WO2001020325A1

WO2001020325A1 - Membrane immunoassays for detection of multiple tick-borne diseases

Info

Publication number: WO2001020325A1
Application number: PCT/US1999/021814
Authority: WO
Inventors: Andrew E. Levin
Original assignee: Immunetics Inc
Current assignee: Immunetics Inc
Priority date: 1999-09-16
Filing date: 1999-09-20
Publication date: 2001-03-22
Anticipated expiration: 2002-03-16
Also published as: AU6054099A

Abstract

Membranes for flow-through immunoassay and methods of using same are provided for the simultaneous detection of multiple tick-borne diseases in a single assay. To detect infections and/or coinfections of multiple tick-borne agents, the membrane is provided with at least three different antigens, each capable of detecting antibodies to the agent of one thick-borne disease of interest, for instance, babesiosis, HGE, or borreliosis.

Description

MEMBRANE IMMUNOASSAYS FOR DETECTION OF MULTIPLE TICK-BORNE DISEASES

Technical Field The present invention relates generally to a membrane and diagnostic method for detecting multiple tick-borne and/or other vector-borne diseases, and more specifically, to a membrane immunoassay and method of using same for simultaneously detecting Lyme disease and additional tick-borne diseases.

Background Art

Lyme disease is a progressive, systemic infection caused by the spirochete Borrelia burgdorferi. The disease is transmitted to man by the bite of the deer tick (Ixodes scapularis and other species). Diagnostic tests for Lyme disease rely mainly on the detection of human antibodies to spirochaetal antigens. The principal test used for screening human sera for antibodies to the Lyme spirochete is enzyme-linked immunosorbent assay (ELISA). Due to the significant inaccuracies inherent in ELISA, sera which are ELISA-positive or indeterminate are often subjected to a confirmatory test. The confirmatory test now in most common use and officially recommended by the U.S. Centers for Disease Control (CDC) is the Western blot. In a conventional Western blot assay, antigens of a given pathogen are resolved into discrete bands on the surface of a paper-like nitrocellulose membrane. The serum to be tested is allowed to react with the antigen bands, and serum antibodies which bind specific bands are detected with a labeled anti-human antibody reagent. Typically, results of the Western blot test appear as a series of bands on a membrane strip. The pattern of bands is compared with the band pattern of known positive sera to produce a diagnostic result. The exact position of bands, and the number of bands which correlate with positivity, differ depending on the pathogen.

Considerable effort has been expended to develop new and improved diagnostic tests for Lyme disease. For example, U.S. Patent No. 5,187,065 discloses methods of detecting Lyme disease in mammals that otherwise show seronegativity due to the generation of immune complexes which "hide" the antibodies raised to the spirochete; disassociation of such complexes followed by immunological assay procedures such as ELISA are described. U.S. Patent No. 5,470,712 provides bioassays which incorporate non-flagellar B. burgdorferi proteins, or antibodies raised to such proteins, to create an assay where such proteins or antibodies are bound to a surface and form complexes with certain components of the serum. Similarly, U.S. Patent No. 5,308,753 teaches the formation of assays which may be used to diagnose Lyme and other diseases which induce primary or secondary IgM antibody- mediated immunity. U.S. Patent No. 5,217,872 teaches a method of detecting B. burgdorferi antigens through an assay which utilizes vesicle proteins released from the spirochete, while U.S. Patent Nos. 5,494,797 and 5,324,630 teach the detection of the Lyme spirochete via oligonucleotide probes. U.S. Patent No. 4,888,276 describes a reliable, noninvasive method for detecting antigens of B. burgdorferi from the urine of affected individuals and U.S. Patent No. 5,155,022 teaches an improved method of assaying for Lyme disease by eliminating crossreacting antibodies. Both U.S. Patent Nos. 4,859,419 and 5,100,626 provide apparati that are able to assay multiple samples for a specific disease such as Lyme. However, none of these patents teach a method of simultaneously assaying for Lyme disease and other diseases.

The same Ixodes ticks which transmit Lyme borreliosis disease to humans also transmit babesiosis, an underdiagnosed parasitic infection which may have serious consequences. In the United States, the major pathogen is the protozoan Babesia microti; in Europe and other countries, other species of Babesia including B. divergens, B. bovis and B. bigemina, all known pathogens of cattle, have been implicated. Despommier, D. et al., Parasitic Diseases, Springer- Verlag, New York (1995). Human granulocytic ehrlichiosis (HGE), caused by a rickettsial Ehrlichia species, is another disease which the Ixodes ticks may transmit. It should be noted that individuals at risk for tick-borne diseases may acquire coinfections with Babesia microti, HGE, and Borrelia burgdorferi. In endemic areas of the United States, mainly the northeast and upper midwest, up to 48% of the mice on which deer ticks feed were found seropositive for mor than one tick-borne pathogen, B. burgdorferi, HGE, E. chaffeensis, and B. microti. Magnarelli, L. et al., J. Wildlife Dis., 33(3):466-473 (1997). Up to 25% of ticks in certain geographical regions of the northeastern U.S. were found to contain the HGE agent by PCR, and 4% of the ticks were PCR-positive for both B. burgdorferi and the HGE agent. Chang,Y.-F. et al., J Vet. Diagn. Invest, 10:56-59 (1998). The frequency of coinfections of tick-borne diseases in humans has been reported to be between 4% and 26%. For instance, B. burgdorferi/RGE coinfections were detected in 4% of a group of Danish patients, Lebech, A.-M. et al., Scand. J. Infect. Dis., 30:173-176 (1998), and 5% of patients were detected with Lyme borreliosis in the upper midwest of the United States. Mitchell, P.D. et al, J. Clin. Microbiol, 34:724-727 (1996). In the same group of midwestern patients, 2% were found to be seropositive for the three pathogens B. burgdorferi, HGE and B. microti. An earlier study in the northeastern U.S. found B. burgdorferi/HGE/B. microti coinfections in 1% of patients in a highly endemic region. Benach, J.L. et al., J. Infect. Dis., 152:473-477 (1985). In Connecticut, 26% of a group of patients with symptoms of HGE infection were found seropositive for more than one tick-borne pathogen, including B. burgdorferi, HGE, E. chaffeensis and 5. microti. Magnarelli, L. et al., J. Clin. Microbiol, 36(10): 2823-2827 (1998). Another study in Connecticut found approximately 10%of patients with Lyme disease to be seropositive for B. microti. Krause, P.j. et al., J Clin. Microbiol, 29: 1-4 (1991). In addition, a babesiosis-like illness in the northwestern United States has been attributed to an unidentified Babesia-like organism, thus far termed WA1. Quick, R. et al, Annals of Intern. Med., 119:284-290 (1993). Less information, however, is available on coinfections in European foci of borreliosis. Preliminary seroprevalence studies on Borrelia/HGE coinfections on the eastern border of Germany suggest a frequency approaching 10% amongst human borreliosis patients. Levin, A.E. and T. Talaska, Serologic Evidence of Coinfection with HGE and Borrelia burgdorferi in Germany, abstract presented at the First Congress of the European Society for Emerging Infections, Budapest, (1998).

The clinical symptoms and severity of babesiosis cover a wide spectrum. While the disease is subclinical in most cases, it may be severe to fatal in others. Telford, S.R. Ill et al., Topley and Wilson's Microbiology, in press; Herwaldt, B.L. et al., Am. J. Trop. Med. Hyg. 53(2):146-151; Garcia, L.S. et al., Diagnostic Medical Par asitology, Washington, D.C., p. 131-135 (Second Edition, 1993); Boustani, M.R. et al., Am. J. Respir. Crit. Care. Med. 149:1689-1691 (1994); Horowitz, M. et al., Chest 106(4):1299-1301 (1994); Rosenbaum, G.S. et al, Clin. Infect. Dis. 20:203-204 (1995); Machtinger, L. et al., J. Clin. Apheresis 8:78- 81 (1993). Splenectomy, immunosuppression, and advanced age are significant negative prognostic indicators. Early symptoms may appear within one to several weeks post- infection, and typically include malaise, anorexia, and fatigue. In susceptible individuals, these progress quickly to more serious symptoms, including fever up to 40 °C, sweating, myalgia, nausea, vomiting, headache, shaking chills, emotional lability and depression, hemoglobinuria, hyperesthesia, and pulmonary edema. Blood analyses may reveal anemia, thrombocytopenia, and low white blood cell count, while lactic dehydrogenase, bilirubin and transaminases may appear at elevated levels. The wide range in symptoms makes the clinical diagnosis of babesiosis difficult, and additionally so, in view of the possibility of either confusion or coinfections with Lyme disease and/or ehrlichiosis. Co-infected patients may be subject to more severe illness than caused by either pathogen alone. Significant frequencies of co-infection have been reported in areas endemic for babesiosis and Lyme disease. Mitchell, P.D. et al., J. Clin. Microbiol. 34:724-727 (1996). Both diseases appear to be rising in incidence, perhaps due to changes both in public awareness of tick-borne diseases and in the interactions of man and the surrounding natural environment. Because therapeutic treatment and prognosis differ for the various diseases, accurate diagnosis is essential for successful clinical management of the patient.

Babesia infects and multiplies within the erythrocytes of the host and thus laboratory testing for babesiosis has traditionally been based on examination of Giemsa-stained blood smears. Telford, S.R. Ill et al., Topley and Wilson's Microbiology, in press; Diagnostic Medical Par asitology, Washington, D.C., p. 131-135 (Second Edition, 1993). Babesia may be visualized in parasitized erythrocytes as pear-shaped piroplasms (hence the common name for babesiosis, "piroplasmosis") or rings, and infrequently as tetrads (maltese cross forms) which are considered as definitive evidence of infection. Parasitemia in infected individuals may vary between l-20%>, while in splenectomized patients, it may attain 85%>. Nevertheless, low level parasitemia is common enough so that failure to observe the parasite in blood smears does not prove the absence of infection with Babesia. Babesia may also be revealed by inoculation of patient blood samples into hamsters, which develop high levels of parasitemia; however, this technique may require up to 6 weeks to yield detectable results.

Serology provides a useful diagnostic approach for babesiosis. Telford, S.R. Ill et al., Topley and Wilson's Microbiology, in press; Diagnostic Medical Parasitology, Washington, D.C., p. 131-135 (Second Edition, 1993). IgM and IgG antibodies to Babesia are produced by infected individuals, and may be detected where direct visual evidence of parasitemia is lacking. An indirect immunofluorescence assay has been developed and has been applied as a diagnostic method. Krause, P.J. et al., J. Infect. Dis. 169:923-926 (1994); Chisholm, E.S. et al., Am. J. Trop. Med. Hyg. 27:14-19 (1978); Chisholm, E.S. et al., Am. J. Trop. Med. Hyg. 35:921-925 (1986). Immunofluorescence testing of babesiosis patient sera has shown very little crossreactivity between B. microti and the WA1 strain found in some Washington State patients. Telford, S.R. Ill et al., Topley and Wilson's Microbiology, in press; Quick, R.E. et al., Ann Intern. Med. 119(4):284-290 (1993).

In another diagnostic method, Babesia DNA is detected by polymerase chain reaction (PCR). Persing, S. et al, BioTechniques 17:788-791 (1994). While the sensitivity of PCR is one of its main advantages, PCR at present remains a technique beyond the expertise of the average diagnostic laboratory. In inexperienced hands, PCR may lead to an inaccurate diagnosis.

Human ehrlichiosis, a potentially life-threatening disease, on the other hand, is caused by infection with rickettsiae of the genus Ehrlichia. Bakken, J.S. et al., JAMA, 275:199-205 (1996). Ehrlichiae are obligate intracellular gram-negative bacteria. Two types of ehrlichiosis have been identified, monocytic, caused by E. chaffeensis, and granulocytic, caused by HGE. First identified as a distinct ehrlichial disease in 1994, Dumler, J.S. and J.S. Bakken, Clin. Infect. Dis., 20:1102-1110 (1995), HGE is caused by an as yet unidentified organism of the E. equi/E. phagocytophila genogroup, which primarily infect horses, dogs and cattle. In the United States, approximately 200 cases of HGE have been diagnosed since 1994. Chen, S.M. et al., J. Clin. Microbiol., 32:589-595 (1994). The geographic distribution of human infection closely parallels that of Lyme borreliosis, with greatest concentration in the northeast, upper midwest and northern California. Closely related or antigenically crossreactive Ehrlichiae have been reported to be associated with infections of humans and animals in Switzerland, Sweden, Norway, Germany and other countries.

Upon infection of the host, the agent of HGE invades granulocytes in the blood and forms morulae, or optically dense intracellular inclusions. Goodman, J.L. et al., N. Engl. J. Med., 334:209-215 (1996). Various organs and tissues may be foci of infection, frequently liver, spleen, bone marrow and lymph nodes. The exact mechanism of pathogenesis remains unclear. The host immune defense systems appear to be impaired in susceptible individuals, many of whom contract lethal opportunistic infections with pathogens including Aspergillus, Cryptococcus, Candida, and Herpes. Chen, S.M. et al., J. Clin. Microbiol., 32:589-595 (1994). As opposed to Lyme disease, typically a chronic illness, ehrlichiosis is frequently manifest as an acute, febrile illness appearing within days of infection. Clinical symptoms include fever, chills, malaise, myalgias, headaches, nausea, vomiting, cough, and confusion, in approximate order of frequency. Leukopenia, anemia or thrombocytopenia are observed in most cases. With an estimated fatality rate as high as 7-10%, Bakken, J.S. et al., JAMA, 275:199-205 (1996), HGE represents one of the most dangerous tick-borne diseases in North America. Prompt therapy with oral or intravenous doxycycline has been shown effective in combatting acute ehrlichiosis, generally resulting in defervescence within 24-48 hours of initiation of treatment. The fact that the swift downhill course of disease can be reversed with prompt antibiotic therapy provides the rationale for rapid diagnosis of suspected cases. Several test methodologies are presently available for HGE. Humans infected with HGE develop an immune response which can be detected by serologic assays including immunocytology, immunofluorescence and immunoblotting. Immunofluores-cence tests based on surrogate antigens, e.g., E. equi and E. phagocytophila, Walker, D.H. and Dumler, J.S., Emerging Infectious Diseases, 2(1): 18-29 (1996), have been developed based on the crossreactivity of these species with HGE. These tests require a fourfold rise in titer with a cutoff of 1 : 80 as criteria for positivity. Until recently, true HGE antigen has been largely unavailable, impeding the development of other tests. With the advent of a cell culture method relying on production ofEhrlichia in HL60 cells, additional tests, including immunoblot have become accessible. Patient sera have been shown to react consistently with an antigen band of approximately 44 kDa on HGE immunoblots, providing diagnostic criteria of high sensitivity and specificity. Beyond serology, PCR has been used to detect infection based on amplification of 16s rRNA sequences in blood obtained during acute illness. Confirmation of HGE infection may require a combination of methods, given the rapidity and varying clinical presentation of the disease.

While individual diagnostic methods are available for each of Lyme disease, Babesiosis and HGE, such diagnostic methods tend to be technically difficult, labor intensive, time-consuming, expensive and potentially inaccurate. Moreover, diagnosis of coinfections can require performance of multiple assays, exacerbating these problems.

It would thus be desirable to provide a screening method which can detect coinfections by simultaneously detecting babesiosis, HGE, Lyme disease and additional diseases. It would be preferable to provide a rapid and accurate screening method for simultaneously detecting at least babesiosis, HGE, Lyme disease. It would also be desirable that such a method have the capability of being automated.

Summary of the Invention The present invention, in accordance with one embodiment, provides a membrane immunoassay and method of using same for the simultaneous detection of antibodies to multiple tick-borne agents of, for example, babesiosis, HGE, and Lyme disease. The immunoassay, in an embodiment, is based on recombinant protein antigens specific to certain tick-borne agents, which protein antigens have been applied to a nitrocellulose membrane, or other applicable membrane made from, for instance, nylon, polyvinyldifluoridene or a mixture thereof. In one embodiment of the invention, the protein antigens may be applied to the nitrocellulose membrane as individual stripes containing a B. microti-specific antigen, an HGE-specific antigen, or a B. burgdorferi-specific antigen. In the event there are several B. microti-specific antigens, several HGE-specific antigens or several B. burgdorferi-spccific antigens, a mixture of the species-specific antigens may be provided in one stripe. In general, a nitrocellulose membrane is prepared with one stripe containing B. burgdorferi antigens, one stripe containing B. microti antigens, and one stripe containing HGE antigens.

The membrane, in an embodiment, may be provided within an apparatus for use in a flow-through assay. Such an apparatus includes an upper plate having a plurality of channels extending from a first surface to a second surface of the upper plate, a lower plate for receiving the upper plate, and a wicking member positioned between the upper and lower plate. The membrane is designed to be placed between the upper plate and the wicking member, such that when fluid, such as serum samples and reagents, are added on to the membrane, the wicking member may act to distribute and absorb the liquid substantially uniformly through the membrane. The apparatus, in one embodiment, includes a vacuum port coupled to the lower plate, so that vacuum pressure may be used to facilitate the flow- through process.

The membrane produced in accordance with an embodiment of the invention, may be used in a flow-through immunoassay, in which samples and reagents are incubated briefly on the membrane, then permitted to flow through the membrane. If subsequently there is an appearance of a colored stripe on the membrane surface, such a stripe is indicative of a positive test result for the corresponding antigen.

Brief Description of the Drawings

The various advantages of the present invention will become apparent to one skilled in the art by reading the following specification and appended claims and by referencing the following drawings in which:

Fig. 1 illustrates an immunoassay membrane in accordance with an embodiment of the present invention.

Figs. 2A-C illustrate representative results from tests of serum samples, some of which are positive for various combinations of B. burgdorferi (Lyme), HGE and Babesia antigens.

Fig. 3 illustrates a cassette for use with one embodiment of an immunoassay of the present invention. Fig. 4 illustrates an instrument for performing rapid membrane flow-through immunoassays.

Fig. 5 illustrates the specificity results for normal serum samples.

Fig. 6 illustrates the sensitivity of detection results for HGE and B. burgdorferi antibodies.

Fig. 7 illustrates the sensitivity of detection results for 5. microti antibodies.

Detailed Description of Specific Embodiments A membrane panel for the simultaneous detection of antibodies to the agents of multiple tick-borne and/or other vector-borne diseases, and methodologies for detecting same are provided.

In Fig. 1, a membrane 10 for use in connection with a flow-through membrane immunoassay is shown. The membrane 10, in a preferred embodiment, includes at least three recombinant protein antigens for the detection of antibodies to the agents of babesiosis, HGE, and Lyme disease respectively. These recombinant protein antigens, namely Babesia microti, HGE and Borrelia burgdorferi, can be immobilized as separate colorless stripes 12 directly on the membrane using methods known in the art (discussed below). An additional stripe containing, for instance, an anti-human IgG antibody, may be provided on the membrane as a control band (not shown). The anti-IgG antibody in this control band, when bound to antibody in the sample, serves as an indicator showing whether the sample contains IgG antibody in sufficient quantity to be detected. The band of antibody may also serve as verification that the assay procedure functioned properly (e.g., that the reagents have been added and that they are active). The band of antibody may further serve to verify that a sample has indeed been added to the membrane. It should be noted that the stripes 12 and the control band are generally colorless prior to performing the immunoassay. In addition, although individual striping patterns are discussed in connection with the membrane of the present invention, the antigens used herewith may be applied to the membrane in any applicable pattern, for example, a dot or a square.

The membrane 10 of the present invention may be a single strip of nitrocellulose paper for testing one serum sample, or a panel 14 having a plurality of strips 16 for testing multiple samples at once. Other materials, of course, may be used for making the membrane 10, examples of which include but not limited to, nylon, polyvinyldifluoridene, or a mixture thereof. In accordance with an embodiment of the present invention, the membrane may be provided with eight strips 16 to permit, for example, eight different samples to be tested at a time.

The membrane immunoassay, in one embodiment, is similar in principle to conventional ELISA, with the exception that a membrane is substituted for the plastic microplate, and the antigen is applied to the membrane as, for example, a stripe. The application of the antigen stripes to the membrane is known in the art. For example, the antigens may be solubilized in a Tris-HCl/SDS/Bromophenol Blue buffer and applied in stripes to a nitrocellulose membrane using a BioDot™ instrument, an Immunetics Miniblotter®, or manually with a calligraphy pen. Other methods are disclosed in Aeppli et al., J. Immunol. Methods 120: 93-98, (1989), UK Patent Application GB 2 099 578 A (Gordon et al.), U.S. Patent No. 4,031,197 (Marinkovich), Pollet, et al, Clin. Chem., 37(10):1700-1707 (1991), Hickman et al., J Virol. Methods, 72: 43-49 (1998), U.S. Patent No. 5,656,503 (May et al. ). Aeppli, for instance, makes use of an instrument from Camag (the Linomat IV) to spray antigens on to the membrane under nitrogen pressure. Marinkovich and Hickman, on the other hand, use multichannel devices for passive adsorption of antigens onto the membrane in stripes. Hickman, in particular, uses an Immunetics Miniblotter® for this purpose. May et al. disclose a variety of techniques for striping reagents on to nitrocellulose membranes for use in lateral flow immunochromatography assays.

The membrane immunoassay of the present invention generally comprises initially introducing a serum sample, which may contain antibody specific to the antigen of interest, directly to a membrane 10. The membrane 10 is then washed to remove unbound antibody. Antibody which is bound to the antigens on the membrane is detected by incubation with a second, anti-human immunoglobulin antibody. This second antibody is linked to an enzyme, such as alkaline phosphatase, which reacts with an applied substrate to yield a colored product from a colorless one, or by converting a nonfluorescent substrate into a fluorescent one. In using this immunoassay, the presence of a particular antibody is indicated by deposition of a colored, insoluble reaction product on the membrane corresponding to the position at which the antigen is placed. In other words, the results appear as a colored stripe (Figs. 2A-C), where antigen is applied on to the membrane as a stripe. Although immunoassays are typically based on unfractionated antigens and may not provide the means to distinguish between antibody reactions with antigen fractions, which are either more or less specific to a given pathogen, the membrane immunoassay of the present invention is sufficiently accurate, as will be discussed hereinafter in detail, to be used in the screening and detection of multiple tick-born diseases. In particular, the analysis of iføbeszα-positive, HGE- positive, and B. burgdorferi-positive samples to obtain sensitivities of the present immunoassay, as compared to traditional Western Blot, provides sensitivity of detection results of 100% for HGE, 96%> for Babesia, and 88% for B. burgdorferi. Moreover, as the agents of babesiosis, HGE, and Lyme disease may be transmitted by vectors other than ticks, for instance, mosquitos, the present invention is also designed for detection of such agents.

Looking now at Figs. 3-4, in accordance with one embodiment of the invention, the immunoassay is performed by placing an eight-strip membrane panel 10 into a cassette 20 having eight channels 22 corresponding to the eight strips on the membrane panel 10. The cassette 20, in an embodiment, includes an upper plate 24, a lower plate 26 capable of receiving the upper plate 24, and a sheet of wicking paper 28 positioned between the upper and lower parts. The membrane 10 is placed over the wicking paper 28 and the cassette upper plate 24 placed within recess 29 of the lower plate 26. To initiate the immunoassays, the assembled cassette 20 is wetted thoroughly by dipping into a tray containing distilled water or a wash buffer, such as phosphate-buffered saline with 0.05%> Tween-20 detergent.

Once the wicking paper 28 and membrane 10 have become uniformly wet (in several minutes), the cassette 20 may be transferred to between panels 32 and 33 of instrument 30 (Fig. 4) for performing rapid membrane flow-through immunoassay. One such instrument is the CodaXcel™ from Immunetics, Inc. in Cambridge, Massachusetts. The instrument 30 can either include a mechanical system or a vacuum locking system, which can be activated to exert a downward pressure against the cassette 20. The pressure exerted by the instrument 30 on the cassette 20 effectively causes the cassette 20 to form a liquid-tight seal between the cassette channels 22. A rocking platform 34 on the instrument 30 may also be activated at this time, if desired, to rock the cassette 20. Although a rocking motion can be used, the present immunoassay may be performed without the activation of the rocking platform 34.

Samples of serum, plasma, or other fluids are then added to each channel 22 with a pipet. Typically, serum or plasma is diluted 1 : 100 in a buffer containing phosphate-buffered saline and 0.05%> Tween-20, but dilutions can vary between 1:1 and 1 :10,000 depending on the type of sample and the relative concentration of antibodies in the test sample. Cerebrospinal fluid, for instance, may be tested at a dilution of 1 : 1 , since antibody concentrations are generally much lower than those of serum, while a sample of purified polyclonal or monoclonal antibody may require dilution in excess of 1:10,000 to yield a concentration appropriate for the assay. The volume of test sample, and of all other solutions and reagents used in the following steps, is approximately 0.5 ml per cassette channel. Smaller or larger volumes may also be used, with moderate effects upon the assay performance. The test samples (e.g., serum or plasma) are next incubated in the cassette for about 4 minutes, after which they may be permitted to flow through the membrane. It should be noted that the time period disclosed hereinafter may be varied in all steps. However, such variance may affect the assay sensitivity. Additionally, the use of wicking member 28 can permit substantially uniform distribution of liquid through the membrane 20. Specifically, the wicking member 28, which may be a filter paper or other absorbent material, can act to pull liquid substantially uniformly along the surface of the membrane 20 and through the membrane 20. To further facilitate the flow-through process, the sample may be aspirated through the membrane 10 by the use of a vacuum. In such a case, the lower plate 26 is designed to permit coupling of the cassette 20 to a vacuum source. Specifically, the lower plate 26 may include a plurality of channels (not shown) similar to channels 22 of the upper plate 24, or a vacuum port (not shown) to permit engagement of the lower part to a vacuum source.

Wash buffer, such as phosphate-buffered saline with 0.05%> Tween-20, or similar solution, is then added to the cassette channels 22 using a pipette or an 8-chaιmel manifold. The buffer is thereafter aspirated immediately through the membrane 10, and replaced with fresh buffer twice. After the final aspiration, a detection reagent is added to the cassette channels 22. The detection reagent may be, for example, horseradish peroxidase-conjugated goat anti-human IgG, alkaline phosphatase-conjugated goat-anti-human IgG, horseradish peroxidase-conjugated Protein A or Protein G, or similar enzyme conjugates using various enzymes and specific for different immunoglobulin types. In addition to enzyme conjugates, the detection reagent may also be a colloidal gold-antibody or other colloidal conjugate, a radioactively labeled antibody, or a wide variety of other reagents. In accordance with one embodiment, alkaline phosphatase-conjugated goat anti-human IgG is added and incubated for about 4 minutes, then aspirated through the membrane. Following this step, wash buffer is added once and aspirated immediately, followed by addition of distilled water. After the distilled water has been aspirated, an enzyme substrate is added.

When alkaline phosphatase is used, the enzyme substrate that is added may be 5- bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. When horseradish peroxidase is used, the enzyme substrate that is added may be tetramethylbenzidene or 4-chloro-l- napthol, both of which are available commercially as stabilized solutions. The substrate is incubated for about 2 minutes, then aspirated through the membrane. Following a final wash with distilled water and aspiration, the cassette 20 is removed from the instrument 30 and opened to release the membrane 10. The membrane 10 is removed and allowed to dry at room temperature.

A complete immunoassay can be performed in approximately 15 minutes with this method, instead of several hours, as seen with conventional Western Blot methodology.

In another embodiment, the immunoassay may be performed without the use of the instrument 30 for performing rapid flow through immunoassay. The following protocol provides an example of such a method. Place a single strip of membrane into each channel of the cassette and exerting pressure on the cassette to effectively cause the cassette to form a liquid-tight seal between the cassette channels. Dispense 0.5-1.0 ml of wash buffer into each channel. Thereafter, place the cassette on a rocking platform and incubate for about 1 minute to thoroughly wet the strips. All incubations on the rocking platform are performed at the speed of 10-15 cycles/min. Aspirate the liquid completely from individual channels using a disposable pipet tip connected to a vacuum system (a water aspirator with a trap is sufficient). Tilting the cassette to empty channels should be minimized, as this could cause liquid to cross over and mix with adjacent samples during the subsequent steps. Load samples to be tested immediately, in order to avoid drying of the strips. Include Positive and Negative Controls in each assay run. Pipet about 0.5-1.0 ml of diluted IgG Negative Control Serum and about 0.5- 1.0 ml of diluted IgG Positive Control Serum, including antibodies reactive with B. burgdorferi, HGE, and B. microti antigens present on the membrane into separate channels. Pipet about 0.5-1.0 ml of diluted serum sample into the appropriate numbered channel corresponding to the sample sequence in the protocol. Incubate on the rocking platform for about 30 minutes. Aspirate liquid from each channel by vacuum as described above.

Rinse the strips three times by adding approximately 0.5-1.0 ml wash buffer to each channel, rocking for about 3 minutes, then aspirating the liquid by vacuum. To avoid cross- contamination, be careful not to overfill the channels. Alternatively, use an eight-channel Immunowash (Immunetics LD-0512S, Nunc or equivalent). This will also speed up the procedure. Load about 0.5-1.0 ml of diluted Enzyme Conjugate into each channel and incubate on the rocking platform for about 15 minutes. Aspirate the conjugate and rinse the strips once with wash buffer as described above. Rinse the strips twice with about 0.5-1.0 ml of deionized water per channel and incubate on the rocking platform for about 3 minutes each rinse. Aspirate all liquid from the channels. Add about 0.5-1.0 ml of Enzyme Substrate Solution containing BCIP/NBT to each channel (a multichannel pipettor is suggested for this step to start the enzymatic reaction at the same time in all channels). Place the cassette on the rocking platform to initiate color reaction. Allow the color reaction to develop until the positive control serum immunoreactive bands are clearly visible, usually 6-7 minutes. Do not incubate longer than 10 minutes. Stop color development by aspirating substrate from the cassette and rinsing the strips immediately with two brief changes of distilled or deionized water. Transfer the strips face up to a paper towel and let air dry. Do not dry the strips between paper towels or pat dry and do not attempt to interpret results until blot is completely dry.

Reactivity of antibodies in the test samples with specific antigens is indicated by the appearance of colored bands over the position of the corresponding antigen stripes. Thus, a sample can be tested simultaneously for the presence of antibodies to Borrelia burgdorferi , HGE and Babesia microti using this assay. A detectable band is interpreted as a positive assay result. In one embodiment of the invention, an intensity indicator stripe may be provided on the membrane, so as to permit determination of a threshold level for positive results of each pathogen. Band results may be read manually, for instance, by eye, or be read by an automated device. For example, the bands on the membrane may be scanned by a CCD camera or other device for digital image acquisition and interpretation by software algorithms.

The presence of antibodies to one of the above pathogens suggests exposure to the pathogen, and may be indicative of an active or previous infection. Test results, in conjunction with other clinical data, may thus be used to aid in the diagnosis of Lyme disease, HGE, babesiosis, or combinations of these diseases in a human or animal patient.

The present invention also provides kits comprising a membrane of the present invention. The kits may also include reagents, as well as apparatus for performing the methods described herein. Examplifϊcation

The invention, now being generally described, will be more readily understood by reference to the following specific examples, which are provided merely for purposes of illustration of certain embodiments of the present invention. These examples are not intended in any way to limit the scope of the claimed invention.

Materials and Methods Recombinant Antigens

Purified recombinant antigens from Borrelia burgdorferi, Human Granulocytic Ehrlichiae (HGE), and Babesia microti were obtained for use in connection with the immunoassays and methods of the present invention.

A. Recombinant Babesia and HGE

Recombinant antigens derived from Babesia microti and human granulocytic Ehrlichiae (HGE) were provided by Corixa, Inc. Babesia microti recombinant antigens represented immunodominant sequences of unknown relationship to native B. microti proteins. The recombinant HGE antigen was a 44 kDa protein. The strategy employed to isolate these novel immunodominant antigens relied on creation of genomic expression libraries from pathogen-infected cells (Corixa, Inc.). Sheared DNA from infected HL60 (HGE) or hamster red blood cells (Babesia) was incorporated into the Lambda Zap π/EcoRl/CIAP vector. Each library was screened using either human Babesia-positive serum or HGE-infected mouse serum. Phagemid was obtained from positive plaques and sequenced using forward, reverse, and internal primers. Twenty (20) Babesia clones and eighteen (18) HGE clones were obtained and expressed in pET 17b constructs and purified by affinity chromatography using nickel affinity columns. Purified protein was used in ELISA and Western Blot format to determine immunological reactivity with Babesia- and HGE-positive sera. Three Babesia-spεcific recombinant antigens, BMNI-2, BMNI-17, and MN-10, and two HGE-specific recombinant antigens, HGE-1 and HGE-3 were evaluated.

A recombinant 44-kDa major outer membrane protein of HGE was obtained from Dr. Yasuko Rikihisa at Ohio State University. This protein has been shown to be the major antigen recognized by sera of HGE-infected humans. To produce this antigen, purified HGE genomic DNA was digested and ligated into the Lambda ZAPII vector. The gene library was constructed by infecting E. coli Xll-Blue MRF with recombinant phage. Clones expressing Ehrlichia proteins were identified with equine anti-HGE serum. Positive recombinant phagemids were used to transform E. coli SOLR cells, and positive clones were analyzed by Western Blotting with the equine anti-HGE serum. DNA and amino acid analysis indicated the isolation of the 44-kDa protein (p44) of the HGE agent. A 219 amino acid polypeptide from the NH₂ terminus of p44 was subcloned into the pET30a expression vector. This recombinant antigen was used for the tick-borne disease panel test.

B. Recombinant B. burgdorferi.

Borrelia burgdorferi antigens included recombinant OspC, OspA, p39, flagellin (p41), and p93. OspC and p41(internal fragment), recombinant antigens were obtained from Mikrogen GmbH (Munich, Germany). Both OspC and p41 (flagellin) are well-studied antigens used in various commercial and non-commercial diagnostic immunoassays.

Recombinant p35-VlsE was obtained from Dr. Steven Norris at Texas A&M University. This antigen appears to be expressed in vivo but not in spirochetes grown in vitro, and according to recent reports may be a promising candidate antigen for serodiagnosis of Lyme disease.

Native Antigens

In addition to the recombinant antigens above, native B. burgdorferi, HGE, and B. microti antigens were also obtained.

A. Native HGE

Native HGE antigen was obtamed from Dr. J. Stephen Dumler. The antigen was prepared by culturing a human isolate of HGE in HL60 cells followed by renograffm gradient purification of the Ehrlichiae. The resulting material was solubilized in SDS sample buffer for electrophoresis and Western Blotting.

B. Native Babesia

Native Babesia microti antigen was provided by Dr. Sam Telford in the form of parasitemic hamster erythrocytes. Hamsters were infected with B. microti (strain GI) and maintained until maximal parasitemia (30-70%), then bled by cardiac puncture. Erythrocytes were harvested by centrifugation, washed and resuspended in Hank's Buffered Salt Solution as for preparation of immunofluorescence slides. For SDS-PAGE and Western Blotting, erythrocytes were solubilized in SDS sample buffer. C. Native B. burgdorferi

B. burgdorferi antigen, strain B31 (low passage) was provided from Immunetics, Inc.'s stock. Spirochetes grown in BSK-II medium were harvested, washed in phosphate-buffered saline, and sonicated. The resulting suspension was solubilized in SDS sample buffer for SDS-PAGE and Western Blotting.

Preparation of Membranes

Recombinant antigens solubilized in a Tris-HCl buffer containing SDS and Bromophenol Blue were applied in stripes to nitrocellulose membranes (e.g., 0.2 nitrocellulose membranes) obtained from Schleicher & Schuell. The membranes were formatted with a size of approximately 5 X 10 cm to fit within a cassette for use with a CodaXcel™ instrument. The CodaXcel™ is an instrument designed for performing rapid membrane flow-through immunoassays, and can be obtained from Immunetics, Inc. in Cambridge, Massachusetts. In general, the nitrocellulose membranes were prepared with one stripe containing B. burgdorferi antigens, one stripe containing an HGE antigen, and one stripe containing B. microti antigens. Individual stripes, however, may contain mixtures of B. microti-specific, HGE-specific or 5. burgdorferi-specific antigens. An additional stripe containing an anti-human IgG antibody, as a control band, was also added to the membrane. The control band was intended to act as an internal control, for verifying to the user that the test sample used contained detectable levels of antibody and that the assay functioned as expected. Stripes were applied to the membranes using, for example, a BioDot™ instrument, an Immunetics Miniblotter® , or manually with a calligraphy pen. The membranes were dried at room temperature and stored for later use.

Western Blotting

Native antigens of Babesia, HGE, and B. burgdorferi were used in the preparation of Western Blots using modifications of the Laemmli SDS-PAGE and electroblotting methods. Western Blots were used to confirm/validate the results of the immunoassays of the present invention. In general, the sample is applied to a polyacrylamide gel and the proteins separated through the technique of gel electrophoresis. The proteins, which have been separated into discrete bands, are subsequently transferred to a sheet (e.g., nitrocellulose) by way of a blotting chamber. Once the protein bands have been transferred, the blot is treated with antibody specific to the particular antigen of interest (e.g., Babesia, HGE, and B. burgdorferi); if the antigen is present, the antibody will bind to the antigen. Free antibody is washed away, the blot is treated with a second antibody which is capable of binding to a site on the first antibody, and the blot is rinsed again to remove excess antibody. In order to detect binding, the second antibody may carry a radiolabel or may be linked to an enzyme as in an ELISA technique. The enzyme linked to the antibody may then in turn react with a substrate applied to the blot which, for example, generates a colored product. In the case of a radiolabel, the bands may be visualized through the technique of autoradiography, where the radioactive blot is exposed to photographic film for a time sufficient to visualize the protein band or bands of interest. The presence of very small quantities of antigen may thus be detected due to the highly sensitive nature of the Western blotting technique, and hence its value as a confirmatory test.

Assay Optimization

Serum samples of known immunoreactivity to Babesia, HGE, and B. burgdorferi- specific antigens were used to optimize the immunoassay. Optimal concentrations of Babesia, HGE, and B. burgdorferi antigens, as well as serum and enzyme conjugates were determined by varying these parameters independently in test runs on positive and negative serum standards. Antibody detection was effected using a mixture of goat anti -human IgG and goat anti-human IgM, both conjugated to alkaline phosphatase. IgG- and IgM-specific conjugates were mixed to maximize the likelihood of detecting an antibody-positive sample irrespective of isotype. Isotype specificity could be determined, if necessary, by re-testing a positive specimen on an isotype-specific immunoblot .

Membranes were examined visually for optimal signal to background ratio. Sensitivities and specificities were determined by testing a limited number of sera against each individual protein antigen. Combinations of species-specific antigens in a single stripe were then tested to increase sensitivity in some cases. Results of sensitivity and specificity values based on combinations of species-specific antigens for detection of antibodies to B. burgdorferi and Babesia, and a single antigen for HGE were obtained. The assay conditions selected were as follows:

• Serum dilution = 1 : 100

• Alkaline phosphatase conjugated goat anti -human IgG dilution = 1 :8000

• Alkaline phosphatase conjugated goat anti-human IgM dilution = 1 :4000

• Antigen composition and concentrations: Babesia antigen combination: BMNI-2 2 g/ml

BMNI-17 2 g/ml

MN-102 g/ml

HGE antigen:

44 kDa40 g/ml

B. burgdorferi antigen combination:

OspC 100 g/ml p35-VlsE 5 g/ml ifabeszα-specific proteins were combined into one stripe, as were B. burgdorferi-specific proteins.

Serum Samples

Thirty-eight (38) serum samples, including twenty four (24) sera from babesiosis patients which tested positive by IF A, PCR, or blood smear were provided by Drs. Peter Krause and Raymond Ryan of the University of Connecticut Hospital (Krause/Ryan Serum Samples).

Twenty-one (21) serum samples which tested positive by Western Blot for Lyme disease were purchased from New York Biologies of Southampton, NY (NYB Serum Samples).

Twenty (20) presumed HGE -positive serum samples were provided by Dr. J. Stephen Dumler of Johns Hopkins University (Dumler Serum Samples). Of these, nine (9) were positive by PCR, four (4) by IF A, six (6) by 4-fold seroconversion (tested by IF A), and one (1) by culture.

Normal human sera were obtained from blood donors at Massachusetts General Hospital in Boston, MA and BioClinical Partners, Franklin, MA (Normal Serum Samples). EXAMPLE 1

The membranes of the present invention were used to test representative serum samples from (i) Krause/Ryan Serum Samples, (ii) NYB Serum Samples, and (iii) Dumler Serum Samples, for the presence of antibodies to the agents of babesiosis (Babesia microti), HGE, and Lyme disease (Borrelia burgdorferi). To detect the antibodies, one membrane was inserted into an eight-channel cassette and placed on to an instrument for performing rapid membrane flow-through immunoassay, and the serum samples added, as described above.

Results from several runs of these serum samples, including some which were positive for various combinations of the three pathogens are shown in Figs. 2A-C. Visible bands in each channel indicate a positive reaction of the antibody with one or more of the antigens. The absence of any band denotes a negative result overall. Positive immunoreaction was verified by re-testing that sample on a conventional strip Western Blot, as shown. In other words, if the sample was a positive for Lyme (sample 2 in Fig. 2 A), a Western Blot was performed to verify the presence of antibody to B. burgdorferi in the sample. Similarly, if the sample was positive for Lyme, HGE and Babesia (sample 3 in Fig. 2A), a separate Western Blot was performed to verify the presence of antibody to each of B. burgdorferi, HGE and B. microti in the sample. Negative samples were also re-tested (results not shown). In general, a positive result on the membrane of the present invention is confirmed by the Western Blot.

EXAMPLE 2

Normal serum samples from Massachusetts General Hospital in Boston, MA and BioClinical Partners in Franklin, MA were tested, as controls, using the protocol of Example 1, and the membranes of the present invention. For the forty (40) samples tested, none reacted with any of the three antigens on the membrane of the present invention, providing an overall specificity of 100%. The results obtained with the membrane are indicated under the heading "Tick Screen" in Fig. 5.

EXAMPLE 3

Immunoassays were performed on the Dumler Serum Samples using the membranes of the present invention and the protocol of Example 1 to detect the presence of antibodies to the agents of babesiosis, HGE, and Lyme disease. The results of the assays obtained with the membrane of the present invention are illustrated in Fig. 6 under the heading "Tick Screen". Western Blots were also performed on the Dumler Serum Samples for confirmation of both positive and negative results to HGE and Lyme antibodies.

As shown in Fig. 6, for the twenty (20) Dumler Serum Samples tested, sensitivity of detection by the membrane for HGE antibodies versus that of the Western Blot was 100%. In addition, agreement between the membrane of the present invention and the Western Blot for both positive results and negative results (4) was 100%>. Sensitivity of detection was calculated by dividing the number of positive results detected using the membrane (16) by the number of positive results detected using the Western Blot (16). The agreement, on the other hand was calculated by dividing the total number of agreements (20) between the membrane and the Western Blot by the total number of samples (20).

Sensitivity of detection by the membrane for Lyme antibodies in the Dumler Serum Samples versus that of the Western Blot was 88%. Overall agreement between the membrane and the Western Blot for both positive and negative results was about 85%.

EXAMPLE 4

Immunoassays were performed on the NYB Serum Samples using the membranes of the present invention and the protocol of Example 1 to detect the presence of antibodies to the agents of Lyme disease, HGE and babesiosis. The results of the assays obtained with the membrane of the present invention are illustrated in Fig. 6 under the heading "Tick Screen".

Western Blots were also performed on the NYB Serum Samples for confirmation of both positive and negative results to Lyme antibodies.

As shown in Fig. 6, for the twenty one (21) NYB Serum Samples tested, sensitivity of detection by the membrane for Lyme antibodies versus that of the Western Blot was 86%. In addition, agreement between the membrane of the present invention and the Western Blot for both positive and negative results was 86%>.

EXAMPLE 5

Immunoassays were performed on the Krause/Ryan Serum Samples using the membranes of the present invention and the protocol of Example 1 to detect the presence of antibodies to the agents of babesiosis, Lyme disease and HGE. The results of the assays obtained with the membrane of the present invention are illustrated in Fig. 7 under the heading "Tick Screen". Western Blots were also performed on the Krause/Ryan Serum Samples for confirmation of both positive and negative results to the B. microti antibodies obtained by the membrane immunoassay of the present invention.

As shown in Fig. 7, for the thirty three (33) Krause/Ryan Serum Samples tested, sensitivity of detection by the membrane for B. microti antibodies versus that of the Western Blot was 96%o. In addition, agreement between the membrane of the present invention and the Western Blot for both positive and negative results was 94%>.

Additional confirmation tests, specifically Blood Smear, PCR, and IFA were performed with the Krause/Ryan Serum Samples. For the twenty nine (29) Krause/Ryan Serum Samples used in the Blood Smear confirmation test, the results of which are also shown in Fig. 7, sensitivity of detection by the membrane for B. microti antibodies versus that of the Blood Smear was 100%. The agreement between the two tests was 55%. For the thirty three (33) samples used in the PCR confirmation test, the results of which are also shown in Fig. 6, sensitivity of detection by the membrane for B. microti versus that of PCR was 90%. The agreement between the two tests was 79%>. For the samples used in the IFA confirmation test (not shown), sensitivity of detection by the membrane for B. microti versus that of IFA was 100%) and overall agreement was 97%>.

Discussion

The results shown in the examples above suggest that problems associated with the diagnosis and detection of tick-borne diseases can be overcome with the membrane and method of the present invention. With sensitivity of detection for HGE antibodies at 100%, for B. microti antibodies at 96%, and for B. burgdorferi at 88%>, when compared to that observed with the Western Blot, and specificity of 100% for normal population, the membranes and methods of the present invention have demonstrated that an immunoassay can be provided to satisfy the relatively stringent performance criteria necessary for positive detection of tick-borne diseases.

The membrane, when used in connection with a flow-through immunoassay, as described above, provides a test that is as substantially accurate as the Western Blot, and thus may be used effectively in the serodiagnosis of at least Lyme disease, HGE and babesiosis, the three principal tick-borne pathogens. The membrane can also provide a basis for an immunoassay which can quickly and accurately test multiple serum samples for the presence of multiple tick-borne and/or other vector-borne pathogens. In particular, as there may be instances where pathogens similar to the one disclosed above are transmitted by vectors other than ticks, for example, by mosquitos, the membrane can also be used to detect for the presence of such pathogens. The immunoassay, when performed in accordance with an embodiment of the invention, can be completed in about 15 minutes.

While the membrane and method of the present invention have been described in detail in connection with the tick-borne pathogens B. microti, HGE and B. burgdorferi, it will be appreciated that it is within the scope of the present invention that the membrane and method may be modified to detect antibodies to other pathogens, including Ehrlichia chaff eensis, the agent of Human Monocytic Ehrlichiosis, and Rickettsia rickettsia, the agent of Rocky Mountain Spotted Fever. Other antigens unrelated to tick-borne pathogens, for instance, autoimmune, rheumatoid, viral and other infections, may also be incorporated as appropriate for the differential diagnosis of conditions for which distinction between tick- borne and other etiologies is desired. A combination of antigens for detecting various diseases may be further chosen based on similarity of resulting disease symptoms, known antigenic cross-reactivity or other criteria.

Furthermore, it will be understood that this application is intended to cover any variations, uses, or adaptations of the invention, and including such departures from the present disclosure as come within known or customary practice in the art to which the invention pertains, and as fall within the scope of the invention and the limits of the appended claims.

Claims

What is claimed is:

1. An apparatus for use in a flow-through assay, the apparatus comprising: an upper plate having at least one channel extending from a first surface to a second surface of the upper plate; a lower plate for receiving the upper plate, the lower plate capable of engaging a vacuum source; a membrane positioned between the upper plate and the lower plate, and having a plurality of binding members immobilized thereon at location corresponding to the channel, each binding member being specific for a distinct analyte of interest; and a wicking member positioned between the membrane and the lower plate, the wicking member being capable of distributing and absorbing a liquid substantially uniformly through the membrane.

2. An apparatus as set forth in claim 1 , wherein the binding members are immobilized on the membrane by striping.

3. An apparatus as set forth in claim 1, wherein the membrane includes at least three distinct binding members.

4. An apparatus as set forth in claim 3, wherein each of the binding members is directed to an analyte associated with a tick-borne disease.

5. An apparatus as set forth in claim 4, wherein each binding member includes antigen derived from one of Babesia microti, human granulocytic Ehrlichiae, and Borrelia burgdorferi.

6. An apparatus as set forth in claim 3, wherein at least one of the binding members is directed to an analyte associated with a disease transmitted by a vector other than tick.

7. An apparatus as set forth in claim 1, wherein the membrane further includes a control band having a binding member which when binds to an analyte of interest in a sample serves as an indicator for verifying that a sample has indeed been added to the membrane.

8. An apparatus as set forth in claim 1, wherein the membrane further includes an intensity indicator, so as to permit determination of a positive result threshold level for each analyte of interest.

9. A flow-through assay method comprising: a) providing an apparatus having: an upper plate having at least one channel extending from a first surface to a second surface of the upper plate; a lower plate for receiving the upper plate; and a membrane positioned between the upper plate and the lower plate, and having a plurality of binding members immobilized thereon at a location corresponding to the channel, each binding member being specific for a distinct analyte of interest; b) introducing a test sample through the channels to a surface of the membrane; c) flushing the test sample through the membrane to remove any analytes in the sample not bound to the binding members; and d) analyzing the results presented on the membrane.

10. A method as set forth in claim 9, wherein, in the step of providing, the membrane includes at least three distinct binding members.

11. A method as set forth in claim 10, wherein, in the step of providing, each of the binding members is directed to an analyte associated with a tick-borne disease.

12. A method as set forth in claim 11 , wherein each binding member includes antigen derived from one of Babesia microti, human granulocytic Ehrlichiae, and Borrelia burgdorferi.

13. A method as set forth in claim 10, wherein at least one of the binding members is directed to an analyte associated with a disease transmitted by a vector other than tick.

14. A method as set forth in claim 9, wherein the step of flushing further includes flushing under vacuum.

15. A membrane for use in a flow-through assay, the membrane comprising: at least three binding members immobilized thereon, each binding member being specific for a distinct analyte of interest; and an intensity indicator, so as to permit determination of a positive result threshold level for each analyte of interest, wherein a positive result is indicative of coupling between a binding member to the specific analyte of interest.

16. A membrane as set forth in claim 15, wherein the binding members are immobilized on the membrane by striping.

17. A membrane as set forth in claim 15, wherein each of the binding members is directed to an analyte associated with a tick-borne disease.

18. A membrane as set forth in claim 17, wherein each binding member includes antigen derived from one of Babesia microti, human granulocytic Ehrlichiae, and Borrelia burgdorferi.

19. A membrane as set forth in claim 15, wherein at least one of the binding members is directed to an analyte associated with a disease transmitted by a vector other than tick.

20. A membrane as set forth in claim 15, further including a control band having a binding member which when binds to an analyte of interest in a sample serves as an indicator for verifying that a sample has indeed been added to the membrane.

21. A membrane as set forth in claim 15, wherein the membrane is a nitrocellulose membrane.

22. A method for simultaneously detecting multiple tick-borne diseases in a sample comprising: a) applying the sample to a membrane comprising: at least three different tick-borne antigens immobilized thereon, each antigen being specific for a distinct antibody of interest; and an intensity indicator, so as to permit determination of a positive result threshold level for each antibody of interest; and b) simultaneously detecting binding of each of the tick-borne antigens to the corresponding antibody in the sample.

23. A method as set forth in claim 22, wherein the sample is human serum.

24. A method as set forth in claim 22, wherein in the step of applying, each of the at least three different tick-borne antigens on the membrane includes antigen derived from one of Babesia microti, human granulocytic Ehrlichiae, and Borrelia burgdorferi.

25. A method as set forth in claim 22, wherein in the step of applying, the at least three different tick-borne antigens on the membrane are immobilized thereon by striping.

26. A method as set forth in claim 22, wherein the membrane in the step of applying further includes a control band having a binding member which when binds to an analyte of interest in a sample serves as an indicator for verifying that a sample has indeed been added to the membrane.

27. A diagnostic kit comprising a membrane having at least three binding members immobilized thereon, each binding member being specific for a distinct analyte of interest, and an intensity indicator, so as to permit determination of a positive result threshold level for each analyte of interest, wherein a positive result is indicative of coupling of a binding member to the specific analyte of interest.

28. A diagnostic kit as set forth in claim 27, wherein each of the binding members is directed to an analyte associated with a tick-borne disease.

29. A diagnostic kit as set forth in claim 28, wherein each binding member includes antigen derived from one of Babesia microti, human granulocytic Ehrlichiae, and Borrelia burgdorferi.

30. A diagnostic kit as set forth in claim 27, wherein at least one of the binding members is directed to an analyte associated with a disease transmitted by a vector other than tick.

31. A diagnostic kit as set forth in claim 27, further including on the membrane an intensity indicator, so as to permit determination of a positive result threshold level for each analyte of interest.

32. A diagnostic kit as set forth in claim 27, further including on the membrane a control band having a binding member which when binds to an analyte of interest in a sample serves as an indicator for verifying that a sample has indeed been added to the membrane.