum, while not including blood cells. Although the pre-filter  108  is configured for loading of an assay sample that includes erythrocytes, it is understood throughout this specification that the lateral-flow immuno-chromatographic assay devices that are disclosed herein may be utilized for carrying out an immuno-chromatographic assay on an assay sample that does not include erythrocytes but that includes either or both of blood plasma and blood serum. For example, the assay sample to be utilized may be a sample of blood plasma or of blood serum. 
     Configuring the pre-filter  108  for loading of an assay sample including erythrocytes and either or both of blood plasma and blood serum onto the pre-filter may include, for example, forming the pre-filter  108  with a first surface  120  selected as suitable for loading such an assay sample onto the surface  120 . The pre-filter  108  is also configured for allowing passage of blood plasma or serum through the pre-filter  108  and configured for causing lateral flow of blood plasma or serum within the pre-filter  108 . Configuring the pre-filter  108  for allowing such passage of blood plasma or serum and for causing such lateral flow may include, for example, selecting a pre-filter  108  having a fibrous structure, or having a structure including pores forming pathways communicating between the first surface  120  and a second surface  122 . 
     In an example, the plasma separation membrane  106  may have a first surface  112  facing toward the pre-filter  108  and a second surface  114  facing toward the conjugate pad  104 . Configuring the plasma separation membrane  106  for allowing passage of blood plasma or serum through the plasma separation membrane  106  and for trapping erythrocytes may include providing the plasma separation membrane  106  with a fibrous structure, or having a structure including pores forming pathways communicating between the first surface  112  and the second surface  114 . In another example, the plasma separation membrane  106  may include a plurality of passageways  115  each communicating with both of the first and second surfaces  112 ,  114 , and wherein a plurality of the passageways  115  each has a first opening  116  at the first surface  112  and a second opening  118  at the second surface  114 , the second opening  118  being smaller than the first opening  116 . Each of a plurality of the passageways  115  may have a frustoconical shape, the plurality of passageways  115  being laterally spaced apart from each other within the plasma separation membrane  106 . The frustoconical shape of each of the plurality of passageways  115  may be configured for trapping and immobilizing erythrocytes. 
     The pre-filter  108  may, for example, have a random structure configured for allowing omni-directional passage of an assay sample, such as an assay sample including whole blood, or otherwise including erythrocytes and either or both of blood plasma and blood serum, or an assay sample including either or both of blood plasma and serum, through the pre-filter  108 . The pre-filter  108  may have a random fibrous structure. The pre-filter  108  may be configured for causing selective passage of blood plasma or serum through the pre-filter  108  and for trapping erythrocytes. The pre-filter  108  may be configured for trapping at least a minimum proportion of the erythrocytes from an assay sample sufficient to significantly reduce a tendency of the plasma separation membrane  106  to become blocked by erythrocytes, thereby improving the plasma separation membrane&#39;s performance in trapping erythrocytes and in allowing blood plasma or serum to flow through the plasma separation membrane  106 . For example, the pre-filter  108  may be configured for trapping at least about 10%, or at least about 30%, of a quantity of erythrocytes from an assay sample. The pre-filter  108  may be configured for causing substantial lateral flow of an assay sample, such as an assay sample including whole blood, or otherwise including erythrocytes and either or both of blood plasma and blood serum, or an assay sample including either or both of blood plasma and serum, within the pre-filter  108 . Throughout this specification, the term “substantial lateral flow” as applied to flow of blood plasma or serum through the pre-filter  108  means that the blood plasma or serum exits from the pre-filter  108  through a portion of the second surface  122  having an area at least about 5% larger than an area on the first surface  120  of the pre-filter  108  through which the blood plasma or serum enters the pre-filter  108 . Throughout this specification, the term “substantial lateral flow” as applied to flow of an assay sample including erythrocytes through the pre-filter  108  means that the assay sample exits from the pre-filter  108  through a portion of the second surface  122  having an area at least about 5% larger than an area on the first surface  120  of the pre-filter  108  through which the assay sample enters the pre-filter  108 . 
     The pre-filter  108  may, for example, have an exposed first surface  120  and a second surface  122  facing toward the plasma separation membrane  106 . The pre-filter  108  may have a structure including a plurality of passageways  123  having first openings  124  communicating with the first surface  120  and second openings  126  communicating with the second surface  122 . The pre-filter  108  may be configured for causing an assay sample, such as an assay sample including whole blood, or otherwise including erythrocytes and either or both of blood plasma and blood serum, or an assay sample including either or both of blood plasma and serum, to flow out of a larger quantity of second openings  126  than a quantity of first openings  124  through which the assay sample enters the pre-filter  108 . The pre-filter  108  may cooperate with the plasma separation membrane  106  to convey a greater portion of the assay sample, such as an assay sample including whole blood, or otherwise including erythrocytes and either or both of blood plasma and blood serum, to the conjugate pad  104  than the plasma separation membrane  106  would be capable of so conveying without the pre-filter  108 . In this regard, the pre-filter  108  may cause the assay sample to laterally spread over an enlarged portion of the first surface  112  of the plasma separation membrane  106 . The assay sample, such as an assay sample including whole blood, or otherwise including erythrocytes and either or both of blood plasma and blood serum, then passes into an enlarged portion of the plasma separation membrane  106 , allowing blood plasma or serum to flow through the plasma separation membrane  106  and allowing erythrocytes to be trapped in a larger quantity of the passageways  115 . As trapped erythrocytes are spread over a larger quantity of the passageways  115 , blockage to flow of blood plasma or serum through the plasma separation membrane  106  is accordingly reduced. Further, the blood plasma or serum may then likewise flow onto an enlarged portion of the conjugate pad  104 , allowing the detection antibodies to make contact with the blood plasma or serum over an enlarged area. As another example, the pre-filter  108  may have an asymmetric structure wherein an average spacing between the second openings  126  is larger than an average spacing between the first openings  124 . 
     The pre-filter  108  and the plasma separation membrane  106  may, for example, be collectively configured for trapping at least about 90% of a quantity of erythrocytes from an assay sample. The lateral-flow immuno-chromatographic assay device  100  may be configured for conveying a substantial portion of the blood plasma or serum from an assay sample to the migration membrane  102 . It is understood throughout this specification that the term “substantial portion” means that at least about 60% by volume of the blood plasma or serum from an assay sample is conveyed to the migration membrane  102 . For example, the lateral-flow immuno-chromatographic assay device  100  may be configured for conveying between about 60% by volume and about 80% by volume of the blood plasma or serum from an assay sample to the migration membrane  102 . The lateral-flow immuno-chromatographic assay device  100  may be configured for being capable of utilizing a single drop of whole blood, such as hanging drop for example, as the assay sample. A drop of blood may have a volume within a range of between about 20 microliters (μl) and about 65 μl. A hanging drop of blood may have a volume within a range of between about 60 μl and about 65 μl. The blood plasma or serum in a hanging drop of whole blood may have a volume within a range of between about 32 μl and about 42 μl. Where an assay sample includes either or both of blood serum and blood plasma, but does not include erythrocytes, an assay sample volume within that range, i.e. between about 30 μl and about 42 μl, may for example be utilized. 
     The lateral-flow immuno-chromatographic assay device  100  may be capable of carrying out a diagnostic assay on a small sample of whole blood, such as a single drop of whole blood, without a need to separate the blood plasma or serum from erythrocytes before loading the whole blood sample onto the lateral-flow immuno-chromatographic assay device  100 . For example, centrifugation of whole blood before loading a drop of whole blood onto the pre-filter  108  may not be needed. Accordingly, the lateral-flow immuno-chromatographic assay device  100  may facilitate carrying out a diagnostic assay under circumstances where external cellular component-separating equipment for pre-treatment of an assay sample, such as a centrifuge, may be unavailable. For example, the lateral-flow immuno-chromatographic assay device  100  may be suitable for utilization “in the field”, away from any hospital or clinic, as a stand-alone portable diagnostic assay device. The capability of utilizing the lateral-flow immuno-chromatographic assay device  100  to carry out a diagnostic assay on a single drop of whole blood also facilitates utilization of the lateral-flow immuno-chromatographic assay device  100  by a layman, who may be able to self-draw the small needed sample of whole blood from a capillary by a routine finger prick with a lance, and to then himself carry out the diagnostic assay. Further for example, a layman who suspects that he or she has had or is having a heart attack may be able to successively self-administer a plurality of diagnostic assays utilizing a plurality of lateral-flow immuno-chromatographic assay devices  100 , in order to monitor his or her own physical condition over a period of time as well as to provide ongoing status information to a remotely-located cardiologist. 
     The migration membrane  102  may have a longitudinal axis  127 . The plasma separation membrane  106  may have a midpoint  128  tangentially located over a first point  130  along the longitudinal axis  127 . The conjugate pad  104  may have a midpoint  132  tangentially located over a second point  134  along the longitudinal axis  127 , and wherein the second point  134  is nearer to the test line  110  than is the first point  130 . This relative orientation of the midpoints  130 ,  134  may serve to bias flow of blood plasma or serum toward the migration membrane  102  as the blood plasma or serum passes from the plasma separation membrane  106  to the conjugate pad  104  and then onto the migration membrane  102 . The migration membrane  102  may also include a control line  136 , on which a control test may be carried out. For example, antibodies selected as capable of binding the detection antibodies may be loaded at the control line  136  to verify proper assay functionality, including flow of the detection antibodies together with blood plasma or serum from the conjugate pad  104  to the test line  110 . The lateral-flow immuno-chromatographic assay device  100  may further include a substrate  138 , an absorption pad  140 , and a housing  142 . The substrate  138  may include an adhesive layer (not shown) for securing the migration membrane  102 , conjugate pad  104 , and absorption pad  140  on the substrate  138 . The housing  142  may include an opening  144  for assay sample introduction and an opening  146  for reading of assay results. In an example, the opening  144  may be centered over a mid-point  147  of the pre-filter  108 . During utilization of the lateral-flow immuno-chromatographic assay device  100  to carry out an assay, this orientation of the opening  144  may enable an assay sample to spread out in all directions over the first surface  120  of the pre-filter  108 , to improve flow of blood plasma or serum. 
     When the lateral-flow immuno-chromatographic assay device  100  is utilized to carry out a diagnostic assay, an assay sample loaded onto the pre-filter  108  flows through the pre-filter  108  and then through the plasma separation membrane  106 . Erythrocytes in the assay sample are trapped in the plasma separation membrane  106 ; and may also be trapped on or in the pre-filter  108 . Leukocytes and thrombocytes, if present in the assay sample, may be trapped in the plasma separation membrane  106 ; and may also be trapped on or in the pre-filter  108 . Blood plasma or serum then passes through the conjugate pad  104 . The detection antibodies loaded onto the conjugate pad  104  then specifically bind with target antigen if present in the blood plasma or serum, and the blood plasma or serum then carries the bound detection antibodies laterally across the migration membrane  102  to the test line  110 . The visibly colored agent, bound to detection antibodies specifically bound to the target antigen, is accordingly carried to the test line  110 . The capture antibodies, which may be bound to the migration membrane  102  at the test line  110 , then specifically bind with the target antigen if present in the blood plasma or serum, effectively binding the visibly colored agent to the test line  110 . If the target antigen was present at a detectable concentration in the assay sample, then an accumulation of the visibly colored agent so bound may form a visible mark at the test line  110 , being a positive qualitative assay result indicating the presence of the target antigen in the assay sample. Where the visibly colored agent is colloidal gold, the visible mark so formed at the test line  110  may have, as examples, a reddish, pinkish or brownish colored appearance. 
     As the diagnostic assay takes place, a visibly colored leading edge (not shown) formed by the visibly colored agent bound to detection antibodies specifically bound to the target antigen is carried to the test line  110 . The migration membrane  102  may, for example, be configured for causing the leading edge of the visibly colored agent to be conveyed across the migration membrane  102  at a controlled speed within a range of between about 2.5 minutes per 3 centimeters (min/3 cm) and about 3.75 min/3 cm. The migration membrane  102  may be impregnated with a membrane blocking buffer at a concentration selected for causing the leading edge of the visibly colored agent to be conveyed at the controlled speed. The migration membrane  102  may have an average pore diameter selected for causing the leading edge of the visibly colored agent to be conveyed at the controlled speed. For example, a migration membrane  102  including pores having an average pore diameter of between about 1 micrometer (μm) and about 250 μm may be suitable for causing the leading edge of the visibly colored agent to be conveyed at the controlled speed. The selected migration membrane  102  may be electrically uncharged in furtherance of maintaining the controlled speed. 
     The physical dimensions of the lateral-flow immuno-chromatographic assay device  100  may be selected, for example, by establishing the flow rates of blood plasma or serum through the pre-filter  108 , the plasma separation membrane  106 , and the conjugate pad  104 ; and the flow rate of blood plasma or serum across the migration membrane  102  to the test line  110 . These flow rates may then be utilized to define physical dimensions for the lateral-flow immuno-chromatographic assay device  100  such that a diagnostic assay may be carried out over and completed after a moderate period of time. It is understood throughout this specification that a “moderate” period of time is a time period of less than about 20 minutes. As an example, a moderate period of time may be a time period within a range of between about 7 minutes and about 20 minutes; or a time period within a range of between about 10 minutes and about 15 minutes. An excessively short assay completion time period may lead to inaccurate assay test results, for example because the blood plasma or serum may migrate too rapidly past the test line  110 . In that case, the capture antibodies may not adequately bind with the assay target in the blood plasma or serum. An excessively long assay completion time period detracts from the usefulness of the lateral-flow immuno-chromatographic assay device  100 , and may lead to false positive results. For example, such an excessively long assay completion time period may enable sufficient hemoglobin from ruptured erythrocytes in an assay sample to reach the test line  110  and accumulate there to generate a visible line to mimic the presence of detection antibody-bound target antigen. However, the lateral-flow immuno-chromatographic assay device  100  may also enable an accurate qualitative assay result to be observed over an extended time period, continuing after completion of a diagnostic assay, such extended time period being longer than a moderate period of time. For example, an accurate qualitative assay result may remain visible upon inspection of the lateral-flow immuno-chromatographic assay device  100  at any point over an extended time period within a range of between about 7 minutes and about 90 minutes following initiation of a diagnostic assay. Suitable physical dimensions for the lateral-flow immuno-chromatographic assay device  100  may further be selected according to a particular end-use application, such that a particular diagnostic assay may be effectively carried out. For example, the physical dimensions of the lateral-flow immuno-chromatographic assay device  100  may additionally take into consideration the flow rates of detection antibody-tagged target antigen through the conjugate pad  104  and across the migration membrane  102  to the test line  110 . 
     As an example, the migration membrane  102  may have a length within a range of between about 22.0 millimeters (mm) and about 30.0 mm; or of about 25.0 mm. It is understood throughout this specification that all length dimensions of components in examples of the lateral-flow immuno-chromatographic assay device  100  are defined in directions of the arrow  127 . It is further understood throughout this specification that all dimensions of components in examples of the lateral-flow immuno-chromatographic assay device  100 , including lengths, widths, heights, relative proportions between dimensions, and any other dimensions, are examples for purposes of illustration; and that lateral-flow immuno-chromatographic assay devices  100  having other dimensions and proportions may be fabricated and utilized. The conjugate pad  104  may have a length within a range of between about 9.0 mm and about 12.0 mm; or of about 10.0 mm. The conjugate pad  104  may overlap with the migration membrane  102  over a length within a range of between about 0.5 mm and about 3.5 mm; or of about 2.0 mm. The test line  110  may be spaced apart from a trailing edge  148  of the conjugate pad  104  by a distance along the migration membrane  102  having a length within a range of between about 7.0 mm and about 12.0 mm; or of about 9.0 mm. The control line  136  may be spaced apart from the trailing edge  148  of the conjugate pad  104  by a distance along the migration membrane  102  having a length within a range of between about 12.0 mm and about 20.0 mm; or of about 17.0 mm. The plasma separation membrane  106  may have a length within a range of between about 11.8 mm and about 15.0 mm; or of about 13.0 mm. The pre-filter  108  may have a length within a range of between about 12.5 mm and about 15.0 mm; or of about 14.0 mm. A trailing edge  150  of the plasma separation membrane  106  and a trailing edge  152  of the pre-filter  108  may, for example, be mutually aligned together along the longitudinal axis  127  slightly farther away than the trailing edge  148  from the test line  110 . The absorption pad  140  may have a length within a range of between about 19.0 mm and about 22.0 mm; or of about 20.0 mm. A leading edge  154  of the absorption pad  140  may overlap with the migration membrane  102  over a length within a range of between about 0.5 mm and about 2.0 mm; or of about 1.0 mm. The trailing edge  148  of the conjugate pad  104  may be spaced apart from the leading edge  154  of the absorption pad  140  by a distance along the migration membrane  102  having a length within a range of between about 21.0 mm and about 24.0 mm; or of about 22.0 mm. The substrate  138  may have a length within a range of between about 61.0 mm and about 63 mm; or of about 62.5 mm. The migration membrane  102  may be located along the length of the substrate  138  such that a portion of the substrate  138  defines a dead space  156 . The dead space  156  may have a length within a range of between about 8.5 mm and about 11.5 mm; or of about 10.0 mm, extending away from a leading edge  158  of the conjugate pad  104 . The dead space  156  may serve to orient the midpoint  147  of the pre-filter  108  in a position approximately centered along the longitudinal axis  127  relative to the opening  144  in the housing  142 . 
     The lateral-flow immuno-chromatographic assay device  100  may have a width defined by directions of an arrow  160 , and a height defined by directions of the arrow  109 . The lateral-flow immuno-chromatographic assay device  100  may have a width  160  within a range of between about 8.0 mm and about 8.3 mm; or of about 8.2 mm. The lateral-flow immuno-chromatographic assay device  100  may have a height in directions of the arrow  109  within a range of between about 1.5 mm and about 1.8 mm; or of about 1.7 mm. 
     The pre-filter  108  may, for example, have a selected thickness within a range of between about 355 μm and about 508 μm. The plasma separation membrane  106  may, for example, have a selected thickness within a range of between about 310 μm and about 350 μm, or of about 330 μm. The conjugate pad  104  may, for example, have a selected thickness within a range of between about 355 μm and about 508 μm. The migration membrane  102  may, for example, have a selected thickness within a range of between about 165 μm and about 205 μm. The absorption pad  140  may, for example, have a selected thickness within a range of between about 304 μm and about 370 μm. The substrate  138  may, for example, have a selected thickness including an adhesive layer, within a range of between about 550 μm and about 650 μm 
     The pre-filter  108  is formed of a material having a structure suited for causing blood plasma or serum to laterally flow within the pre-filter  108 , and also for allowing blood plasma or serum to pass through the pre-filter  108 . The pre-filter  108  may have a random structure that is both porous to flow of blood plasma or serum and that causes such lateral flow to occur. The random structure may be a fibrous random structure. Such a material may also have a porosity suitably sized or a fiber density suitable for trapping some of the erythrocytes present in an assay sample. The pre-filter  108  may, for example, be formed of a cellulosic glass fiber material. In further examples, the pre-filter  108  may be formed of borosilicate glass fiber with a polyvinyl alcohol binder, having the grade designation “SMCON64” or “SMCON75”, both of which are commercially available from the Pall Corporation, having a business address at 2200 Northern Blvd., East Hills, N.Y. 11548 USA; www.pall.com. The entirety of the Pall Corporation&#39;s 3-page “Conjugate Pads” product data sheet including the SMCON64 and SMCON75 materials is hereby incorporated herein by reference. 
     The plasma separation membrane  106  is formed of a material having a structure suited for allowing blood plasma or serum to pass through the plasma separation membrane  106 , and for trapping erythrocytes. For example, the plasma separation membrane  106  may be formed of an asymmetric membrane material having large pores  116  at the first surface  112  and smaller pores  118  at the second surface  114 . As an example, the large pores  116  may have diameters of about 220 μm, and the small pores  118  may have diameters of about 2.5 μm. Erythrocytes may then be trapped in the large pores  116 , while blood plasma or serum flows out of the plasma separation membrane  106  through the smaller pores  118 . In an example, the plasma separation membrane  106  may be an asymmetric membrane material formed of a polysulfone and having the trade name “Vivid™ Plasma Separation Membrane” which is commercially available from the Pall Corporation. The entirety of the Pall Corporation&#39;s 6-page product data sheet for the Vivid™ Plasma Separation Membrane is hereby incorporated herein by reference. 
     The conjugate pad  104  is formed of a material having a structure suited for loading onto the conjugate pad  104  of one or a plurality of detection antibodies having specific binding affinity for an assay target. For example, the material may have a structure suited for allowing or causing the one or plurality of detection antibodies to penetrate into and soak the conjugate pad  104 . The conjugate pad  104  may have a random structure that is porous to flow of blood plasma or serum. The random structure may be a fibrous random structure. Such a material may also have a porosity suitably sized or a fiber density suitable for trapping any remaining erythrocytes from an assay sample. The conjugate pad  104  may, for example, be formed of a cellulosic glass fiber material. In further examples, the conjugate pad  104  may be formed of borosilicate glass fibers with a polyvinyl alcohol binder, having the grade designation “SMCON64” or “SMCON75”, both of which are commercially available from the Pall Corporation. Further, for example, the conjugate pad  104  may be formed of a fibrous material having the grade designation “FUSION 5™”, commercially available from Whatman Inc., having a business address at Building 1, 800 Centennial Avenue, Piscataway, N.J. 08854 USA; www.whatman.com. The entirety of Whatman Inc.&#39;s 2-page product data sheet for the Fusion 5™ material is hereby incorporated herein by reference. As another example, a binder- and surfactant-free hydrophilic fibrous material formed of hydroxylated polyester, having a basis weight of about 101 grams per square meter (g/m 2 ), a hold-up volume of about 39 microliters per square centimeter (μl/cm 2 ), a water wicking rate of about 44 seconds per 3 centimeters (44 sec/3 cm), and an absorption capacity of about 38 μl/cm 2 , may be utilized. 
     The materials from which each of the pre-filter  108 , the plasma separation membrane  106 , and the conjugate pad  104  are formed, may take the form of sheets, flat discs, or webs, as examples. The materials may have a high water wicking rate to facilitate flow of blood plasma or serum into and through the pre-filter  108 , the plasma separation membrane  106 , and the conjugate pad  104 . The materials from which each of the pre-filter  108 , the plasma separation membrane  106 , and the conjugate pad  104  are formed may further be selected to minimize binding of a target antigen to such material; and may be treated with a membrane blocking buffer to inhibit protein binding. The assay sample may be treated with an anti-coagulant such as a heparin salt, a citrate such as sodium citrate, or an ethylene diamine tetra-acetic acid (EDTA) salt before loading onto the pre-filter  108 . Alternatively, the material from which the pre-filter  108  is formed may be treated with an anti-coagulant. In addition, the materials from which the pre-filter  108 , the plasma separation membrane  106 , and the conjugate pad  104  are formed may be treated with a surfactant suitable for causing such materials to be hydrophilic. 
     The migration membrane  102  is formed of a material having a structure suited to allow lateral flow of blood plasma or serum across the migration membrane  102  to the test line  110 . As an example, the migration membrane  102  may be formed of a material selected as suitable to allow such lateral flow to occur over a selected period of time. Further, for example, the structure of the material utilized in forming the migration membrane  102  may actively cause the lateral flow of the blood plasma or serum to occur, such as by capillary action or wicking. The migration membrane  102  may be formed of a material having a structure including a generally uniform wicking rate, thickness, tensile strength, and protein binding level. The migration membrane  102  may have a generally uniform surface  111  with minimal scratches, dust and other irregularities. The migration membrane  102  may be a layer of material formed on a backing (not shown), where the backing has sufficient tensile strength to maintain the shape and integrity of the migration membrane  102  during fabrication and use of the lateral-flow immuno-chromatographic assay device  100 . For example, the migration membrane  102  may include a nitrocellulose layer formed on a polyester backing. Further, the migration membrane  102  may be formed on such a backing without an adhesive, to avoid leaching of the adhesive into the blood plasma or serum when an assay is carried out. For example, the migration membrane  102  may be formed of a material selected as having a tensile strength of at least about 12 Newtons. Tensile strength may be measured on a sample of material for forming the migration membrane  102  having a width of 15 mm and a length of 1,000 mm, using the testing protocol in DIN 53 112, part 1; or utilizing ASTM D 828 “Standard Test Method for Tensile Properties of Paper and Paperboard Using Constant-Rate-of-Elongation Apparatus.” In an example, the migration membrane  102  may be a polyester-backed nitrocellulose membrane material having a wicking rate within a range of between about 150 sec/4 cm and about 225 sec/4 cm, a tensile strength of at least about 12 Newtons, and a protein binding (bovine serum albumin) rate within a range of between about 45 micrograms per square centimeter (μg/cm 2 ) and about 59 μg/cm 2 , sold under trade name “Vivid™ 170 Lateral Flow Nitrocellulose Membrane” by the Pall Corporation. The entirety of the Pall Corporation&#39;s 4-page product data sheet for the Vivid™ 170 Lateral Flow Nitrocellulose Membrane is hereby incorporated herein by reference. The migration membrane  102  may be impregnated with a membrane blocking buffer. The migration membrane  102  may be treated with a surfactant suitable for causing the migration membrane  102  to be hydrophilic. 
     The substrate  138  may be formed of a material having a structure suited for physically supporting the pre-filter  108 , the plasma separation membrane  106 , the conjugate pad  104 , the migration membrane  102 , and the absorption pad  140 , and for maintaining such components of the lateral-flow immuno-chromatographic assay device  100  in position within the housing  142 . For example, the substrate  138  may be formed of a rigid sheet material such as a high impact polystyrene sheet having a thickness of about 500 μm and including an adhesive layer. Such a material suitable for forming the substrate  138 , having the grade designation “L-H50” and including an acrylic adhesive layer, is commercially available from Advanced Microdevices Pvt. Ltd., having a business address at 20-21 Industrial Area, Ambala Cantt 133 006, INDIA; www.mdimembrane.com. The entirety of the “Lateral Flow Test” section of the Advanced Microdevices Pvt. Ltd. product catalog, pages 2-5, including data sheet information for the L-H50 material, is hereby incorporated herein by reference. 
     The absorption pad  140  may be formed of a material having a structure suited to be highly absorbent, generating a wicking action facilitating the lateral flow of the blood plasma or serum across the migration membrane  102  to the test line  110 . For example, the absorption pad  140  may be formed of a cellulosic fibrous material. Such a cellulosic material suitable for forming the absorption pad  140 , having a nominal pore size of 3 μm, a thickness of about 330.2 μm, and a basis weight of about 186.3 g/m 2  is commercially available under the grade designation “BSP113PK Cellulose Absorbent 113” from the Pall Corporation. The entirety of the Pall Corporation&#39;s “Cellulose Absorbent Papers” 2-page product data sheet including information regarding BSP113PK Cellulose Absorbent 113 is hereby incorporated herein by reference. 
     The housing  142  may be formed of a material suited for fabricating a rigid protective container for the lateral-flow immuno-chromatographic assay device  100 . For example, an organic polymeric material may be utilized. 
     In an example of a lateral-flow immuno-chromatographic assay device  100 , the one or plurality of detection antibodies on the conjugate pad  104  may have specific binding affinity for a cardiac Troponin-I epitope. The plurality of detection antibodies may include cardiac Troponin-I antibody clone 19C7 together with either or both of cardiac Troponin-I antibody clones 4C2 and M155. Further, for example, the lateral-flow immuno-chromatographic assay device  100  may include one or a plurality of capture antibodies on the test line  110  having specific binding affinity for a cardiac Troponin-I epitope. The plurality of capture antibodies may include both cardiac Troponin-I antibody clone MF4 and cardiac Troponin-I antibody clone 16A11. The detection and capture antibodies may, as further examples, have specific binding affinity for cardiac Troponin-I in its free form, or fragmented forms, or phosphorylated forms, or in forms partially-digested by proteases, or as part of a complex with either or both of Troponin-T and Troponin-C, such as a cardiac Troponin-ITC complex. The detection and capture antibodies may have specific binding affinity for human cardiac Troponin-I epitopes. A threshold of sensitivity for a lateral-flow immuno-chromatographic assay device  100  utilized for qualitatively detecting cardiac Troponin-I in an assay sample including whole blood may be, for example, about 0.001 microgram/milliliter (μg/ml) equivalent to about 1.0 nanogram per milliliter (ng/ml), of cardiac Troponin-I in free form, or fragmented forms, or phosphorylated forms, or in forms partially-digested by proteases, or as part of a complex with either or both of Troponin-T and Troponin-C, such as a cardiac Troponin-ITC complex. As another example, the lateral-flow immuno-chromatographic assay device  100  may include first and second detection antibodies on the conjugate pad  104 , wherein the lateral-flow immuno-chromatographic assay device  100  may include third and fourth capture antibodies on the test line  110 , and wherein each of the first, second, third and fourth antibodies has specific binding affinity for a substantially different cardiac Troponin-I epitope. As examples, the first antibody may be cardiac Troponin-I antibody clone 19C7, the second antibody may be selected from cardiac Troponin-I antibody clones 4C2 and M155, the third antibody may be cardiac Troponin-I antibody clone MF4, and the fourth antibody may be cardiac Troponin-I antibody clone 16A11. 
     Suitable cardiac Troponin-I antibody clones, including clones 19C7, 4C2, M155, MF4 and 16A11, are commercially available under the grade designation 4T21 from HyTest Ltd., having a business address at Intelligate, Joukahaisenkatu 6, 20520 Turku, Finland (www.hytest.fi); and from Abcam plc, having a business address at 332 Cambridge Science Park, Cambridge CB4 0WN, England (www.abcam.com). Further cardiac Troponin-I antibodies that may be utilized are disclosed in “Markers of Cardiovascular Diseases and Metabolic Syndrome—II Troponin-specific Antibodies,” (pp. 14-22, 2009), published by HyTest Ltd. and downloaded from http://www.hytest.fi/data_sheets/Markers%20of%20Cardiovascular%20Diseases%20and%20Metabolic%20Syndrome.pdf, the entirety of which hereby is incorporated herein by reference. The Troponin-I detection and capture antibodies as obtained may be further diluted by their buffers, such as phosphate buffered saline (PBS) with sodium azide. Skeletal Troponin-I in its free form, fragmented forms, or as part of a complex with either or both of Troponin-T and Troponin-C may be utilized as a negative control. The detection and capture antibodies selected for utilization in a cardiac Troponin-I assay may be screened to rule out cross-reactivity with skeletal Troponin-I, by carrying out trials utilizing assay samples including skeletal Troponin-I. Cardiac Troponin-I in its free form, fragmented forms, or as part of a complex with either or both of Troponin-T and Troponin-C may be included in assay samples utilized in trials carried out as positive controls to verify the sensitivity and specificity of binding activity of the selected cardiac Troponin-I detection and capture antibodies. Suitable human cardiac Troponin-ITC complex is commercially available under the grade designation “8T62” from HyTest Ltd., chosen by AACC cTnI Standardization Subcommittee for international reference material. Suitable human cardiac Troponin-I is commercially available under the grade designation “8T53” from HyTest Ltd. Troponin-I—free blood serum may be utilized as another negative control. Suitable Troponin-I—free blood serum, purified by immunoaffinity chromatography, is commercially available under the grade designation “8TFS” from HyTest Ltd. 
     An assay sample to be tested may be pre-treated with an anti-coagulant such as a heparin salt, a citrate such as sodium citrate, or an EDTA salt before loading onto the pre-filter  108 , to prevent coagulation of the assay sample while a diagnostic assay is being carried out. For example, an anti-coagulant—coated pipette or tube may be utilized. Alternatively, the material from which the pre-filter  108  is formed may be treated with an anti-coagulant. The lateral-flow immuno-chromatographic assay device  100  may be utilized, as examples, to qualitatively detect cardiac Troponin-I at a concentration within a range of between about 1 ng/ml and at least about 2,000 ng/ml. 
       FIG. 3  is a flow diagram illustrating an example of an implementation of a method  300 . The method  300  starts at step  305 , and then step  310  includes providing a lateral-flow immuno-chromatographic assay device  100  including a migration membrane  102 , a conjugate pad  104  being on the migration membrane  102 , a plasma separation membrane  106  being on the conjugate pad  104 , and a pre-filter  108  being on the plasma separation membrane  106 ; wherein the conjugate pad  104  is loaded with one or a plurality of detection antibodies having specific binding affinity for an assay target; and wherein the migration membrane  102  has a test line  110  loaded with one or a plurality of capture antibodies having specific binding affinity for the assay target. Step  310  may further include providing a lateral-flow immuno-chromatographic assay device  100  having any of the further features discussed earlier in connection with  FIGS. 1-2 . Step  315  includes loading an assay sample including erythrocytes and either or both of blood plasma and blood serum onto the pre-filter  108 . The assay sample may include, in addition to erythrocytes, either blood plasma or blood serum alone, or blood plasma and blood serum together. Step  320  includes causing blood plasma or serum to laterally flow within the pre-filter  108  and allowing blood plasma or serum to pass through the pre-filter  108  to the plasma separation membrane  106 . Step  325  includes causing erythrocytes to be trapped in the plasma separation membrane  106  and allowing blood plasma or serum to pass through the plasma separation membrane  106  and to flow to the conjugate pad  104 . Step  330  includes allowing blood plasma or serum to pass through the conjugate pad  104  and to then flow onto the migration membrane  102  and to laterally flow across the migration membrane  102  to the test line  110 . Steps  315 ,  320 ,  325  and  330  collectively define a diagnostic assay cycle  335 . The method  300  may then end at step  340 . 
     In an example, step  315  may include loading a chase buffer onto the pre-filter  108  after loading the assay sample onto the pre-filter  108 , and step  330  may include causing the chase buffer to enhance lateral flow of blood plasma or serum across the migration membrane  102  to the test line  110 . As an example, the chase buffer may include bovine serum albumin (BSA). Further, for example, a buffered saline solution including a nonionic detergent and a preservative such as sodium azide may be utilized as the chase buffer. As an example, the chase buffer may include, at a pH of 7.2: 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 135 mM NaCl, 1% w/v BSA, and 50 milliliters per liter (mL/L) Tween 20. As another example, the chase buffer may include, at a pH of 7.8: 0.5% poly(ethylene glycol), 0.5% BSA, 0.1% Tween 20, and 0.1% MgCl 2  in Tris-buffered saline. As an additional example, the chase buffer may include 0.15M NaCl and 0.015M sodium citrate, supplemented with 1.4% Triton X-100 and 0.1% sodium dodecyl sulphate. 
     Step  320  may further include causing the blood plasma or serum to selectively pass through the pre-filter  108 , and causing erythrocytes to be trapped on or in the pre-filter  108 . Causing blood plasma or serum to laterally flow across the migration membrane  102  to the test line  110  at step  330  may include causing the leading edge of a visibly colored agent bound to the detection antibodies to be conveyed across the migration membrane  102  at a controlled speed, such as a controlled speed within a range of between about 2.5 min/3 cm and about 3.75 min/3 cm. 
     As an example, step  310  may include fabricating or obtaining a lateral-flow immuno-chromatographic assay device  100  that either has not yet been loaded with the detection antibodies or has not yet been loaded with the capture antibodies, or has not been loaded with the detection and capture antibodies, and then providing and loading such antibodies onto the lateral-flow immuno-chromatographic assay device  100 . In another example, step  310  may include providing one or a plurality of detection antibodies having, and may include providing one or a plurality of capture antibodies having, specific binding affinity for a cardiac Troponin-I epitope. The plurality of detection antibodies may include cardiac Troponin-I antibody clone 19C7 together with either or both of cardiac Troponin-I antibody clones 4C2 and M155. The plurality of capture antibodies may include cardiac Troponin-I antibody clones MF4 and 16A11. 
     In another example, providing the lateral-flow immuno-chromatographic assay device  100  at step  310  may include loading first and second detection antibodies onto the conjugate pad  104  and loading third and fourth capture antibodies onto the test line  110 ; wherein each of the first, second, third and fourth antibodies has specific binding affinity for a substantially different cardiac Troponin-I epitope. For example, the first antibody may be cardiac Troponin-I antibody 19C7, the second antibody may be selected from cardiac Troponin-I antibody clones 4C2 and M155, the third antibody may be cardiac Troponin-I antibody clone MF4, and the fourth antibody may be cardiac Troponin-I antibody clone 16A11. 
     Step  315  of the method  300  may include utilizing an assay sample that includes whole blood. The assay sample may be a single drop of whole blood, such as a hanging drop. The method  300  may include collecting the whole blood from a human patient suspected of recently having suffered from or suspected of currently suffering from a myocardial infarction, also referred to as a heart attack. 
     In another example, the method  300  may include collecting another assay sample including whole blood or otherwise including erythrocytes together with either or both of blood plasma and serum from the same human patient, repeating step  310  to provide another lateral-flow immuno-chromatographic assay device  100 , and carrying out another diagnostic assay cycle  335  utilizing the other assay sample and the other lateral-flow immuno-chromatographic assay device  100 . 
     EXAMPLES 
     In each of the following Examples, a lateral-flow immuno-chromatographic assay device  100  was fabricated. Each such device  100  included a substrate  138 , a migration membrane  102  on the substrate  138 , a conjugate pad  104  on the migration membrane  102 , a plasma separation membrane  106  on the conjugate pad  104 , and a pre-filter  108  on the plasma separation membrane  106 . The pre-filters  108  and conjugate pads  104  in each of the lateral-flow immuno-chromatographic assay devices  100  were formed of a material including borosilicate glass fibers with a polyvinyl alcohol binder, having the grade designation SMCON64, obtained from the Pall Corporation. The plasma separation membranes  106  in each of the devices  100  were formed of a polysulfone material having the trade name “Vivid™ Plasma Separation Membrane”, also obtained from the Pall Corporation. The migration membranes  102  in each of the devices  100  were formed of a polyester-backed nitrocellulose membrane material sold under trade name “Vivid™ 170 Lateral Flow Nitrocellulose Membrane”, obtained from the Pall Corporation. Each migration membrane  102  included a defined test line  110  loaded with 1.4 μl of a dispersion including equal parts of mouse-derived cardiac Troponin-I antibody clones MF4 and 16A11 at concentrations of 0.9 mg/ml each, in 20 millimolar (mM) sodium phosphate buffer containing 2.5% v/v (2.5 milliliters per 100 milliliters) isopropanol. The migration membrane  102  was impregnated with a membrane blocking buffer dispersion containing 0.01M Na 2 HPO 4 , 0.5% w/v (grams per 100 milliliters) BSA, 0.1% w/v Tween 20, 0.5% v/v polyvinylpyrrolidone, 0.06M sucrose, and 0.05M NaCl. The substrate  138  was formed of L-H50, obtained from Advanced Microdevices Pvt. Ltd. Each conjugate pad  104  was loaded with 48 μl of a dispersion including equal parts of 40 nm colloidal gold-conjugated (at  10 μg/ml) mouse-derived cardiac Troponin-I antibody clones 19C7 and 4C2 each at a concentration of 3 optical density units, in a dilution vehicle at pH 7.3 containing 5% w/v sucrose, 1.25% w/v trehalose, 0.01M disodium tetraborate decahydrate, 1% v/v polyvinyl acetate, 0.2% w/v Tween 20, and 0.2% w/v Triton-X 100. Each migration membrane  102  also included a defined internal control line  136  loaded with 1.4 μl of a dispersion including goat anti-mouse antibodies in sodium phosphate buffer, to verify proper assay functionality. Each of the devices  100  also included an absorption pad  140  formed of a cellulosic fibrous material. The above-identified components for each lateral-flow immuno-chromatographic assay device  100  were assembled together to form a structure as shown in  FIGS. 1-2 , and secured within a housing  142  also as shown in  FIGS. 1-2 . These components of the lateral-flow immuno-chromatographic assay devices  100  were held in position together by being moderately compressed between upper and lower halves of the housing  142 . 
     Each of the lateral-flow immuno-chromatographic assay devices  100  was then utilized to carry out a qualitative assay for the detection of human cardiac Troponin-ITC complex. Each assay sample was prepared by drawing a sample of whole blood having the volume of a hanging drop, supplemented with human cardiac Troponin-ITC complex at the concentrations in ng/ml indicated in Table 1, from a lithium-heparin-salt—containing tube. Each assay sample was loaded through an opening in the housing  142  onto the pre-filter  108 , immediately followed by addition of 100 μl of a chase buffer. The human cardiac Troponin-ITC complex was obtained from HyTest Ltd., having the grade designation 8T62. Photographs of the lateral-flow immuno-chromatographic assay devices  100  showing their test lines  110  and control lines  136  were taken at the elapsed time periods indicated in Table 1 following the loading of the hanging drops of blood. The results are summarized in Table 1 and shown in  FIGS. 4-17 . In each photograph among  FIGS. 4-17 , the test lines  110  and control lines  136  of all of the lateral-flow immuno-chromatographic assay devices  100  shown in the photograph are mutually oriented in the same direction. The control lines  136  are to the left of the test lines  110  in Examples K, S, and AI; and the control line  136  is above the test line  110  in Example AK. Vivid control lines as shown in  FIGS. 4-17  confirmed that the detection antibodies were carried to the test lines  110  in each of the examples. 
     Example B, using an assay sample having the volume of a hanging drop of whole blood including human cardiac Troponin-ITC complex at a concentration of 1 ng/ml, showed a faint positive test line after an elapsed test time period of about 15 minutes. Example C, using an assay sample having the volume of a hanging drop of whole blood including human cardiac Troponin-ITC complex at a concentration of 5 ng/ml, showed a clearly visible positive test line after the same elapsed test time period. Example A, serving as a negative control and carried out over the same elapsed test time period using an assay sample having the volume of a hanging drop of human whole blood and not supplemented with any added cardiac Troponin-ITC complex, did not yield a falsely-positive test line. 
     Example E, using an assay sample having the volume of a hanging drop of whole blood including human cardiac Troponin-ITC complex at a concentration of 1 ng/ml, showed a faint positive test line after an elapsed test time period of about 10 minutes. Examples I, M, Q, U, Y, AC and AG show that the faint positive test line remained visible after total elapsed test time periods of 11, 12, 15, 20, 25, 35 and 70 minutes, respectively. Each of Examples F, J, N, R, V, Z, AD and AH was carried out using an assay sample having the volume of a hanging drop of whole blood including human cardiac Troponin-ITC complex at a concentration of 5 ng/ml, yielding clearly visible positive test lines. Each of Examples G, K, O, S, W, AA, AE and AI was carried out using an assay sample having the volume of a hanging drop of whole blood including human cardiac Troponin-ITC complex at a concentration of 25 ng/ml, yielding vivid positive test lines. None of Examples D, H, L, P, T, X, AB or AF, each serving as a negative control and carried out using an assay sample having the volume of a hanging drop of human whole blood and not supplemented with any added cardiac Troponin-ITC complex, yielded any falsely-positive test lines. 
     Examples AJ through AO were carried out utilizing human whole blood having a concentration of hemolyzed blood cells sufficient to yield blood plasma having a visibly reddish color. Example AJ, serving as a negative control and carried out using an assay sample having the volume of a hanging drop of human whole blood and not supplemented with any added cardiac Troponin-ITC complex, did not yield a falsely-positive test line after an elapsed test time period of about 15 minutes. Examples AK and AL, each using an assay sample having the volume of a hanging drop of whole blood including human cardiac Troponin-ITC complex at a concentration of 1 ng/ml serving as a positive control, showed a faint positive test line after an elapsed test time period of about 15 minutes. Examples AM and AN, each using an assay sample having the volume of a hanging drop of whole blood including human cardiac Troponin-ITC complex at a concentration of 5 ng/ml, showed a clearly visible positive test line after an elapsed test time period of about 15 minutes. Example AO, using an assay sample having the volume of a hanging drop of whole blood including human cardiac Troponin-ITC complex at a concentration of 25 ng/ml, showed a vivid positive test line after an elapsed test time period of about 15 minutes. 
     Example AP, using an assay sample having the volume of a hanging drop of whole blood including human cardiac Troponin-ITC complex at a concentration of 1,000 ng/ml (equivalent to 1 μg/ml), showed a very vivid positive test line after an elapsed test time period of about 2 minutes. 
     In each of Examples AQ and AR, an assay sample was prepared by drawing a sample of whole blood, having the volume of a hanging drop supplemented with human skeletal Troponin-I instead of human cardiac Troponin ITC complex. The concentrations of human skeletal Troponin-I utilized in Examples AQ and AR were 25 ng/ml and 1,000 ng/ml, respectively. Neither of Examples AQ and AR yielded a falsely-positive test line after an elapsed test time period of about 15 minutes. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                 FIG. 
                 ITC 
                 Elapsed 
                   
               
               
                 Example 
                 No. 
                 ng/ml 
                 Minutes 
                 Qualitative Assay Result 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 A 
                 4 
                 0 
                 15 
                 Negative result at test line 
               
               
                 B 
                 4 
                 1 
                 15 
                 Faint positive test line 
               
               
                 C 
                 4 
                 5 
                 15 
                 Clearly visible positive test line 
               
               
                 D 
                 5 
                 0 
                 10 
                 Negative result at test line 
               
               
                 E 
                 5 
                 1 
                 10 
                 Faint positive test line 
               
               
                 F 
                 5 
                 5 
                 10 
                 Clearly visible positive test line 
               
               
                 G 
                 5 
                 25 
                 10 
                 Vivid positive test line 
               
               
                 H 
                 6 
                 0 
                 11 
                 Negative result at test line 
               
               
                 I 
                 6 
                 1 
                 11 
                 Faint positive test line 
               
               
                 J 
                 6 
                 5 
                 11 
                 Clearly visible positive test line 
               
               
                 K 
                 6 
                 25 
                 11 
                 Vivid positive test line 
               
               
                 L 
                 7 
                 0 
                 12 
                 Negative result at test line 
               
               
                 M 
                 7 
                 1 
                 12 
                 Faint positive test line 
               
               
                 N 
                 7 
                 5 
                 12 
                 Clearly visible positive test line 
               
               
                 O 
                 7 
                 25 
                 12 
                 Vivid positive test line 
               
               
                 P 
                 8 
                 0 
                 15 
                 Negative result at test line 
               
               
                 Q 
                 8 
                 1 
                 15 
                 Faint positive test line 
               
               
                 R 
                 8 
                 5 
                 15 
                 Clearly visible positive test line 
               
               
                 S 
                 8 
                 25 
                 15 
                 Vivid positive test line 
               
               
                 T 
                 9 
                 0 
                 20 
                 Negative result at test line 
               
               
                 U 
                 9 
                 1 
                 20 
                 Faint positive test line 
               
               
                 V 
                 9 
                 5 
                 20 
                 Clearly visible positive test line 
               
               
                 W 
                 9 
                 25 
                 20 
                 Vivid positive test line 
               
               
                 X 
                 10 
                 0 
                 25 
                 Negative result at test line 
               
               
                 Y 
                 10 
                 1 
                 25 
                 Faint positive test line 
               
               
                 Z 
                 10 
                 5 
                 25 
                 Clearly visible positive test line 
               
               
                 AA 
                 10 
                 25 
                 25 
                 Vivid positive test line 
               
               
                 AB 
                 11 
                 0 
                 35 
                 Negative result at test line 
               
               
                 AC 
                 11 
                 1 
                 35 
                 Faint positive test line 
               
               
                 AD 
                 11 
                 5 
                 35 
                 Clearly visible positive test line 
               
               
                 AE 
                 11 
                 25 
                 35 
                 Vivid positive test line 
               
               
                 AF 
                 12 
                 0 
                 70 
                 Negative result at test line 
               
               
                 AG 
                 12 
                 1 
                 70 
                 Faint positive test line 
               
               
                 AH 
                 12 
                 5 
                 70 
                 Clearly visible positive test line 
               
               
                 AI 
                 12 
                 25 
                 70 
                 Vivid positive test line 
               
               
                 AJ 
                 13 
                 0 
                 15 
                 Negative result at test line 
               
               
                 AK 
                 13 
                 1 
                 15 
                 Faint positive test line 
               
               
                 AL 
                 14 
                 1 
                 15 
                 Faint positive test line 
               
               
                 AM 
                 14 
                 5 
                 15 
                 Clearly visible positive test line 
               
               
                 AN 
                 15 
                 5 
                 15 
                 Clearly visible positive test line 
               
               
                 AO 
                 15 
                 25 
                 15 
                 Vivid positive test line 
               
               
                 AP 
                 16 
                 1,000 
                 2 
                 Very vivid positive test line 
               
               
                 AQ 
                 17 
                 0 
                 15 
                 Negative result at test line 
               
               
                 AR 
                 17 
                 0 
                 15 
                 Negative result at test line 
               
               
                   
               
            
           
         
       
     
     It is understood that the method  300  may include fabricating, obtaining, or otherwise providing a lateral-flow immuno-chromatographic assay device  100  having any of the features included in the examples of lateral-flow immuno-chromatographic assay devices  100  discussed in this specification. It is further understood that the discussion herein of the lateral-flow immuno-chromatographic assay devices  100  illustrates suitable variations of the method  300 . Likewise, it is understood that the discussion of the method  300  herein illustrates suitable variations of the lateral-flow immuno-chromatographic assay devices  100 . Accordingly, the entire discussion of the lateral-flow immuno-chromatographic assay devices  100  is deemed incorporated into the discussion of the method  300 . In addition, the entire discussion of the method  300  is deemed incorporated into the discussion of the lateral-flow immuno-chromatographic assay devices  100 . 
     The lateral-flow immuno-chromatographic assay devices  100  may be utilized in carrying out qualitative sandwich immunoassays for detection of target antigens that may be found in an assay sample including whole blood, or otherwise including erythrocytes and either or both of blood plasma and blood serum; or an assay sample including either or both of blood plasma and blood serum and not including erythrocytes. Examples of such target antigens include proteins, viruses, bacteria, microbes, drugs of abuse, and other normal and abnormal constituents of human and non-human blood, blood plasma or serum. 
     While the foregoing description refers in some instances to the lateral-flow immuno-chromatographic assay devices  100 , it is appreciated that the subject matter is not limited to these devices, or to the devices discussed in the specification. Devices having other configurations consistent with the foregoing teachings may be fabricated. Further, it is understood that the method  300  may include additional steps and modifications of the indicated steps. It will be understood that the foregoing description of numerous examples has been presented for purposes of illustration and description. This description is not exhaustive and does not limit the claimed invention to the precise forms disclosed. Modifications and variations are possible in light of the above description or may be acquired from practicing the invention. The claims and their equivalents define the scope of the invention.