EP2245186A2 - Test rapide incluant une sonde de séquence génétique - Google Patents

Test rapide incluant une sonde de séquence génétique

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Publication number
EP2245186A2
EP2245186A2 EP09732630A EP09732630A EP2245186A2 EP 2245186 A2 EP2245186 A2 EP 2245186A2 EP 09732630 A EP09732630 A EP 09732630A EP 09732630 A EP09732630 A EP 09732630A EP 2245186 A2 EP2245186 A2 EP 2245186A2
Authority
EP
European Patent Office
Prior art keywords
antigen
test
buffer
rna
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09732630A
Other languages
German (de)
English (en)
Other versions
EP2245186A4 (fr
Inventor
Weidong Xu
Shyam Mohapatra
Arun Kumar
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ultrapid Nanodiagnostics Inc
Original Assignee
Ultrapid Nanodiagnostics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US12/008,861 external-priority patent/US20090181361A1/en
Application filed by Ultrapid Nanodiagnostics Inc filed Critical Ultrapid Nanodiagnostics Inc
Publication of EP2245186A2 publication Critical patent/EP2245186A2/fr
Publication of EP2245186A4 publication Critical patent/EP2245186A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens

Definitions

  • the field is test kits providing rapid detection and diagnosis of an infectious agent, RNA or DNA in a volume of fluid containing enough antibodies, RNA, DNA or fragments thereof for detection of antibodies or sequences of DNA or RNA by the test kit.
  • Blood may be stored for 7-14 days in order to screen for a virus, increasing risks for anaphylactic reactions, increasing potassium concentration, and decreasing its oxygen carrying capacity.
  • the ability to screen bodily fluids, such as blood, saliva and urine, using reliable and rapid test kits is an unfilled and longstanding need.
  • the most common screening test is the enzyme-linked immunosorbent assay (ELISA), sometimes called enzyme immunoassay (EIA).
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • the most often used confirmatory test is the Western blot. If antibodies are being produced in the body, these tests are capable of detecting the antibodies at low levels.
  • the conventional HIV testing protocol starts with a sensitive EIA in a clinical laboratory.
  • the EIA might be performed with serum, plasma, urine, or oral fluid, and the results might be available in 3 to 4 days. If the EIA is negative, the result is considered definitive, and no further testing is indicated.
  • a limitation of any testing is that many viral antibodies take up to 3 months to express after infection occurs, causing a window between the infection and detection using even the most sensitive of assays. If the EIA is repeatedly positive, more specific testing, using the Western blot technique, is done for confirmation. The testing process from the time a specimen is submitted until a final result is available is often a week or even longer. The cost and time required to complete a test make frequent testing, even among high risks groups, impractical.
  • the Western blot test uses an electrical field that separates out the various components of a sample by molecular weight. This allows identification of antibodies to specific viral antigens, which show up as identifiable "bands" on a strip of test paper.
  • This test offers a high degree of specificity, ELISA combines the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily assayed enzyme that possesses a high turnover number. ELISA can provide a useful measurement of antigen or antibody concentration, which is unavailable in rapid test kits.
  • a rapid test is one that provides for a buffered specimen of blood or serum to be used in a test requiring less than five minutes to complete.
  • the window period is the period between the onset of viral infection and the appearance of detectable antibodies to the virus.
  • the window period is about three to four weeks. This period can, however, be longer. Any antibody-based blood test (such as the ELISA, rapid tests and the Western blot) conducted during this window period may give false negative results. The expense and time that these tests take means that testing is conducted infrequently on individuals.
  • the virus is present in the person's blood there may be no (detectable) antibodies in the blood during a screening test for a period up to about three months, but the cost of testing increases this window to a year or more, especially if the individual is in a low risk group.
  • test kits for detecting infectious agents, such as viral and bacterial diseases.
  • test kits marketed for home use that are neither approved nor adequately tested for diseases such as AIDS.
  • the only approved test kit for HIV in the United States takes a sample and sends the sample to a laboratory for analysis, No known rapid test kits that do not require sending a sample to a laboratory are approved for use in screening for HIV in the United States.
  • test kits are available for testing serum samples for disease.
  • test kits are available that include lateral flow tests.
  • Lateral flow tests also called immunochromatographic strip tests, arc used for specific screening or semi-quantitative detection of many analytes including antigens and antibodies.
  • Samples may either be used alone or with an extraction reagent, or running buffer, which is then placed on a sample pad on one end of a test strip,
  • the test strip also includes a membrane.
  • a signal reagent is solubilized and binds to an antigen if present in the sample and moves through the membrane by capillary action.
  • the complex is then captured by a second antibody, which produces a visible line, indicating presence of the antigen.
  • Lateral-flow dipstick test kits are known that can detect DNA, as reported in Glynou K (2003), but the shortcomings of lateral-flow disptick tests for blood and other bodily fluids are not solved, and no detection of RNA has been reported using a lateral-flow dipstick test.
  • Flow through tests may involve kits as individual cassettes with extraction and wash buffers included. These tests involve capturing of an analyte such as antibody or an antigen by a reagent as it flows through a membrane. These test kits often suffer from poor contrast, The protocols may require a user to prepare the sample to be tested, to wash the membrane, to add a signal reagent, and to wash the membrane to clear the membrane of any residue from the sample in an attempt to improve the contrast between the background and any screening line or marker for indicating the presence of an enzyme or antibody. Direct, flow-through test kits are known to be rapid but are seldom used in practice due to the complexity of the protocol required to provide enough contrast between the indicator and the background membrane.
  • Chen in WO 96/21863, describes an immunoassay test device for detection of antibodies to HIV-I and HIV-2 in biological fluid, providing for immediate immunoreaction and detection of the presence of such antibodies, comprising an assembled filter device and reaction cell using a nitrocellulose membrane on which an immunoreaction occurs.
  • Visualizing the antibodies that react with HIV antigenic glycoproteins g ⁇ 41, gp36, gp38 and g ⁇ l20 occurs by conjugating the antibodies with a Protein A colloidal indicator and viewing the membrane for the presence of a red color, indicating the presence of antibodies.
  • Chen teaches a lateral flow and/or filtering of blood through a filtration medium before contacting a nitrocellulose membrane.
  • Mahajan in US Patent Publication No. 2004/0023210, discloses a diagnostic kit for detection of antibodies of Hepatitis C virus in human serum and plasma, which comprises a base, an immunofiltration membrane of nitrocellulose mounted over an absorbent pad disposed on the base, and a top cover removably attached to the base having a central hole conforming to the membrane's circumference.
  • Antigens such as NS3, NS4, and NS5 are immobilized on the membrane and visualized with a Protein A conjugate.
  • This reference teaches that the pore size of the nitrocellulose membrane is 0.8-1.5 microns. The pore size is poorly correlated with specificity and sensitivity, which are correlated with contrast (or color index values as reported herein). Test kits suitable only for use with serum or plasma are not suitable for use as rapid field test kits.
  • Hu in U.S. Patent Publication No. 2003/0165970, teaches a diagnostic device for simultaneously detecting multiple infectious agents, such as HIV antibodies, Hepatitis B and C antibodies and syphilis antibody.
  • the kit disclosed by Hu comprises an immunogold filtration assay device, buffer and a mixture of colloidal gold particles where the device includes a nitrocellulose membrane blotted with HBsAg monoclonal antibody, HCV antigen, syphilitic antigen, HIV antigen, and goat anti-mouse IgG antibody.
  • the test is not rapid and requires a very complicated protocol.
  • Chu in U.S. Patent No. 5,885,526, discloses a flow-through test device having a reaction membrane that includes porous material, such as nitrocellulose. A small pore size is taught to be needed when using nitrocellulose membranes in order to provide a greater area for immobilizing receptor molecules.
  • Chu teaches that larger pore sizes lead to decreased assay sensitivity, as described in col. 5, Ins. 53-56.
  • Chu prefers the porosity of the reaction membrane to be in a range from 0.45 to 3 microns.
  • Chu teaches away from using compression to hold the reaction membrane, as it makes the device less suitable for some immunoassays where quantitative results are needed, as disclosed in col. 3, Ins. 15-32, and Chu fails to disclose any example using whole blood with cellulose filter papers.
  • Chu also teaches that a thick reaction membrane is needed to form an air pocket to prevent lateral flow and direct flow.
  • the working example discloses a thick 800 micron paper-backed nitrocellulose reaction membrane, as disclosed in Example 2.
  • Chu also discloses many disadvantages of prior art devices which have thin reaction membranes such as membranes being less than 0.1 mm thick, as disclosed in col. 7, Ins. 66-col. 8.
  • Chu discloses that a membrane should be capable of immobilizing an antigen and Protein A and he suggests materials such as nitrocellulose and fiberglass as being suitable for immobilizing the antigen and Protein A.
  • Chu requires an inoculation of both Protein A and an antigen at different areas of the membrane before testing of an analyte sample.
  • Chu also requires both protein A and an antigen of interest to be inoculated on the membrane first before a serum sample is absorbed into the membrane and also utilizes an additional step of adding protein A- colloidal gold conjugate to be added after the serum or plasma is absorbed, which makes Chu's preferred protocol, which is necessary to provide adequate contrast, very complex and not at all rapid.
  • Chu discloses inoculation of Protein A to be preferably at an edge of a device, as the central location of the membrane will contain an antigen of interest, such as a Hepatitis C antigen.
  • the test kits of Chu are not rapid test kits and suffer from complicated protocols, and unpredictable results in the hands of less trained staff and individuals. Chen et al., in U, S. Patent Publication No.
  • a porous reaction membrane such as paper-backed nitrocellulose, and a preferred pore size of 0.2 to 0.8 microns, as disclosed in paragraph [0062].
  • the membranes disclosed in Chen must be suitably porous membranes, such as the examples disclosed that use a nitrocellulose backed with porous paper. Testing of nitrocellulose membranes show that flow rate of water through the membranes are very rapid, but nitrocellulose failed in tests conducted by the applicant. While Chen does not exclude cellulose filter paper as a membrane, cellulose filter paper having a flow rate comparable to nitrocellulose is inoperable, as shown by the applicants results. No examples are provided by Chen using cellulose filter paper as a membrane in any test kit. Also, Gelman et al., in U.S. Patent No.
  • Chen discloses that a more complex test kit having a separate blood separation zone is needed, such as one using a glass fiber matrix as the blood separation material, an example provided in paragraph [0089] of U.S. Patent Publication No. 2004/0002063, for example. This complicated procedure is not viable as a field test.
  • Krutzik in U.S. Patent No. 6,653,066, discloses a lateral flow test using a matrix pore size of less than 5 microns and nitrocellulose membranes and discourages the use of larger pore sizes, which tends to have poor results.
  • a vaccine against AIDS usually comprises a complex mixture of HIV-I epitopes (peptides, proteins, DNA expression plasmids, and recombinant viral vectors) and can elicit persistent antibody responses in vaccinated volunteers that are detectable by FDA-licensed HIV-I detection kits.
  • HIV-I epitopes peptides, proteins, DNA expression plasmids, and recombinant viral vectors
  • Vaccine- induced antibodies can cause false positives or indeterminate reactivity when sera of vaccinated volunteers are tested using existing serological detection assays.
  • infants of mothers infected with HIV may test positive for HIV antibodies, because the infant's immune system is influenced by maternal antibodies for an uncertain duration.
  • Nanoscale materials such as single wall carbon nanotubes (CNT) and gold (Au) nanoparticles are known. Furthermore, it is known how to functionalize gold nanoparticles with an oligonucleotide to detect DNA, such as in a lateral-flow dipstick. Also, a gold- nanoparticle-based staining technology was successfully used in genotyping single-nucleotide polymorphisms when combined with a primer extension reaction. A gold-nanowire microfluidics platform for sensitive detection of blood analytes is known, but this method requires an electrochemical device for the readout. None of these methods or materials have been combined with a rapid test kit for use in detecting a disease at the point of care.
  • a rapid test for detecting infection is capable of detecting HIV infection in less than five minutes, and examples of test kits may be used for detecting the presence of antibodies or sequences of RNA or DNA in bodily fluids or both.
  • a single test kit tests for both the presence of antibodies and a viral RNA sequence indicative of a disease.
  • separate test kits are provided for antibody screening and detecting a sequence or sequences of a particular DNA or RNA of a disease using a genetic probe.
  • a test kit using a genetic probe may be used in testing separately from a test kit for use in measuring antibodies presence in a sample of bodily fluids.
  • a test kit including a genetic probe used for testing for a disease only if a subject specimen tests positive for antibodies is a test kit including a genetic probe used for testing for a disease. The tests may be used to qualitatively and/or quantitatively determine a level or concentration of antibodies or sequences of RNA or DNA within the bodily fluid.
  • a comparison is made against a contrast scale to determine the relative level or concentration of detected antibodies and/or sequences of RNA or DNA in the type of bodily fluid tested.
  • a quick indication of the presence of certain sequences of RNA or DNA in bodily fluids tested may provide detection and/or confirmation of an infection, for example, especially an acute infection, such as in the. case of an HIV infection.
  • test kits have comparatively low flow rates and large particle retention size (correlating with pore size) and are capable of completion of a rapid test in less than 3 minutes.
  • a test kit uses an antigen or a combination of antigens immobilized on a membrane, such as a cellulose filter paper, the membrane being selected to immobilize the antingen or antingens and having a flow rate in a range from about 0.04 ml/min/cm 2 to about 0.4 ml/min/cm 2 .
  • the test kit is capable of detecting antibodies by direct deposit, flow-through of a buffered suspension such as PBS buffered blood, serum or plasma, for example.
  • None of the other tested commercial test kits were capable of testing whole blood, which was readily achieved using examples of the present invention without affecting the outcome and with similar contrast to the same test using serum or plasma.
  • the prior art teaches that testing with whole blood is not known to achieve that same results as the use of serum or plasma.
  • a particular portion of an antigen is used to improve the contrast of a positive indication region, especially for whole blood, and the short fragment of the antigen achieved better results than using the entire antigen.
  • a diagnostic kit includes an antigen-immobilizing cellulose filter paper, at least one antigen immobilized on the cellulose filter paper, a staining agent to detect antibodies against the at least one antigen, a destaining buffer to remove non-specific background staining, and a plurality of wicking layers disposed in a bottom portion of the diagnostic kit opposite of the reaction membrane.
  • a cellulose filter paper used as a reaction layer of the test kit may have a particle retention size selected in a range from about 6 to about 25 microns.
  • test results for a variety of particle retention sizes for cellulose filter papers show that papers having particle retention sizes of 6, 11, and 20-25 (Whatman Qualitative/Wet Strengthened grade cellulose filter papers) do not exhibit a large departure in flow rate.
  • a rapid test kit should not have a flow rate unnecessarily low, but there is a correlation between flow rate and a color index value reported in the results, which is related to sensitivity of the test kit for detecting antibodies. Thus, there is a preferred range for selecting cellulose filter paper with an optimum flow rate.
  • a staining agent is Protein A coupled to colloidal gold.
  • a destaining buffer is used, such as phosphate buffered saline (PBS) to improve contrast with the background.
  • PBS phosphate buffered saline
  • a rapid test for detecting infection selects cellulose filter paper or an equivalent that has a phosphate buffer saline (PBS) solution flow rate in a range between about 0.04 to about 0.4 ml/min/cm 2 , more preferably 0.04 to 0.2 ml/min/cm for higher contrast (sensitivity). Flow rate, is more important than pore size in determining assay sensitivity and time to complete the test.
  • a cellulose filter paper can be selected to have a flow rate in a range from about 0.1 to about 0.2 ml/min/cm 2 , providing an optimum trade-off in sensitivity and flow rate for some examples.
  • the cellulose filter paper can be selected to have a PBS flow rate in a range from about 0.2 + 0.05 ml/min/cm 2 to increase flow rate without unduly sacrificing sensitivity (i.e., color index value),
  • the term "about” is used to indicate the manufacturing variances in manufacturing cellulose filter paper and in testing of flow rate according to the modified ASTM method described herein.
  • a person of ordinary skill in the art will be able to measure flow rates and select cellulose filter papers based on the disclosed flow rate testing method and flow rates and those cellulose filter papers having about the same flow rates as the ranges given herein.
  • One advantage of the diagnostic kit using cellulose as a reaction layer is the ability to obtain rapid results for a particular infectious agent or a plurality of infectious agents without complicated user protocols. Indeed, results are provided as readily for whole blood as for serum or plasma in some examples.
  • Another advantage is the cost of a test kit, which substantially reduces the costs associated with screening.
  • a rapid test kit is inexpensively produced and provided at low cost, which is especially necessary for use in remote locations and doctor's offices.
  • a single test may be used to test more than one type of disease detectable from blood.
  • a single diagnostic kit may be used in detecting one or more of a variety of bodily fluids, such as blood, plasma and serum, thus offering greater flexibility in testing. Field tests may be administered without the need of a mobile laboratory or a centrifuge,
  • a genetic probe is included for detecting RNA, DNA or a fragment or sequence of RNA or DNA in a bodily fluid.
  • the probe may include a pair of primers, for example.
  • One of the pair of primers may be immobilized on filter paper, while the other of the pair of primers is coupled to a nanoparticle or nanotube, such as by a thiolation of the other of the pair of primers, and the coupled primer-nanoparicle or primer- nanotube is included in a staining buffer.
  • the genetic probe may provide a qualitive or quantitative analysis of the level or concentration of a sequence of RNA and/or DNA in the volume of a fluid tested, such as urine, blood, edema or saliva, which may be correlated to a viral load, for example.
  • a fluid tested such as urine, blood, edema or saliva
  • the genetic probe may distinguish a vaccinated subject or a subject having maternal antibodies from a subject infected by a disease.
  • a rapid test kit for detection of a DNA, an RNA or a fragment of a DNA or an RNA includes a detection surface, such as a membrane, one or more genetic probes immobilized on a test portion of the detection surface, and a staining agent.
  • the genetic probe is selected to hybridize a genetic sequence to be detected by the test, such as the genetic sequence of viral RNA, for example.
  • the staining agent may include a different genetic probe, such as functionalized nanoparticle or nanotube that binds to the genetic sequence to be detected by the test.
  • the genetic sequence is immobilized preferentially on a region of the detection surface, when a sample containing the genetic sequence is applied to the detection surface or is passed through the detection surface, such as a cellulose filter paper membrane. Then, the staining agent is immobilized by binding to the genetic sequence, providing a contrast between the test portion and a background portion of the membrane.
  • a destaining buffer may be selected to remove at least a portion of any non-specific background staining unrelated to binding between the at least one genetic probe, the DNA, RNA or the fragment of the DNA or the RNA, and the staining agent.
  • the detection surface may be the surface of a glass slide or a membrane.
  • the membrane may be a cellulose filter paper selected such that the membrane has a measured flow rate of a phosphate buffered saline from about 0.04 to about 0.4 mL/min/cm 2 , using a modified ASTM standard flow rate measurement, for example. More preferably, the membrane may have a measured flow rate in a range from about 0.04 mL/min/cm 2 to about 0.2 mL/min/cm in order to increase contrast between the test region and the background.
  • the measured flow rate of the membrane may be limited to a range of at least 0.1 mL/min/cm 2 and no greater than about 0.2 mL/min/cm 2 in order to optimize the time required for testing and the contrast, for example.
  • the staining agent includes at least one type of oligonucleotide-functionalized nanoparticles or nanotubes having at least one oligonucliotide capable of hybridizing, at room temperature, with regions of genetic sequences to be detected by the rapid test kit.
  • the at least one genetic probe may include a complimentary oligonucleotide for hybridization with a specific region of the genetic sequences to be detected by the kit.
  • the complimentary oligonucleotide is conjugated with a chitosan or a chitosan derivative before being applied to the detection surface, such that the complimentary oligonucleotide is immobilized on a cellulose filter paper membrane, for example.
  • chitosan and chitosan derivatives are provided in the prior art, such as a thiolated chitosan derivative in U.S. Pat. Publication US 2007/0036867, chitosan derivatives disclosed in U.S. Pat. Publication US 2008/0087290, and the like.
  • the oligonucleotide-functionalized nanoparticle or nanotube may be a gold nanoparticle functionalized by a thiolated oligonucleotide complementary to a different portion of the genetic sequence than the genetic sequence targeted by the complimentary oligonucleotide immobilized on the membrane, for example.
  • the thiolated oligonucleotide may be a primer selected to hybridize a viral RNA selected from the group consisting of an HIV virus, a Hepatitis B virus, a Hepatitis C virus, a SARS virus and combinations thereof.
  • examples of oligonucleotides may be thiolated and bound to a surface of a gold nanoparticle, and may be selected to hybridize the viral RNA of the HIV virus.
  • the complementary oligonucleotide immobilized on the membrane may be selected to hybridize the same or a different region of the viral RNA of the HIV virus, such as the LTR sequence, in one example.
  • the staining agent comprises carbon nanotubes functionalized by examples of the oligonucleotides.
  • the concentration may be adjusted to provide sufficient contrast under the appropriate lighting conditions in order to observe a positive test result.
  • a plurality of oligonucleotides for attaching to a plurality of regions within a genetic sequence to be detected are provided in a staining agent.
  • Each nanotube or nanoparticle may be functionalized with one or more of the oligonucleotides,
  • a plurality of nanotubes or nanoparticles may be hybridized to one or more regions of a single genetic sequence, providing an amplification in the contrast observed compared to the use of only one oligonucleotide targeting one region of the single genetic sequence.
  • the oligonucleotide or oligonucleotides immobilized on the detection surface may be targeted to only one or a select few regions of the genetic sequence in order to increase the specificity of the test kit to only the genetic sequence or sequences selected for detection.
  • Tables 8A-P (intentionally omitting designators 81 and 80 for clarity) disclose a screening of genetic sequences for locating unique HIV specific genetic sequences that provide specifity for a rapid test, for example.
  • a test may have a very good contrast even with a low viral load, yet remain very selective in the genetic sequences detected, which provides a surprising and unexpected improvement over any known rapid test including the ability to distinguish between the effects of a vaccine on antibodies and a viral load, for example.
  • certain complementary oligonucleotides may be selected that hybridize with the selected regions of the genetic sequences. In a rapid test for use at point of care, it is preferred to select complementary oligonucleotides that hybridize at room temperature, for example.
  • oligonucleotides may be targeted for any genetic sequence, such as viral RNA's consisting of an HIV virus, a Hepatitis B virus, a Hepatitis C virus, a SARS virus and combinations thereof, for example,
  • at least one genetic probe includes a 33-nt oligonucleotide from HIV 89.6 proviral clone, which may be conjugated with a chitosan or a chitosan derivative.
  • the method includes detecting a detectable level of viral load in a sample volume of fluid.
  • the method may include a step of conjugating the at least one genetic probe with a chitosan or a chitosan derivative to form a conjugate, and immobilizing the conjugate on a test region of a membrane.
  • the method includes illuminating the membrane with ultraviolet light to increase the contrast between the test portion and a background portion of the membrane, such that the ultraviolet light causes the test portion to fluoresce.
  • a level or concentration of the viral load may be determined by comparing the contrast or fluorescence to known levels or concentrations, for example.
  • the determination is automated by a detector and processor that compares the signal received by the detector to a look up table, for example.
  • the step of reporting may include comparing the contrast or intensity of at least a portion of the test portion of the membrane to a standard, for example.
  • a staining agent is deposited on the membrane such that a genetic probe in the staining agent binds selectively to a portion of a genetic sequence within a temperature range, which may include room temperature. Room temperature is considered to be a range of temperature from about 15 degrees centigrade to about 25 degrees centigrade, for example.
  • a genetic probe is combined with an antibody test.
  • the antibody test may comprise one or more peptide fragments, such as a gp41 peptide fragment comprising the following sequence: QLQARILAVERYLKDQQLLGIWGCSGKLICTTA VPWNAS.
  • At least one genetic probe is deposited on a detection region of a glass slide, a fluid to be tested is deposited on the detection region, and a suspension of nanotubes or particles is functionalized by a complementary oligonucleotide such that, when the suspension is directly deposited on the detection region of the glass slide, the complimentary oligonucleotide hybridize specific regions of a genetic sequence, if the genetic sequence is present on the detection region.
  • the genetic probe may be fixed on the surface of the slide before placing the fluid onto the surface of the slide. After a fixed period of time, the fluid may be rinsed from the surface.
  • the staining agent may be deposited on the detection region for a fixed period of time within a temperature range, such as room temperature.
  • the staining agent may be rinsed away and the slide may be observed under light, such as an ultraviolet light, to detect a contrast between the detection region and a control region or a background region.
  • the detector may observe the slide for the emission of light or for the absorbtion of light by the detection region.
  • functionalized carbon nanotubes functionalized by complementary oligonucleotides, may be used detecting a fluorescence under ultraviolet light
  • functionalized gold nanoparticles may be used for detection of light absorbed passing the light, such as ultraviolet light, through the detection region. Either fluorescence or phosphorescence may be detected, for example.
  • the detector or system may be capable of reporting a value or outpuot associated with a viral load in the sample measured, for example.
  • a genetic probe includes a 33-nt oligonucleotide from HIV 89.6 proviral clone, for example.
  • a genetic probe may be immobilized on one portion of a test kit, and an antigen for detecting an antibody may be immobilized on another portion of a test kit.
  • the two portions are in the same testing window and react to the same sample of a bodily fluid.
  • the two portion are disposed in separate windows.
  • One or more staining agents may be used, which may include functionalized nanotubes or nanoparticles, for example.
  • a complementary-oligonucleotide-functionlized nanotube or oligonucleotide functionalized particle is provided.
  • the genetic probe may be a complimentary oligonucleotide capable of hybridizing a genetic sequence to be detected, such as a portion of the LTR genetic sequence of the HIV-I virus.
  • the staining agent may comprise a plurality of thiolated oligonucleotides coupled with gold nanoparticles selected such that the plurality of thiolated oligonucleotides are each capable of hybridizing different portions of the RNA of the HIV-I virus, for example.
  • FIG. 1 The drawings describe some examples of a rapid diagnostic kit and a method for preparing and using the diagnostic kit.
  • Figure IA illustrates an example of a cross section of a diagnostic kit 100.
  • Figure IB illustrates another example of a cross section of a diagnostic kit 110.
  • Figure 1C depicts a top plan view of a diagnostic kit such as those shown in Figures IA and IB.
  • Figures 2A-B provide illustrations of top views of examples of test kits that (A) tested negative for the presence of an antibody and (B) tested positive for the presence of an antibody.
  • Figures 3A-B provide illustrations of a comparison of (B) an example of a diagnostic kit using a cellulose filter paper and (A) a glass fiber membrane, which resulted in failure when tested with blood.
  • Figures 4A-C illustrate comparisons of examples using a cellulose filter paper membrane for a diagnositc kit with a nitrocellulose membrane.
  • Figure 5 graphs color index value versus flow rate of PBS, as measured using a modified ASTM flow rate procedure with 7 cm circles of the cellulose filter papers used in the tests.
  • Figure 6 illustrates a color index chart for determining color index values where any marker discernable over background is given a value of 1 , anything darker than 1 is 2, anything darker than 2 is 3, and anything darker than 3 is deemed a 4, quantifying color intensity of test samples.
  • Figure 7 shows measured flow rate versus particle retention size for 6 different cellulose filter papers.
  • Figure 8 discloses a graph color index value by sample number for various results including testes using blood and plasma with a test kit having a PBS flow rate of about 0.1 mil/min/cm 2 , and also showing color index of control spots.
  • Figures 9 is an illustration of test results using blood.
  • Figure 10 graphically compares test results for plasma using a rapid test kit of the examples using a cellulose filter paper having a flow rate of about 0.1 ml/min/cm 2 and a commercially available test kit (Reveal ® G3) 1 .
  • Figures 11 is an illustration of test results using whole blood.
  • Figures 12 is an illustration of test results using plasma.
  • Figures 13 is an illustration of test results using plasma.
  • Figures 14A-C illustrate possible outcomes of a test kit having a control test spot and an antibody test spot for both a genetic probe and antibodies with (A) two separate test windows; (B) a single test window; and (C) graphical representation of all outcome for the two test spots (i.e. assuming controls visible).
  • Figure 15 illustrates an example of a process for using a rapid test kit including a genetic probe.
  • Figure 16 illustrates, schematically, functionalization of a gold nanoparticle.
  • Figures 17 illustrates a perception of contrast between (1) ssDNA-gold nanoparticles hybridized by complementary DNA; (2) ssDNA-gold nanoparticles with ssDNA; and (3) ssDNA-gold nanoparticles alone.
  • Figure 18 graphs the ultraviolet absorbance spectra as shown and disclosed.
  • Figure 19 illustrates, schematically, functionalization of a carbon nanotube.
  • Figure 20 graphs ultraviolet light absorbance spetra for (A) carbon nanotubes (CNT), alone, without any ssDNA strand or fragment; (B) CNT functionalized by a single strand oligonucleotide; (C) CNT funtionalized by a single strand oligonucleotide hybridized with a non- complementary single strand oligonucleotide; and (D) CNT functionalized by a sngle strand nucleotide after hybridization with a complementary oligonucleotide fragment.
  • CNT carbon nanotubes
  • B CNT functionalized by a single strand oligonucleotide
  • C CNT funtionalized by a single strand oligonucleotide hybridized with a non- complementary single strand oligonucleotide
  • D CNT functionalized by a sngle strand nucleotide after hybridization with a complementary oligonucleotide fragment.
  • Figures 21 A - 21 C are atomic force microscopy micrographs of (A) carbon nanotubes (CNT), alone, without any single strand oligonucleotides; (B) CNT functionalized by a single strand oligonucleotide; and (C) CNT functionalized by a single strand oligonucleotide and hybridization with a complementary single strand oligonucleotide fragment.
  • CNT carbon nanotubes
  • Figures 22A-B illustrate a control 784, 794 with a non-complementary 786, 796 and a complementary 788, 798 combination of single strand oligonucleotides (e.g. ssDNA or a fragment of ssDNA) for comparison.
  • the regions having complementary 788, 798 single strand oligonucleotides fluoresce, while the non-complementary 786, 796 oligonucleotides fail to hybridize and are not subject to fluorescence. '
  • Figure 23 illustrates a range of concentrations of carbon nanotubes using a range of picomoles (pmol).
  • Figure 24 contrasts a oligonucleotide that is not immobilized on a test region to an oligonucleotide immobilized on a test region after conjugating with a chitosan or a chitosan derivative.
  • Figure 25 illustrates a detector for measuring emitted or transmitted light from a test region on a slide.
  • a rapid diagnostic assay provides a quick and inexpensive screening test for detecting antibodies resulting from disease-causing organisms, such as a viruses, bacteria, fungus, mold and other disease-causing organisms that are detectable through an antibody assay.
  • the diagnostic assay is a rapid assay meaning that the time to conduct the test from drawing of a bodily fluid to completing the test is rapid (e.g. less than ten minutes) and the time to obtain a test result after preparing a buffered suspension is rapid (e.g. less than one minute). Rapid test kits are not known that have both the sensitivity and the specificity of test kits used in the examples.
  • test kits known to the inventors are able to provide a result in less than 1 minute from the time that PBS buffered samples are ready to be used, such as shown for test kits obtaining strong positives in high titer tests and excellent results in low titer tests, also. Rapid is meant to mean both time scales (test preparation to completion and time for the test kit to provide a result after the test sample is mixed in buffer solution). Furthermore, examples of test kits provide rapid diagnostic assay using whole blood, serum or plasma as testing material. Whole blood is particularly problematic for all of the commercial test kits tested.
  • a method of rapid diagnostic assay uses the test kit of the examples in the field without any need of medical or laboratory facilities.
  • Ability to distribute to remote locations makes testing convenient and inexpensive.
  • Various types of antigens may be used in a rapid diagnostic assay.
  • the antibodies detected by a rapid diagnostic assay may be produced in response to bacteria, fungi, parasites, or viruses, for example.
  • a wide variety of antigens may be used separately or together in a screening array.
  • the rapid diagnostic assay may also detect antibodies or antigens in non-infectious diseases such as cancer, Alzheimer's disease, or other non-infectious diseases,
  • Bacterial antigens Bacterial pathogens may be detected by a rapid test kit.
  • an antigen is selected from a major outer membrane protein within strains of the genus Actinobacillus.
  • the antigen is disclosed in U.S. Patent No. 6,541,011.
  • a bacterial antigen may be from any of the following: Actinomyces, such as an ornithine-rich antigen from Actinomyces naeslundii, or Actinomyces viscosus as disclosed in U.S. Patent No.
  • bronchiseptica with molecular masses of 70 and 68 kDa respectively; Bartonella; Borrelia, such as Borrelia recurrentis or OspA of the Lyme disease Borrelia burgdorferi, as mentioned in U.S. Patent No. 6,541,011; Brucella, such as Brucella abortus or Brucella melitensis, such as Omp29 on Brucella melitensis as mentioned in U.S. Patent No. 6,541,011 or Brucella suis; Campylobacter, such as Campylobacter pylori as mentioned in U.S. Patent No.
  • Chlamydia such as Chlamydia traqchomatis or Chlamydia psittaci, such as 80-90 kDa protein and 110 kDa protein, chlamydial exoglycolipid (GLXA), Chlamydia pneumoniae species-specific antigens in the molecular weight ranges 92-98, 51-55, 43-46 and 31.5-33 kDa and genus-specific antigens in the ranges 12, 26 and 65-70 kDa, as mentioned in U.S. Patent No.
  • Clostridium such as Clostridium botulinum or Clostridium perfingens or Clostridium tetani or a C fragment from C. tetani as mentioned in U.S. Patent No. 5,527,529; A, B, C, and D toxoids from C. perfringens, such as a B toxoid as mentioned in U.S. Patent No. 6,524,592 or toxin A from C. difficile, as mentioned in U. S, Patent No. 6,503,722 or LT and HT toxins from C. sordellii disclosed in U.S. Patent No. 6,849,715 or an alpha toxin from C. septicum of U.S.
  • Corynebacterium such as Corynebacterium diptheria
  • Coxiella Dermatophilus
  • Enterococcus Enterococcus
  • Ehrlichia Echinococcus granulosus antigen 5
  • the antigen is from Mycoplasma hominis.
  • the antigen is from Mycoplasma pneumoniae.
  • the bacterial antigen is from Neisseria.
  • the antigen is from Neisseria gonorrhea.
  • the antigen is from Neisseria meningitidis.
  • the antigen is Por, Rmp or a LOS protein of Neisseria gonorrhoeae.
  • the antigen may include PorA, Por B, Rmp, Ope, FrpB, TbpB or Nsp may be used, as mentioned by U.S. Patent No.
  • Neorickettsia Nocardia; Pasteurella, such as Pasteurella pestis; Peptococcus, such as Peptostreptococcus; Pneumococcus, such as Diplococcus pneumonia; Proteus; Pseudomonas; P. gingivalis, such as the 43-kDa and the fimbrilin (41 kDa) proteins of P. gingivalis, as disclosed in U.S. Patent No.
  • Rickettsia such as Rickettsia australis or Rickettsia burnetii or Rickettsia conori or Rickettsia mooseri or Rickettsia prowazekii or Rickettsia tsutsugamushi
  • Rochalimaea Salmonella, such as Salmonella choleraesus or Salmonella typhimurium or Salmonella typhosa or O, H, and Vi antigens of Salmonella or SEFl 4 fibrial antigen of Salmonella enteriditis and flagellar (G) antigens observed on Salmonella enteritidis and S. pullorum, disclosed in U.S.
  • Patent No. 6,541 ,011 Shigella, such as Shigella arabinotardo or Shigella boydii or Shigella dysenteria or Shigella flexneri or Shigella schmitzii or Shigella sonnei or O-antigens disclosed by U.S. Patent No. 5,958,686 or S. dysenteria, disclosed in U.S. Patent No, 5,204,097; Staphylococcus, such as Staphylococcus aureus or Staphylococcus albus or type 5, type 336, type 4, K73 antigens of S. aureus, disclosed by U.S. Patent No.
  • Shigella such as Shigella arabinotardo or Shigella boydii or Shigella dysenteria or Shigella flexneri or Shigella schmitzii or Shigella sonnei or O-antigens disclosed by U.S. Patent No. 5,958,686 or S. dysenteria, disclosed in U.S. Patent No,
  • Streptococcus such as Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), or Streptococcus pneumoniae, as disclosed in U.S. Patent No.
  • streptococcus pyogenes include group carbohydrate antigen, C-substance, fimbrial proteins, fibronectin- binding proteins (e.g., Protein F) , a cell bound streptokinase, A, B, and C streptococcal pyrogenic exotoxins, alpha C protein, beta C protein, Rib and Sip proteins, or group B carbohydrate antigens, as disclosed in U.S.
  • Patent Publication 2006/0269541 purified capsular polysaccharide of 7 serotypes of S, pneumoniae (4,9V, 14, 19F, 23F, 18 C and 6B); pneumococcal surface protein A, pneumococcal surface adhesion A , choline binding protein A, LytB glucosaminidase, LytC muramidase, PrtA serine protease, PhtA (histidine triad A) and pneumococcal vaccine antigen A, as mentioned in WO/2004/092209; Group B streptococcal Etna (extracellular matrix adhesion protein polypeptides) EmaA, EmaB, EmaC, EmaD and EmaE from U.S.
  • Group B streptococcal Etna extracellular matrix adhesion protein polypeptides
  • Fungal antigens Fungal pathogens may also be detected by the kit and the methods disclosed.
  • the antigen is from Candida albicans.
  • the antigen is a fungal adhesion molecule, such as a phosphomannoprotein, from Candida albicans, as mentioned in U.S. Patent No. 6,630, 146.
  • the antigen is a fungal antigen from Absidia.
  • the antigen is from Absidia corymbifera.
  • the antigen is a fungal antigen from Acremonium, In one example, the antigen is a fungal antigen from Alternaria, In one example, the antigen is a fungal antigen from Aspergillus, In one example, the antigen is a fungal antigen from the species Basidiobolus. In one example, the antigen is a fungal antigen from the species Bipolaris. In one example, the antigen is a fungal antigen from the species Blastomyces. In one example, the antigen is a fungal antigen from the species Blastomyces. In one example, the antigen is a fungal antigen from Candida.
  • an antigen is a fungal adhesion molecule, such as a phosphomannoprotein, from Candida albicans, as mentioned in U.S. Patent No. 6,630, 146.
  • the antigen is a fungal antigen from Candida.
  • the antigen is a fungal antigen from Coccidioides.
  • the antigen is from Coccidioides immitis.
  • the antigen is a fungal antigen from Conidiobolus.
  • the antigen is a fungal antigen from Cryptococcus.
  • the antigen is a fungal antigen from Conidiobolus.
  • the antigen is a fungal antigen from Cryptococcus.
  • the antigen is a fungal antigen from Cryptococcus.
  • the antigen is from Cryptococcus neoformans. In one example, the antigen is a fungal antigen from Curvalaria. In one example, the antigen is a fungal antigen from Epidermophyton. In one example, the antigen is a fungal antigen from Exophiala. In one example, the antigen is a fungal antigen from Geot ⁇ chum. In one example, the antigen is a fungal antigen from Histoplasma. In one example, the antigen is from Histoplasma capsulatum. In one example, the antigen is a fungal antigen from Madurella . In one example, the antigen is a fungal antigen from Malassezia.
  • the antigen is a fungal antigen from Microsporum. In one example, the antigen is a fungal antigen from Moniliella. In one example, the antigen is a fungal antigen from Mortierella. In one example, the antigen is a fungal antigen from Mucor. In one example, the antigen is a fungal antigen from Paecilomyces. In one example, the antigen is a fungal antigen from Penicillium. In one example, the antigen is a fungal antigen from Phialemonium. In one example, the antigen is a fungal antigen from Phialophora. In one example, the antigen is a fungal antigen from Protoiheca.
  • the antigen is a fungal antigen from Pseudallescheria. In one example, the antigen is a fungal antigen from Pseudomicrodochium. In one example, the antigen is a fungal antigen from Pythium. In one example, the antigen is a fungal antigen from Rhinospo ⁇ dium. In one example, the antigen is a fungal antigen from Rhizopus. In one example, the antigen is a fungal antigen from Scolecobasidium. In one example, the antigen is a fungal antigen from Sporoth ⁇ x. In one example, the antigen is a fungal antigen from Stemphylium. In one example, the antigen is a fungal antigen from Trichophyton. In one example, the antigen is a fungal antigen from Trichosporon. In one example, the antigen is a fungal antigen from Xylohypha.
  • Parasital antigens Parasital pathogens may also be detected by the kit and the methods disclosed.
  • the antigen is a protozoan parasite and the antigen is from Babesia.
  • the antigen is a protozoan parasite and the antigen is from Balantidium.
  • the antigen is a protozoan parasite and the antigen is from Balantidium.
  • the antigen is a protozoan parasite and the antigen is from Besnoitia.
  • the antigen is a protozoan parasite and the antigen is from Cryptosporidium.
  • the antigen is a protozoan parasite and the antigen is from Eimeria.
  • the antigen is a protozoan parasite and the antigen is from Encephalitozoon, In one example, the antigen is a protozoan parasite and the antigen is from Entamoeba. In one example, the antigen is a protozoan parasite and the antigen is from Giardia. In one example, the antigen is a protozoan parasite and the antigen is from Hammondia. In one example, the antigen is a protozoan parasite and the antigen is from Hepatozoon. In one example, the antigen is a protozoan parasite and the antigen is from Isospora.
  • the antigen is a protozoan parasite and the antigen is from Leishmania. In one example, the antigen is a protozoan parasite and the antigen is from Microsporidia. In one example, the antigen is a protozoan parasite and the antigen is from Neospora. In one example, the antigen is a protozoan parasite and the antigen is from Neospora. In one example, the antigen is a protozoan parasite and the antigen is from Pentatrichomonas . In one example, the antigen is a protozoan parasite and the antigen is from Plasmodium. In one example, the antigen is a protozoan parasite and the antigen is from Plasmodium.
  • the antigens may include P. falciparum circumsporozoite (PfCSP), sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver state antigen 1 (PfLSAl c-term), and exported protein 1 (PfExp-1).
  • PfCSP P. falciparum circumsporozoite
  • PfSSP2 sporozoite surface protein 2
  • PfLSAl c-term carboxyl terminus of liver state antigen 1
  • PfExp-1 exported protein 1
  • the antigen is from a protozoan parasite Pneumocystis.
  • the antigen is from a protozoan parasite Sarcocystis.
  • the antigen is from a protozoan parasite Schistosoma.
  • the antigen is from a protozoan parasite Theileria.
  • the antigen is from a protozoan parasite Toxoplasma.
  • the antigen is from a protozoan parasite Trypanosoma. In other examples, the antigen is from helminth parasites. In one example, the antigen is from Acanthocheilonema. In one example, the antigen is from Aelurostrongylus . In one example, the antigen is from Ancylostoma. In one example, the antigen is from Angiostrongylus. In one example, the antigen is from Ascaris. In one example, the antigen is from Brugia. In one example, the antigen is from Bunostomum. In one example, the antigen is from Capillaria. In one example, the antigen is from Chabertia. In one example, the antigen is from Cooperia.
  • the antigen is from Cooperia. In one example, the antigen is from Crenosoma. In one example, the antigen is from Dictyocaulus. In one example, the antigen is from Dioctophyme. In one example, the antigen is from Dipetalonema. In one example, the antigen is from Diphyllobothrium. In one example, the antigen is from Diplydium. In one example, the antigen is from Dirofilaria. In one example, the antigen is from Dracunculus. In one example, the antigen is from Enterobius. In one example, the antigen is from Filaroides. In one example, the antigen is from Haemonchus.
  • the antigen is from Lagochilascaris, In one example, the antigen is from Loa. In one example, the antigen is from Mansonella. In one example, the antigen is from Muellerius, In one example, the antigen is from Nanophyetus. In one example, the antigen is from Necator. In one example, the antigen is from Nematodirus. In one example, the antigen is from Oesophagostomum. In one example, the antigen is from Onchocerca. In one example, the antigen is from Opisthorchis. In one example, the antigen is from Ostertagia. In one example, the antigen is from Parafllaria, In one example, the antigen is from Paragonimus.
  • the antigen is from Parascaris. In one example, the antigen is from Physaloptera. In one example, the antigen is from Protostrongylus. In one example, the antigen is from Setarla. In one example, the antigen is from Spirocerca. In one example, the antigen is from Spirometra. In one example, the antigen is from Stephanofilaria. In one example, the antigen is from Strongyloides . In one example, the antigen is from Strongylus, In one example, the antigen is from Thelazia. In one example, the antigen is from Toxascaris , In one example, the antigen is from Toxocara. In one example, the antigen is from Trichinella.
  • the antigen is from Trichostrongylus. In one example, the antigen is from Trichu ⁇ s. In one example, the antigen is from Uncinaria. In one example, the antigen is from Wuchereria. In one example, the antigen may include the schistosome gut-associated antigens CAA (circulating anodic antigen) and CCA (circulating cathodic antigen) in Schistosoma mansoni, S. haematobium or S. japonicum. In one example, the antigen may include a multiple antigen peptide (MAP) composed of two distinct protective antigens derived from the parasite Schistosoma mansoni.
  • MAP multiple antigen peptide
  • the antigen may include Leishmania parasite surface molecules third-stage larval (L3) antigens of L. loa (Akue et al. (1997), Tamsl-1 and Tamsl-2, encoding the 30-and 32-kDa major merozoite surface antigens of Theileria annulata (Ta) and Plasmodium falciparum merozoite surface antigen 1 or 2.
  • the antigen is Plasimodium falciparum antigen Pfs230.
  • the antigen may include Plasimodium falciparum apical membrane antigen (AMA-I); Plasmodium falciparum proteins Pfs28 and Pfs25 ; Plasimodium falciparum merozoite surface protein, MSPl ; the malaria antigen Pf332; Plasmodium falciparum erythrocyte membrane protein 1; Plasmodium falciparum merozoite surface antigen, PfMSP-I; Plasmodium falciparum antigens SERA, EBA-175, RAPl and RAP2 ; Schistosoma japoni cum paramyosin (Sj97) or fragments; and Hsp70 in parasites.
  • AMA-I Plasimodium falciparum apical membrane antigen
  • Pfs28 and Pfs25 Plasimodium falciparum merozoite surface protein
  • MSPl Plasimodium falciparum merozoite surface protein
  • PfMSP-I Plasmodium falciparum
  • the antigen is a viral antigen from an adenovirus. In one example, the antigen is a viral antigen from an alphavirus. In one example, the antigen is a viral antigen from a calicivirus . n one example, the antigen is a viral antigen from a calicivirus capsid antigen . In one example, the antigen is a viral antigen from a coronavirus. In a specific example of a coronavirus, the antigen is a SARS coronavirus. In one example, the antigen is from a cytomegalovirus.
  • the antigen may include cytomegalovirus glycoprotein gB or glycoprotein gH.
  • the antigen is a Dengue virus
  • the antigen may include a Dengue virus envelope (E) and premembrane antigens .
  • the antigen is a viral antigen from a distemper virus
  • the antigen is a viral antigen from an Ebola virus
  • the antigen is from an Epstein-Barr virus.
  • the antigen is an Epstein-Barr virus (EBV) gp340 protein
  • the antigen is the Epstein-Barr virus (EBV) latent membrane protein LMP2.
  • the antigen is Epstein-Barr virus nuclear antigens I and 2.
  • the antigen is measles virus nucleoprotein (N).
  • the antigen is a viral antigen from an enterovirus ,
  • the antigen is a viral antigen from a flavivirus.
  • the antigen is from Hepatitis A.
  • the antigen is from Hepatitis B.
  • the antigen is a viral antigen from a hepatitis B core or surface antigen.
  • the antigen is Hepatitis B virus core and E antigen.
  • the antigen is a hepatitis B surface antigen fused to a core antigen, core-preS2 particles.
  • the antigen is from Hepatitis C.
  • the antigen is a Hepatitis C virus nucleocapsid protein in a secreted or a nonsecreted form,
  • the antigens may include the hepatitis C virus antigens: the core protein (pC); El (pEl) and E2 ( ⁇ E2) alone or as fusion proteins.
  • the antigen is from Herpes simplex, types I and II, In one example, the antigen is a viral antigen from a herpes simplex virus or varicella zoster virus glycoprotein. In one specific example, the antigen may include ICPO, ICP4, ICP27, ICP47, gB, gD, gE, gG ,gH, and gl of the herpes simplex virus. In one example, the antigen is a viral antigen from an infectious peritonitis virus. In one example, the antigen is a viral antigen from HIV.
  • the antigen may include a HIV antigen such as Gag, Pol, Vif, Nef, p24, gp 120, gp 160 , gp41 or gp36.
  • the antigen is a viral antigen from an influenza virus, In one example, the antigen is from an influenza A, B or C viruses. In one specific example, the antigen is a viral antigen from an influenza A hemagglutinin, neuraminidase, or nucleoprotein. In one specific example, the antigen is N2 neuraminidase of an influenza A virus. In one example, the antigen is a viral antigen from a leukemia virus.
  • the antigen is a viral antigen from a Marburg virus. . In one example, the antigen is from a measles virus. In one example, the antigen is from the mumps virus. In one example, the antigen is a viral antigen from an orthomyxovirus. In one example, the antigen is a viral antigen from a papilloma virus. In one specific example the antigen may include the El, E2, E3, E4, E5, E6 and E7 proteins of human papillomavirus. In one example, the antigen is a viral antigen from a parainfluenza virus.
  • the antigen is a hemagglutinin or a neuraminidase.
  • the antigen is a viral antigen from a paramyxovirus.
  • the antigen is a viral antigen from a pestivirus.
  • the antigen is a viral antigen from a picorna virus.
  • the antigen may come from a coxsackievirus.
  • the antigen may come from an echovirus.
  • the antigen may come from a poliovirus.
  • the antigen may come from a rhinovirus.
  • the antigens may include a poliovirus capsid antigen, or a pox virus antigen.
  • the antigen is a viral antigen from a rabies virus, In one specific example, the antigens include rabies virus glycoproteins.
  • the antigen is a viral antigen from a reovirus.
  • the antigen is from a respiratory syncytial virus.
  • the antigen is a respiratory syncytial virus fusion protein (PFP-2).
  • the antigen is from a rubella virus.
  • the antigen is a viral antigen from a rotavirus.
  • the antigen may include rotavirus antigen VP4, VP7, or VP7sc.
  • the antigen may include proteins encoded by the VP6 and VP7 genes of rotaviruses.
  • the antigen may be from vaccinia.
  • the antigen is from human T-lymphotropic virus.
  • the antigen may include a human T-lymphotropic virus type I gag protein.
  • an antigen is selected to detect a non-infectious disease, such as cancer, Alzheimer's disease or other non-infectious diseases.
  • the cancer may be prostate cancer, and the antigen selected may be a prostate specific antigen (PSA).
  • PSA prostate specific antigen
  • the antigen is an Alzheimer's disease antigen, i.e., A68, or a recombinant human tau, as described in U.S. Patent No. 6,864,062, for example.
  • Figures 1 A-C show a schematic example of a test kit assembly cross section.
  • a plurality of layers 42 of an absorbent material and a membrane 22 are compressed between a cassette top 60 and a cassette bottom 62, which are represented in the drawin in an exploded view, for clarity.
  • a rapid test kit 100 comprises a cassette top 60 having an opening 63 and a cassette bottom 62.
  • a wall 61 of port 63 may be angled or may be straight as shown.
  • connection part 65 which may provide a snap or press fit, for example,
  • a cellulose filter paper 22 may be loaded with one or more antigens.
  • a plurality of absorbent layers 42 may be the same as the filter paper 22 or may be different.
  • the absorbent layers 42 may have the same physical and chemical characteristics or may differ from each other, including length, absorbancy and thickness. In one example of the filter paper 22 and plurality of absorbant layers 42 have a dimension of 1 inch squares.
  • the layers may be of uneven length, width and thickness.
  • the plurality of absorbant layers 42 may be two or more depending on their thick ⁇ ess and the dimensions of the cavity formed by the top 60 and the bottom 62.
  • the top 60 and the bottom 62 compress the layers 42 to achieve intimate physical contact one to the other.
  • the layers 42 are of a filter paper and include five to ten layers, depending on the characteristics of the filter paper and the cassette.
  • a cassette top 60 may be press or snap fitted onto the cassette bottom 62.
  • a central opening or port 63 through which plasma, serum, blood, saliva or other body fluids pass through the device, includes antigens for detecting antibodies.
  • the antigen or antigens may be loaded before testing either before or after assembly of the kit.
  • a wicking pad 24 replaces one or more absorbant layers 42 of a test kit 110.
  • FIG. 1C a top plan view of a diagnostic kit is illustrated.
  • the cassette top 60 includes an angular wall 61 defining a port 63.
  • the length of the wall 61 may be increased by a collar 67 extending above the top 60 and providing a greater volume within the port 63.
  • 1 ⁇ l of an antigen or antigen mixture is added at a position T (i.e., a test position) of the flow through device and 1 ⁇ l of protein A (1 mg/ml) is added at a different position C (i.e., a control position) of the test device.
  • the test device is dried. For example, 6-8 hours of air drying is sufficient for drying most test kits.
  • a test sample such as blood, serum or plasma, may be tested for presence of an antibody using a staining buffer.
  • the staining buffer is Protein A coupled to colloidal gold.
  • a staining buffer may be freeze-dried for later use and may be rehydrated using a buffer solution, such as IX Dulbecco's Phosphate Buffer Saline (DPBS), for example.
  • DPBS IX Dulbecco's Phosphate Buffer Saline
  • 10 ⁇ l of serum, plasma, or whole blood of a test sample may be diluted with 150 ⁇ l of dilution buffer,
  • the dilution buffer is ACK Lysis Buffer, Cat # 1683, obtained from Invitrogen.
  • the now diluted sample is deposited into a port 63 of a test device and onto the reaction layer 22, which may be comprised of an antigen test spot on a cellulose filter paper.
  • a staining buffer may be added.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer may be added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer (IX) Saline (DPBS), which is also an example of phosphate buffer solution, for example.
  • IX Dulbecco's Phosphate Buffer
  • DPBS Dulbecco's Phosphate Buffer
  • results may be read immediately, without further delay, resulting in a rapid test. When both test position T and control position C appear red, a test result is positive for the presence of antibodies indicative of a particular disease, such as HIV, for example.
  • control position C has a red dot
  • the test result is negative for the presence of antibodies associated with the disease detected by the antigen. If no dot is visible or if the control position has no dot visible, then the test is invalid. The control dot C should always be visible, if the test is properly performed.
  • a blood sample is diluted ten fold with a lysing buffer. Samples testing positive for a specific antibody have two red dots.
  • a silver enhancing buffer is used to improve contrast.
  • a first C spot 102 of a first test device 120 and second C spot 112 of a second test device 140 serve as control spots, which help to confirm that the test device is functioning properly.
  • a first T spot 104 of the first test device 120 and a second T spot 114 of a second device 140 are test spots for detecting the presence of a specific antibody or antibodies. None of the tests performed resulted in false negatives.
  • the first T spot 104 has no red spot, indicating the absence of any detectable level of antibodies in the particular test sample.
  • the T spot 114 shows a red spot in addition to control spot 112, positively indicating infection of the specimen with antibodies for HIV.
  • the following examples illustrate various types of antigens that may be used in a rapid test kit.
  • An antibody or antibodies present in a sample may bind to the specific antigen.
  • the examples are not intended to limit the type of antibody tested by the test kit, as any antibody that is capable of being tested in bodily fluid, such as blood, serum or plasma or other bodily fluids may be tested.
  • the antigen is Actinomyces.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is Aerobacter aerogens.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is Bacillus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is Bacteroides.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • 150 ⁇ l of a staining buffer is added.
  • the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is from the species Bartonella.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected from a species of Borrelia.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected from a species of Brucella.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the, now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected from a species of Campylobacter, or detection of an antibody or antibodies specific to the antigen
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold
  • the staining buffer is Protein A coupled to colloidal gold
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected from a species of Chlamydia.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected from a species of Clostridium.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is selected from a species of Corynebacte ⁇ um.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be H. pylori.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be Heliobacter.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example; Once the destaining buffer flushes the system, results may be read immediately. When both test position T and control position C appear red, a test result is positive for the presence of antibodies indicative of a particular disease, the antibodies specific for the antigen.
  • the antigen is selected to be Hemophilus influenzae.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Klebsiella.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • it is advised to wait for about three minutes or until a diluted sample is a clear red, before going on to the next step of loading the diluted sample.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Leptospira icterohemorrhagiae.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from Leishmania major.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • 150 ⁇ l of a staining buffer is added, In one example, the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example, Once the destaining buffer flushes the system, results may be read immediately. When both test position T and control position C appear red, a test result is positive for the presence of antibodies indicative of a particular disease, the antibodies specific for the antigen.
  • IX Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from Leptospira
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Listeria.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Moraxella.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Mycobacteria.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device, For a blood sample, it is advised to wait for about three minutes or until a diluted sample is a clear red, before going on to the next step of loading the diluted sample.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Neisseria.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Pasteurella.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is added.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Pneumococcus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Rickettsia.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Salmonella
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is selected to be from a species of Shigella.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Staphylococcus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Streptococcus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from Treponema pallidum.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • 150 ⁇ l of a staining buffer is added.
  • the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from Yersina.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device, For a blood sample, it is advised to wait for about three minutes or until a diluted sample is a clear red, before going on to the next step of loading the diluted sample.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from Candida albicans.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from Absidia.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from Acremonium.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from Alternaria.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold
  • the staining buffer is Protein A coupled to colloidal gold
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from Basidiobolus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is selected to be from Blastomyces.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold
  • the staining buffer is Protein A coupled to colloidal gold
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Coccidioides.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Cryptococcus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted' sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is selected to be from a species of Curvalaria.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold
  • the staining buffer is Protein A coupled to colloidal gold
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Epidermophyton.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Exophiala.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Geotrichum.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer, The 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • 150 ⁇ l of a staining buffer is added.
  • the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Histoplasma capsulatum.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Madurella.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is selected to be from Malassezia.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from Microsporum,
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from Moniliella.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from Mortierella.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is added.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from Phialemonium.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from Phialophora.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is selected to be from Prototheca.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from Pseudallescheria.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is selected to be from Pseudomicrodochium.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold
  • the staining buffer is Protein A coupled to colloidal gold
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Phythium.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • 150 ⁇ l of a staining buffer is added.
  • the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Rhinosporidium.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device, For a blood sample, it is advised to wait for about three minutes or until a diluted sample is a clear red, before going on to the next step of loading the diluted sample.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Rhizopus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Scolecobasidium.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device, For a blood sample, it is advised to wait for about three minutes or until a diluted sample is a clear red, before going on to the next step of loading the diluted sample.
  • the staining buffer is Protein A coupled to colloidal gold
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is selected to be from a species of Sporothrix.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Stemphylium.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Trichophyton.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is selected to be from a species of Trichosporon.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Xylohypha.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Babesia.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is selected to be from a species of Balantidium,
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining ' buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is selected to be from a species of Balantidium.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is selected to be from a species of Besnoitia.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l .of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Cryptosporidium.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • 150 ⁇ l of a staining buffer is added.
  • the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Eimeria.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Encephalitozoon.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example, Once the destaining buffer flushes the system, results may be read immediately, When both test position T and control position C appear red, a test result is positive for the presence of antibodies indicative of a particular disease, the antibodies specific for the antigen, Example 72- Entamoeba
  • the antigen is selected to be from a species of Entamoeba.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Giardia.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device, For a blood sample, it is advised to wait for about three minutes or until a diluted sample is a clear red, before going on to the next step of loading the diluted sample.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Hammondia.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example, Once the destaining buffer flushes the system, results may be read immediately. When both test position T and control position C appear red, a test result is positive for the presence of antibodies indicative of a particular disease, the antibodies specific for the antigen.
  • IX Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Hepatozoon.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is selected to be from a species of Isospora.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Leishmania.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Microsporidia.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is selected to be from a species of Neospora.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Pentatrichomonas.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Plasmodium.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Pneumocystis.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • 150 ⁇ l of a staining buffer is added.
  • the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Sarcocystis.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer, The 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Theileria.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Toxoplasma.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Trypanosoma.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold
  • the staining buffer is Protein A coupled to colloidal gold
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Schistosoma.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of Schistosoma.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is selected to be from a species of an adenovirus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer. The 150 ⁇ l of the
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of a coronavirus.
  • the coronavirus antigen may be a SARS antigen, for example.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example. Oncejhe destaining buffer flushes the system, results may be read immediately. When both test position T and control position C appear red, a test result is positive for the presence of antibodies indicative of a particular disease, the antibodies specific for the antigen.
  • IX Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of cytomegalovirus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer, The 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of a Dengue virus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of an Ebola virus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of an Epstein-Barr virus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is from a species of a measle virus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • 150 ⁇ l of a staining buffer is added.
  • the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is from a species of a chickenpox virus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is selected to be from a species of an enterovirus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is a Hepatitis A antigen.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is a Hepatitis B antigen.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is a Hepatitis C antigen.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device, For a blood sample, it is advised to wait for about three minutes or until a diluted sample is a clear red, before going on to the next step of loading the diluted sample.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is a Herpes simplex virus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer, The 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the antigen is a HIV 1 antigen such as p24 for detecting HIV- 1 .
  • the p24 antigen also works for detecting a HIV-2 antigen,
  • the p24 antigen consists of the following sequence:
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device. For a blood sample, it is advised to wait for about three minutes or until a diluted sample is a clear red, before going on to the next step of loading the diluted sample.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the HIV antigen is a HIV-I gp 41 partial protein, which consists of the following sequence:
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen used to detect HIV infection is a gp41 peptide fragment which consists of the following sequence:
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • an antigen preparation for a diagnostic kit comprises at least two HIV antigens, such as gp41 and p24 in a 1 :1 ratio.
  • g ⁇ 41 at a concentration of 1.6 mg/ml and a p24 concentration of 1.47 mg/ml may be prepared to a final concentration of 0.8 mg/ml gp41 and 0.735 mg/ml p24.
  • an HIV-I antigen an HIV-2 antigen or both are used.
  • an antigen mixture may be prepared.
  • Peptide antigens gp41 and g ⁇ 36 are dissolved in distilled H 2 O at concentration of 2 mg/ml each.
  • a ⁇ 24 purified protein is suspended at a concentration of 2 mg/ml in 20 mM carbonate buffer (pH of 9.6).
  • An antigen cocktail is prepared at ratio of gp41 :gp36:p24 at a molar ratio of 5:2:3. The prepared antigen cocktail is then distributed into aliquots and kept in -20° C degree.
  • the antigen cocktail is immobilized on a filter paper made of a cellulose having a substantial ⁇ -cellulose content.
  • the cellulose content is 98%, for example.
  • a cellulose filter having a particle retention size of 20-25 ⁇ m and an ash percentage of 0.06% is used, for example.
  • a frozen antigen cocktail may be thawed before loading to the cellulose filter paper.
  • One microliter ( ⁇ l) of antigen (about 2 ⁇ g) is loaded on the filter paper and is air-dried and stored at room temperature before assembling the antigen loaded filter paper in a test device.
  • an antigen cocktail is prepared using peptide antigens gp41 and gp36 dissolved in distilled H 2 O at concentration of 2 mg/ml each.
  • a p24 purified protein is suspended at a concentration of 2 mg/ml in 20 mM carbonate buffer (pH of 9.6).
  • an antigen cocktail is prepared at ratio of gp41 :gp36:p24 of 5:2:3.
  • the prepared antigen cocktail may be distributed into aliquots and kept in -20° C degree.
  • Frozen antigen cocktail may be thawed before loading onto a cellulose filter paper.
  • One ⁇ l of antigen (about 2 ⁇ g) is loaded on a cellulose filter paper, which is air-dried and stored at room temperature before assembling the loaded filter paper in a test device.
  • two HIV-I antigens are used.
  • the antigens are expressed in bacteria and purified using standard molecular biology methods. They may be a HIV-I p24 protein, as previously discussed, and a HIV-I gp41, which may be either be the whole protein, partial protein or peptide fragment.
  • a homologous sequence exhibits more than 80% identity with an amino acid sequence of a g ⁇ 41 peptide, for example.
  • the antigen is an influenza viral antigen such as an influenza A, B or C antigen, for example.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is from a leukemia virus
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is from a Marburg virus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is a papilloma virus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is a species of paramyxovirus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is a species of pestivirus.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is a picorna viral antigen.
  • the antigens may include a poliovirus capsid antigen, or a pox virus antigen.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is a rabies viral antigen.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • the antigen is a reovirus antigen.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • 150 ⁇ l of a staining buffer is added.
  • the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is a respiratory syncytial viral antigen.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example, Once the destaining buffer flushes the system, results may be read immediately. When both test position T and control position C appear red, a test result is positive for the presence of antibodies in a particular disease, the antibodies specific for the antigen.
  • IX Dulbecco's Phosphate Buffer Saline
  • the antigen is a rubella viral antigen.
  • 10 ⁇ l of scrum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is a rotavirus antigen.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is a vaccinia viral antigen.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is a human T-lymphotropic viral antigen.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold
  • the staining buffer is Protein A coupled to colloidal gold
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is from a non-infectious disease, such as cancer.
  • the cancer is prostate cancer and the antigen is a prostate specific antigen (PSA)
  • PSA prostate specific antigen
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • the antigen is an A68 antigen, from Alzheimer's disease.
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then added to the center of the test device.
  • a staining buffer is Protein A coupled to colloidal gold.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • IX Dulbecco's Phosphate Buffer Saline
  • DPBS Dulbecco's Phosphate Buffer Saline
  • two or more antigens may be detected by the test kit.
  • the two different antigens may be a viral and a bacterial antigen, for example.
  • the bacterial antigen may be a Mycobacterium Tubercolis.
  • the viral antigen may be a Hepatitis antigen or a HIV antigen, for example,
  • 10 ⁇ l of serum, plasma, or whole blood of the test sample is first diluted with 150 ⁇ l of dilution buffer.
  • the 150 ⁇ l of the now diluted sample is then ' added to the center of the test device.
  • the staining buffer is Protein A coupled to colloidal gold.
  • 200 ⁇ l of destaining buffer is added.
  • the destaining buffer may be Dulbecco's Phosphate Buffer Saline (IX) (DPBS) solution, for example.
  • Combinations that are tested using the test kit are not merely a combination of viral and bacterial antigens, In another example, a combination of two or more viral antigens may be tested. In another example, a combination of viral, parasital, bacterial and fungal antigens may be selected. In one example of type of combination of viral, parasital, bacterial and fungal antigen, a combination of fungal and viral infections may be tested. The combinations described herein are not limited to the specific examples disclosed.
  • test dots will be present, indicating that the test result is positive for the presence of antibodies for two or more particular diseases.
  • two test dots testing for a combination of viral and bacterial antigens indicate that the person has antibodies for a viral disease and a bacterial disease
  • FIGs 4A-C an illustration of a plasma sample tested using a nitrocellulose membrane 200 is compared with a sample tested using a cellulose filter paper 220.
  • the nitrocellulose membrane as presented in the sketch of Figure 4A fails, providing poor contrast and requiring a longer time to perform the test than for the rapid test kit of Figure 4C, using cellulose reaction layer 220.
  • a red residue of colloidal gold solution remains in the testing region of Figure 4A that did not pass through the nitrocellulose reaction membrane.
  • the nitrocellulose membrane 200 obtained from Bio-Rad Laboratories, had a pore size of 0.45 microns.
  • a device membrane 205 uses a nitrocellulose mixed ester membrane having a pore size of 5 microns. Plasma fails to flow through, causing this membrane to fail also.
  • a flow rate of PBS is measured using a modified ASTM Standard for measuring flow rate through a 7 cm circle of filter paper folded in quarters and suspended in a ring. Then, several filter papers were used to make test kits using the same antigens and loading. Tests were performed using HIV positive samples, and the color intensity of test spots were determiend using the color index value chart of Figure 6.
  • Figure 5 shows a graph of color index value versus DPBS flow rate. Measurements are shown for water and phosphate buffered saline, using qualitative filter paper and wet strengthened filter paper having various ratings for particle retention size. Data for Figure 5 is found in Table 2. The error bars in Figure 5 represent high and low data values. High titer samples are shown with squares and low titer with circles.
  • Flow rate is correlated with color index values.
  • the low flow rates are more sensitive than higher flow rate membranes.
  • six different types of WhatmanTM filter paper were tested; each having particle retention sizes ranging from 2.5 microns to 30 microns.
  • the flow rate showed a plateau region between about 6 to 20 microns with a flow rate of about 0.1 to 0.2 mL/min/cm 2 , The plateau region corresponded to an optimal combination of sensitivity and flow rate for testing samples, whether based on blood, serum or plasma, in one example of an HIV antibody sensitive test kit.
  • a modified ASTM Standard measurement was used to determine the flow rate of each membrane.
  • Filter paper was dimensioned to a 7 cm diameter circle, The paper was placed in filtering solution (both PBS and water were tested) for a time sufficient, for the paper to be completely soaked. Then the paper was placed flat in a funnel, except for edges, which were folded upwards. Then, 5 ml of filtering solution was added to the center of the funnel and time was measured using a stopwatch. When an amount of the filtering solution had passed through the filter, the time was recorded.
  • a filter paper of qualitative type had pore sizes of 2.5, 6, 11 and 20-25 microns (which we have graphed as 20 microns), A wet strengthened filter paper had a particle retention size of 23 and 30 microns.
  • the flow rates in water for a filter paper with a particle retention range from 2.5 microns to 23 microns is in the range of about 0.04 mL/min/cm 2 to about 0.4 mL/min/cm 2
  • the flow rates in DPBS are also in the range of about 0,04 mL/min/cm 2 to 0.4 mL/min/cm 2 . It is not clear that there is any statistical significances in the measured differences between water and PBS.
  • the nitrocellulose mixed ester membrane filters used were Magna Nitrocellulose mixed ester membrane filters, manufactured by GE Infrastructure Water and Process Technology.
  • the pore size used in the example is 5 microns.
  • the flow rate of nitrocellulose mixed ester membrane in water was measured in mL/min/cm 2 measured at 520 mmHg (10 psi), at 20 degrees Celsius.
  • the air flow rate is measured in units of L/min/cm 2 of filtration area, measured at 520 mmHg (10 psi), at 20 degrees Celsius (68 degrees Fahrenheit).
  • the Bubble Point pressure occurs at which air is first forced through pores of water-wet membrane.
  • Table 1C Properties of cellulose filter papers is shown in Table 1C.
  • Table 1C obtained from a Whatman web site shows typical properties of cellulose filters tested, such as particle retention liquid, and airflow rate. Such properties may be used to select for a particular filter paper. Grades 1, 3, 4, 5, 113 and 114, as reported in Table 1C, were utilized in preparing test kits. Wet strengthened qualitative cellulose filters contain a small quantity of a chemically stable resin to give improved wet strength. For these tests, filter paper is cut down into circles with a diameter of 7 cm for flow rate measurements, and the filter papers were dimensioned to 1 inch by 1 inch squares for use in test kits.
  • Table 2 shows the respective color index values for each low titer and high titer sample tested at a respective particle retention size and flow rate.
  • Figure 5 shows mean color index and the color index bars show high and low values of the color index. Samples having a color index value of 1 are considered to be low titer samples, while samples having a color index value greater than 2 considered to be high titer samples. The cellulose filter paper with about a 1.2 mL/min/cm flow rate failed on each low titer sample.
  • Figure 6 illustrates, schematically in a black and white line drawing, a color index for semi-quantitative determination of the sensitivity by measuring color index values.
  • the background associated with 0 indicates that no contrast is visible between a test spot and the background. Anything darker than background is a 1 , which is represented by light shading in Figure 6.
  • a value of 1 or greater is deemed a positive test result.
  • a color index value greater than 2 corresponds to the high titer samples and is represented in Figure 6 by darker shading.
  • a higher contrast between background and the test spot is represented by cross hatching 3, and the highest contrast is represented by double cross hatching 4.
  • This schematic representation relates to actual colors that are shown in the disclosure of United States Patent Application 12/008,861, which is incorporated by reference.
  • Figure 6 is an example of a color index chart, the scale runs from 0, which is negative for the presence of an antibody or antibodies specific for a given antigen, to a 4, which is the highest semi-quantitative value.
  • An index value of 0 indicates that pink staining of the background may occur but does not indicate presence of a discernable dot.
  • An index value of 1 is distinguishable from the background, but is not darker than the color represented by 1.
  • An index value of 2 indicates a clearly visible dot darker than 1, but not darker than 2.
  • a value of 3 is a highly intense dot darker than 2 but not darker than the reference provided at 3.
  • a color index value of 4 is darker than the reference labelled 3.
  • comparison of plasma and blood samples obtained from the same donor sample are shown in Figure 9, for example.
  • Blood tests (a), (b), have control spots 312, 332 and test spots 314, 334 comparable in color index value to the control spots 322, 342 and test spots 324, 344 of plasma samples (c), (d). Both whole blood and plasma may use the same test kit with the same color index value chart.
  • Figure 8 shows a comparison of tests using samples of blood and plasma, The color index values are measured.
  • the results for blood and plasma tests are remarkably similar which is very surprising and unexpected.
  • Most tests kits cannot be used to test whle bood.
  • all of the others are inoperative when used with the blood rather than plasma or serum.
  • Use of whole blood allows testing to be conducted in the field where centrifuges are not easily available, and represents a substantial advantage over other test kits.
  • Table 3 shows data reported graphically in Figure 8. Some samples were tested twice, while other samples were tested once. Table 3 compares data for samples using blood and samples using plasma, from the same source and using the same type of celulose reaction layer.
  • Plasma, serum and blood samples all have similar visual results. For example, a comparison of plasma and blood samples is shown in Figures 9, 11, 12 and 13. Examples using whole blood ( Figures 9, 11) and blood plasma ( Figures 12, 13) are schematically represented and tested positive for HIV. These images are represented by test sample 84, as reported in the tabulated data of Table 3. Positive tests spots for presence of HIV are indicated by test spots 314, 324, 334, 344 and control spots 312, 322, 332, 342. Test samples 260 and 262 were obtained from the same donor sample. One used blood while the other used plasma. Similarly, test samples 270 and 272 were obtained from the same donor sample, with one for blood and one for plasma. Both blood and plasma samples tested 3 on the color index scale,
  • Figure 10 shows a bar graph representing color index values for various samples using a rapid test kit with a flow rate of about 0.1 mL/min/cm 2 in DPBS and a commercial assay, using the Reveal ® G3. Most of the plasma samples using the test kit had better contrast than plasma samples using MedMira ® Reveal ® G3 test kit. 2 In Figure 11 , some representative comparisons of a rapid test kit with a Reveal ® G3 kit are shown. Rapid test kits having cellulose filter paper with a flow rate of about 0, 1 mL/min /cm 2 in DPBS were tested.
  • the procedure for using a rapid test kit includes adding 150 microliters of Phosphate Buffer Saline (PBS) solution is added to a freeze dried staining buffer. 10 microliters of plasma are diluted with 150 microliters of PBS solution, The kit is loaded with the diluted plasma, 150 microliters of staining buffer, and 200 microliters of PBS solution in succession. The test duration is less than two minutes, qualifying as a rapid test.
  • PBS Phosphate Buffer Saline
  • blood and serum alternatively, may be used, in addition to plasma. This is not the case for other commercial test kits.
  • the Reveal ® G3 kit used 3 drops of Universal Buffer added to the kit, followed by 1 drop of plasma. Then 3 drops of Universal Buffer are added to the kit. Then an instant gold cap was added on the kit, with 12 drops of Universal Buffer added through the cap. Optionally, an additional 3 drops of Universal Buffer may be added. The test duration is less than three minutes.
  • the term "Universal Buffer” is used in the instructions for the Reveal ® G3 kit. Test kit 400 tested HIV positive, which is the same result as the test kit 300 of Reveal ® G3. Both kits tested 1 on the color index scale.
  • the Reveal ® G3 of Figure l l(f) shows a G3 test kit 320 that tested patient sample BBI #10 as negative.
  • Rapid test kit 420 for sample BBI #10 tested positive, having a color index of 1.
  • the rapid test kit 420 indicated HIV-positive even though the Western blot showed indeterminate.
  • sample BBI #10 was from an Anti-HIV-1 PRB204 performance panel
  • MedMira ® and Reveal ® are registered trademarks of MedMira Laboratories, Inc., Toronto, Canada. purchased from BBI Diagnostics, which had tested the panel on different kits. A comparison of BBI #10 with other competing kits showed that sample BBI #10 is positive using an Abbott DetermineTMHIV-l/2. Other kits such as OraQuick ® and Uni-GoldTM tested negative. 3 A Western blot test of BBI #10 was indeterminate. BBI refers to screening assay PRB 204, which is shown in Tables 4 and 5. While Western blot is the gold standard, an indeterminate Western blot fails to identify either a positive or negative test result for HIV.
  • this test determined that the test kit sample 460 was low titer with a color index value of 1.
  • a rapid test kit may be used for screening, and using a color index scale, may also serve as a qualitative assay of antibody titer.
  • Reveal ® G3 (a) kit has a nitrocellulose membrane; therefore, a test with blood using the Reveal ® G3 kit failed, while a rapid test kit successfully found the sample to be negative for HIV. Blood did not flow through, but instead coagulates, in a Reveal G3 test kit,
  • the rapid test kit that successfully tested the blood contained filter paper with*a flow rate of about 0.1 mL/min /cm 2 in DPBS.
  • a glass fiber membrane was tested. Blood did not flow through the glass fiber membrane but instead coagulated on the surface.
  • the glass fiber membrane that was used was a Whatman ® GF/C. While Chan, in U. S Patent Publication No. 2004/0002063, taught the use of glass fiber membranes for use with blood samples, these tests clearly showed that using glass fiber membranes in test kits failed for tests using whole blood, without using the complex procedures of Chen.
  • the Reveal ® G3 test kit also cannot utilize blood samples, completely failing in that regard.
  • the test kit of Mahajan in U.S. Patent Publication No. 2004/0023210 ultilizes a nitrocellulose membrane, and also limits its use to serum and plasma.
  • a rapid test kit is capable of better contrast using blood, plasma and serum, which is a significant and important improvement for a rapid test kit.
  • Nitrocellulose is well-known in the art for binding proteins, which is why it is routinely used in Western blots and other assays.
  • none of these nitrocellulose assays use cellulose reaction membranes, and none are suitable as a rapid assay for use with whole blood.
  • Alternatives to nitrocellulose are seldom considered for use in test kits.
  • nitrocellulose fails, while cellulose selected in an operative flow rate range, such as 0.04-0.4 mL/min/cm works as well with blood as with plasma and serum.
  • the added flexibility makes the test suitable for use as a field test. Surprisingly, there is no loss of sensitivity or specificity with the use of blood in some example test kits used for testing HIV-positive samples.
  • Table 6 shows characteristics of a glass fiber membrane and shows data for glass fiber membranes.
  • particle retention the following is assumed: 2% initial penetration values using solid particulates dispersed in water. (Represents complete retention in normal laboratory analysis.).
  • flow rate the following is assumed: Vacuum filtration of prefiltered water through 2 1/16 in. (5.5cm) flat filter at 100 mmHg (1.9psi). Water absorbance assumes that there is an equilibrium volume of water absorbed by filter.
  • example rapid kits are compared to Reveal® G3 kits using high and low titer samples of blood plasma
  • AU test kits shown in the examples use a cellulose filter paper selected with a PBS flow rate of about 0.1 mL/min /cm 2 , unless otherwise specified herein.
  • Example test kits 500, 502, 504, 506, 508, 510 used specimens #80, #81, #82, #83, #84, and #91, respectively, and had better visual contrast than corresponding Reveal® kits 488, 490, 492, 494, 496 and 498.
  • Color index values for samples are represented in Table 7. The data show that all the test kits, except for one, sample #81, had better visual contrast than Reveal® G3 kits for the same plasma samples tested.
  • Figures 14A-C illustrate, schematically, examples of a test kit having both an antibody-based test spot 496 and a genetic probe test spot 498, in addition to the control test spots 494, 495, for example.
  • a single test kit has two testing windows 1, 2, which contain ' test regions for an antibody test 1 and a genetic probe 2, respectively.
  • a single test kit has both an antibody test region 496 and a genetic probe region 498 in a single test window.
  • the kit in Figure 14B may have a procedure that uses a single staining step or may have a sequence including a staining agent for the antibody test separate from application of the staining agent for the genetic probe.
  • a staining buffer may have a viral- specific genetic probe coupled to a nanotube or a particle, such as a colloidal gold particle, gold nanoparticle, silver nanoparticle, carbon nanotube or the like.
  • Figure 14C graphically illustates four possible outcomes of a test kit combining both antibody and genetic probe test regions 496, 498, when it is assumed that the control spot is properly demonstrated.
  • a positive antibody spot 496 and a negative genetic probe spot 498 produces a first result 504 indicating the presence of antibodies but having no indication of the virus. This HIV-negative indication would suggest innoculation or the presence of maternal antibodies.
  • a second negative result 510 could be negative for both antibodies and RNA.
  • Two possible positive results 506, 508 might be demonstrated with either a positive indication for the genetic probe test region 498.
  • the presence or absence of antibodies may be a significant indication, leading to a different course of treatment or clinical monitoring regime, for example.
  • a patient might indicate positive for the presence of the virus prior to indicating positive for the presence of antibodies to the virus, due to a delay in being able to detect the presence of antibodies in the blood, for example.
  • a probe for an RNA or DNA compatible sequence may be immobilized onto blotting paper or filter paper, which may be cellulose filter paper or nitrocellulose filter paper, for immobilizing RNA or DNA having the compatible sequence, as illustrated in Figure 15 (a), for example,
  • cellulose filter paper is used for detecting the presence of a specific RNA, DNA or fragment thereof in a volume of bodily fluid passing through the filter paper, which allows for the passing of a significant volume through the surface of the filter paper in a short period.
  • a primer or a pair of primers may be used as a genetic probe with one primer being attached to a marker, such as a gold nanoparticle, and being included in the staining buffer, and another primer being immobilized on the paper in a spot or other indicator region of the paper to capture a specific RNA or DNA sequence.
  • a marker such as a gold nanoparticle
  • Each of the pair of primers are complementary to a specific RNA or DNA sequence, such as a viral RNA or single stranded DNA, for example.
  • the specific RNA or DNA is captured by the primer immobilized on the paper, as illustrated in Figure 15 (b) and (c), for example.
  • the primer attached to a gold nanoparticle, or other particles or nanotubes is immobilized on the specific RNA or DNA that is immobilized on the paper, as illustrated in Figure 15(d), providing a visual contrast compared to a portion of the paper having no immobilized primer. Contrast may be enhanced by a chemical reaction, such as in photodevelopment, fluroescence, such as under an ultraviolet light, or electrical properties, such as conductance, resistance or the luck.
  • the marker which may be a nanoparticle or nanotube, fluoresces or phosphoresces, such as when exposed to ultraviolet light, for example.
  • a rinsing solution may be used to wash residual staining buffer from the paper to provide enhanced contrast between the spot and the background, because the rinsing solution removes staining buffer only from the background and not the immobilized RNA or DNA complexes captured on the test spot. Then, the contrast between the spot and the background may be analyzed to determine the presence of a sequence of RNA or DNA in the bodily fluid, and in some examples, a relative level or concentration of the sequence of RNA or DNA in the bodily fluid may be determined, either qualitatively or quantitatively.
  • the resulting contrast on the test kit may be compared to a plurality of known viral loads, such as concentrations of 5000, 10,000 and 15,000 viral copies per milliliter.
  • the plurality of known viral loads may be used to provide a standard intensity of a marker region or of a contrast between a marker region and a background of the test kit.
  • the comparative concentration range of the viral load may be determined qualitatively or quantitatively.
  • Figure 16 schematically illustrates a gold nanoparticle before and after thiolization and functionalization with an oligomer, such as a single strand oligonucleotide.
  • an LTR oligonucleotide is used as an example for detecting viral load using a complementary pair of primers.
  • Primers may be derived from the LTR region of HIV-I isolates, for example. Contrast is visible between the spot (1) and the background under ultraviolet light, because there is an interaction with ssDNA-Au-RNA and complementary DNA (cDNA) to immobilize the nanoparticles, which are gold in this example, to a spot on the filter paper, as illustrated schematically in Figure 15.
  • spot (2) results from an interaction only between ssDNA-Au and ssDNA
  • spot (3) results from ssDNA only, without genetic probe interactions.
  • An absorbance (abs.) spectrum is shown in Figure 18 that differentiates a sample of sDNA-gold nanoparticels -RNA with complementary DNA from both ssDNA-gold nanoparticles alone and ssDNA-gold nanoparticles with ssDNA, as a control. Contrast is good for a a sample of blood having HIV-I present.
  • the genetic probe is capable for use in detecting the presence of HIV using the complementary DNA as a target for binding a marker to the marker region of a test kit.
  • An example of a test using a flow-through rapid test kit detects viral RNA using an LTR-specific DNA genetic probe attached to gold nanoparticles.
  • preparation of DNA-gold nanoparticle complexes may proceed as described by Mirkin et al. using citrate- stabilized gold nanoparticles and thiol modified DNA oligomers.
  • 5'- fluorescein, 3 '-thiol labeled oligonucleotides may be used for determining surface coverages.
  • a bifunctional DNA-gold nanoparticle conjugate may be prepared by adding a mixture containing the desired amount of oligonucleotides to an aqueous nanoparticle solution as reported by JJ. Storhoff, R.
  • Functionlized gold nanoparticles conjugated with a single strand oligonucleotide may be mixed with 0.5% of a 10% bovine serum albumin in phosphate buffer (BSA) solution (e.g. pH 7.4, BD), which may be dropped on a glass plate for spectral analysis using a ultraviolet spectrophotometer, for example.
  • BSA phosphate buffer
  • the ultraviolet spectrophotometer results in Figure 18 are capable of distinguishing the plate with the functionlized gold nanoparticles conjugated with single strand oligonucleotide, without or without additional single stranded oligonucleotide, from a similar plate with a complementray oligonucleotide added by dropping 25 microliters of the complementary oligonucleotide on the functionalized gold nanoparticles conjugated with single strand oligonucleotide.
  • an HIV genetic probe is prepared using functionalized gold nanoparticles conjugated with a single strand oligonucleotide for detecing the presence of HIV.
  • a single stranded DNA primer with a functionally identical sequence of HIV-I LTR is synthesized and immobilized in a spot on the cellulose filter paper of a rapid test kit of the present invention.
  • a bodily fluid such as raw blood, blood plasma, urine, saliva, or the like, is v added to a buffer solution or placed directly on the filter paper of a rapid test kit for a flow - through test.
  • the HIV RNA e.g.
  • HIV-I LTR hybridizes with the DNA primer on the filter paper
  • a staining buffer including the functionlized gold nanoparticles conjugated with a DNA probe is added to the filter paper, such that the functionalized gold nanoparticles conjugated with the DNA probe hybridize with the HIV RNA.
  • the gold nanoparticles concentrate at the HIV RNA, providing a red-tinted contrast to the background.
  • a destaining buffer may be used to further rinse the staining buffer from the background, while having no effect on the gold nanoparticle complexes hybridized to the HIV RNA.
  • a plurality of different DNA probes are selected to be complementary to a plurality of different regions of HIV RNA, such that one HIV RNA molecule has the possibility of binding several gold nanoparticles, improving contrast and senstivity of the rapid test kit for each type of HIV RNA detected by the test kit.
  • An important advantage of using a DNA probe is that it is the HIV RNA, itself, that is detected; therefore, vaccinated individuals will be negative in a test using a DNA probe and the level or concentration of HIV in a volume of bodily fluid may be compared and analyzed, directly.
  • a test kit detecting only antibodies for HIV could indicate a positive test for a vaccinated individual and could only be used to show the level of antibodies produced by the individual, not the level of the virus, itself.
  • a rapid test kit testing for both antibodies in one spot and using a DNA probe for another spot may provide both detection of antibodies and either a qualitative or quantitative analysis of an HIV viral load in the bodily fluids of an individual.
  • Figure 19 illustrates schematically an example of coupling carbon nanotubes with an oligonucleotide.
  • carbon nanotubes such as single walled carbon nanotubes or herringbone carbon nanotubes, are dispersed in a dimethylfromamide under ultrasonic agitation. Two hours of agistation is adequate for a sample of 1 milligram of carbon nanotubes in 2 millileters of dimethylfomamide, for example, This provides a carbon nanotube suspension with a density of 5 milligrams per milliliter having a black color.
  • the carbon nanotubes in suspension are thiolated by thoroughly mixing 2 milliliters of a 1 mM solution of a thiol having a thiol group (positively charged), such as a mercaptan, into the carbon nanotube (negatively charged) suspension. Centrifugation at 18,000 rpm for 15 minutes is adequate to collect the thiol-functionalized (or thiolated) carbon nanotubes by removing the the supernatant liquid. Distilled water may be used to wash the thiolated carbon nanotubes, at least three times in one example, to rinse away any of the unbound thiol molecules.
  • a mass of 10 milligrams of single stranded DNA may be added to 1 milligram of thiolated carbon nanotubes in 1 millileter of PBS (pH 7.0) at 4 degrees centigrade for twelve hours, for example. Afterwards, the suspension is centrifuged and the ssDNA-sulfur-carbon nanotube complexes are collected by removing the supernatant. Again, the DNA functionlized carbon nanotubes may be rinsed thoroughly to remove all of the unbound molecules of ssDNA.
  • carbon nanotubes alone have a different ultraviolet absorbance spectrum from carbon nantoubes conjugated with single strand oligonucleotides (B), which have a different absorbance spectrum compared to (C) non-complementary oligonucleotide (DNA) fragments and (D) complementary fragments of single strand oligonucliotides hybridized with carbon nanotubes conjugated with single strand oligonucleotides.
  • a genetic probe using carbon nanotubes conjugated with ssDNA may be used to detect the presence of complementary DNA, qualitatively or quantitatively, for example.
  • single strand DNA (ssDNA) functionlized carbon nanotubes may be included in a staining buffer that is added to a rapid test kit of the present invention after a bodily fluid, with or without dilution in a buffer solution, is deposited on the filter paper of the test kit.
  • the filter paper is prepared by including a spot with an immobilized genetic probe on the spot that is capable of immobilizing viral RNA or the DNA to be detected.
  • the hybridizing of viral RNA or the complementary DNA with the ssDNA functionlized carbon nanotube binds the sDNA functionalized carbon nanotube preferentially at the test spot of the rapid test kit, providing a contrast between the test spot and the background, for example.
  • the spectra in Figure 20 were obtained by preparing a glass slide with ssDNA functionalized carbon nanotube dropped on the surface of the glass plate, pre-coated with bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the ssDNA functionlized carbon nanotubes were allowed to dry overnight at room temperature (about 25 degrees centigrade).
  • a fluorescein isothiocyanate (FITC) label was added to complimentary oligonucleotides, which were dropped on the ssDNA functionalized carbon nanotubes, which were heated to 60 degrees centigrade for 50 seconds. Afterwards the samples were washed and observed under a microscope, and hybridization is observed within 25 seconds.
  • FITC fluorescein isothiocyanate
  • Atomic force microscopy was used to compare carbon nanotubes (A) before and (B) after ssDNA functionlization and after subsequent hybridization with (C) non-complementary DNA; and (D) hybridization with complementary DNA (cDNA), which shows that ssDNA functionalization and hybridization with cDNA takes place, when carbon nanotubes are used as a genetic probe (or marker),
  • cDNA complementary DNA
  • Figure 21A - 21C illustrate atomic force microscopy micrograph images of (A) carbon nanotubes (CNT), alone, without any ssDNA strand or fragment; (B) CNT functionalized by ssDNA; and (C) CNT functionalized by ssDNA and hybridization with a complementary DNA fragment, providing evidence of the hybridization process for preparing carbon nanotubes as a contrast marking agent for genetic probe test spots, for example.
  • any oligonucleotide having a complementary oligonucleotide may be hybridized with single wall carbon nanotubes according to the process dislcosed,
  • a rapid test kit is capable of detecting samples of DNA, RNA or fragments thereof without the need of polymerase chain reactions to amplify the amount of DNA or RNA within a sample of bodily fluids.
  • a small sample size may first be processed using a polymerase chain reaction technique to provide a sufficient concentration of DNA or RNA for detection by a rapid test kit.
  • no PCR is used, and the rapid test kit is capable of determining a qualitatitive comparison to a standard or quantitative viral load based on a measurable intensity, contrast or other physical property based on the concentration of particles, nanotubes or the like on a test region.
  • Figures 22A-B provide another example comparing non-complementary 786, 796 and complementary 788, 798 single strand oligonucleotides to a control 784, 794,
  • the complementary oligonucleotide 788, 798 hybridizes the DNA, RNA or fragment of the DNA or RNA, resulting in fluorescence.
  • Figure 23 illustrates a comparison of intensity with concentration of functionalized carbon nanotubes. A concentration of at least 750 pmol is preferred, but concentrations as low as 250 pmol are discernable.
  • a genetic probe is selected as a DNA primer for a viral RNA sequence, such as one of the viral RNA sequences for the viruses listed in the detailed disclosure.
  • one DNA primer may be coupled to a nanotube or nanoparticle, such as by thiolation and another DNA primer may be immobilized on a membrane, such ⁇ as a cellulose filter membrane for one of the examples of rapid test kits using an antigen,
  • both an antigen and a vrial RNA sequence may be detected to determine whether an individual has developed antibodies and a level or concentration of the viral load in the fluid sample volume tested,
  • a test kit may quickly determine if a regimen or treatment plan is not controlling an HIV infection, requiring immediate medical care or modification of a treatment plan.
  • oligonucleotids may be selected having complementary oligonucleotides with oligonucleotide sequences to be detected, such as some of the following, with underling and double underline indicating oligonucleotide sequences for binding with complementary single strand oligonucleotides,
  • a genetic probe is used in a point of care test.
  • the point of care test used a rapid test kit that provides results of the test at room temperature.
  • an HIV test kit uses genetic probes for hybridizing one or more oligo nucleotides with HIV specific regions of the HIV RNA.
  • the LTR region of HIV has regions that are conserved among a wide variety of HIV strains, are unique to HIV (as compared to human DNA, for example), and are hybridizable at room temperature by an oligonucleotide probe. Some unique regions are identified in the following examples of HIV viral RNA sequences. Portions that are unique are identified below using a single underline. HIV Unique Regions in Sequence Context:
  • TTGAGTGCTTC A test kit usable at room temperature is most suitable for field and point of care testing.
  • hybridization of a genetic probe and viral RNA is not conducted at room temperature.
  • the following portions of HIV RNA indicate in double underline certain portions identified as hybridizable at room temperature, for example.
  • oligonucleotide sequences suitable for point of care test kits are provided in the following examples: Sequence 1-44 (Before GAG region) GGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGC
  • Sequence 762-929 (Sequence in the GAG region)
  • Sequence 951-1113 (Sequence in the GAG region)
  • Sequence 2873-2930 (Sequence in the GAG-POL region)
  • HIV viral RNA Some additional examples of unique portions of HIV viral RNA are identified in the following sequence by single and double underline for use in hybridization and detection of HIV viral RNA with a genetic probe: GGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCC TCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGA
  • specimen were prepared on glass slides for absorption spectral analsysis.
  • a selected single strand oligonucleotide was conjugated with gold nanparticles by adding a mixture containing the desired amount of single strand oligonuclotide to an aqueous nanoparticle suspension or solution containing a nanoparticle precursor.
  • the oligonucleotide functionalized gold nanoparticles are mixed with 0.5 % BSA and are deposited onto a glass pate for absorption spectral analysis.
  • the oligonucleotide functionalized gold nanoparticles may be deposited onto a filter paper for hybridization with complementary oligonucleotides sequences to be detected.
  • a detection kit for viral RNA is prepared using a 33-mer oligonucleotide from the HIV-I LTR sequence.
  • the LTR sequence is relatively conserved among several HIV-I strains, including a clade C HIV 1084i and is not included in any vaccine constructs since none of them are live- attenuated vaccines. Thus, a test positive for this LTR sequence indicates the presence of live HIV-I strains and does not test positive for antibodies or innoculants used in vaccines.
  • env, or gag/pol may be selected to detect viral RNA of HIV-I ; however, some of these are included in vaccine constructs, which may cause a positive indication of the genetic probe, due to the presence of the vaccine constructs, especially if being used in vaccine clinical trials for detection of viral loading in a sample of bodily fluid, such as blood, serum, plasma or any other bodily fluid having a sufficient concentration of viral RNA for detection by the test kit.
  • the presence of oligonucleotides used in vaccine constructs may not distinguish HIV-infected individuals from those who have been vaccinated with a candidate vaccine including the oligonucleotides selected for detection within the vaccine constructs. Thus, it is preferred to avoid oligonucleotides used in such vaccine constructs for a test to detect the presence of viral RNA from a live virus.
  • a single stranded oligonucleotide complementary to a 33-mer HIV-I LTR RNA sequence is synthesized and tested for hybridization.
  • it may be used to funtionalize carbon nanotubes or gold nanoparticles, such as by thiolating the oligonucleotide.
  • absorbtion spectral analysis hybridization with the specific sequence of HIV LTR RNA may be tested.
  • a strong absorbtion spectra shows hybridization of the complementary oligonucleotide, while missing or very weak absorbtion spectra indicate that hybridization failed.
  • oligonucleotide is a non-complementary oligonucleotide.
  • Figures 22A and 22B illustrate regions 784, 794 before adding oligonucleotides coupled with single walled carbon nanotubes.
  • a complementary oligonucleotide may be immobilized on the matrix of a cellulose filter membrane, for example, such as the membrane used in an antibody test kit as illustrated in Figures 14A-14B,
  • a cellulose filter membrane for example, such as the membrane used in an antibody test kit as illustrated in Figures 14A-14B
  • an HIV-I -infected sample is added to the test cassette, such as by deposting bodily fluids (with or without a diluent)
  • the complementary oligonucleotide will hybridize with the RNA (HIV-I LTR) from the infected sample and will become fixed on the matrix of the membrane.
  • Carbon nanotubes or gold nanoparticles may be functionalized by a second single-stranded oligonucleotide complementary with another region of HIV RNA. If added to the surface of the membrane of the test kit, the functionalized carbon nanotubes or gold nanoparticles hybridize with the HIV RNA, binding the carbon nantobues or gold nanoparticles to a test region on the membrane. When a sufficiently large number of nanotubes or nanoparticles accumulate at a test spot, a contrast between the background and the test spot will become apparent.
  • Figure 23 illustrates a screening test for determining the concentration of functionalized single wall carbon nanotubes for use in a rapid test kit.
  • a test spot may be visualized during such a test in less than five to ten minutes.
  • Contrast may be enhanced by functionalizing the nanotubes or nanoparticles with a plurality of different complementary oligonucleotide probes targeting different regions of the HIV RNA sequence, such as different regions of the LTR sequence, for example.
  • the binding of the HIV RNA to the membrane to specific sequences not found in vaccines, no contrast will be apparent, unless the virus is present in the bodily fluid tested.
  • one HIV RNA strand or sequence may bind multiple functionalized nanotubes or nanoparticles, if such nanotubes or nanoparticles are functionalized with a plurality of complementary oligonucleotides, improving contrast.
  • the signal i.e. contrast
  • the concentration of RNA such as by using a PCR technique
  • only an HIV-infected sample will give a positive result. Therefore, the positive result in the viral RNA test will indicate a true HIV-I infection, and samples collected from those individuals who are vaccinated with any candidate vaccine constructs will be negative in the viral RNA test.
  • the complementary oligonucleotides used for functionlizing a nanotube or nanoparticle are thiolated at the 5 '-end and are mixed with gold nanoparticles.
  • the complementary oligonucleotides may be complementary to a region of the HIV virus, such as the LTR region .HIV RNA, for example.
  • different types of nanoparticles or nanoparticles functionalized with different markers are added to specific complementary oligonucleotides. Then, the presence of a concentration of the specific type of nanoparticles or the markers associated with specific nanoparticles will indicate the presence of one type of complimentary oligonucleotide. Since one complimentary oligonucleotide may be selected to hybridize with a specific type or strain of HIV, a test kit is able to detect specific types and strains of FIIV RNA present in a sample of a bodily fluid, for example.
  • complementary oligonucleotides are selected that are common to many different strains of HIV and/or to both HIV-I and HIV-2.
  • Several different complementary oligonucleotides may be immobilized on a membrane, such as a cellulose filter paper, in one or more than one area, and each of the plurality of complementary oligonucleotides may be selected to immobilize one or more different HIV types or strains.
  • a protocol described in Glynow, K et al, Oligonucleotide- functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for DNA analysis by hybridization, Anal. Chem., 75(16), (2003) p. 4155-60 is used to prepare a functionalized genetic probe using gold nanoparticles added to a stain used in an HIV antibody test kit.
  • the test kit includes a single window, as illustrated in Figure 14B, for example, having test spots for detecting the presence of both antibodies and HIV RNA.
  • the oligonucleotide probe may be synthesized and thiolated.
  • 0.9 nmol of the thiolated DNA probe is added to a 10 ⁇ l suspension of gold nanoparticles (about 1 ,5 pmol) at 4 0 C for 24 hrs.
  • a sodium chloride solution is added to the mixture to a concentration of 90 nmol/L, and is allowed to stand for another 24 hrs at 4°C.
  • the oligonucleotide-functionalized gold nanoparticles may be centrifuged at 2800 g for 45 min and may be suspended in 600 ⁇ l of 30% sucrose with 45 nmol/L NaCl.
  • the functionalized gold nanoparticle probes are lyophilized and stored at room temperature for prolonged periods.
  • RNA in one method for detection of viral RNA, 50 ⁇ l of an HIV-infected blood is diluted in 150 ⁇ l hybridization buffer.
  • the sample may be added to a preassembled test cassette having an LTR- complementary oligonucleotide immobilized on a test spot.
  • 200 ⁇ l of the functionalized gold nanoparticles are added to the test well as a genetic probe.
  • the genetic prove hybridizes the gold nanoparticles to complementary regions of the viral RNA.
  • 200 ⁇ l of washing buffer such as a saline solution, is added to reduce the background color and to increase contrast between the background and the test spot.
  • the entire viral RNA test can be performed in less than 5 to 10 minutes. Color of a test kit may remain stable for 48 hrs at room temperature.
  • a 33-nt oligonucleotide from HIV 89.6 proviral clone is immobilized on the membrane test spot of an HIV test kit.
  • This immobilized DNA primer is capable of hybridizing and fixing HIV LTR RNA on the test spot, because it is complementary to the HIV LTR region, including a clade C HIV 1084i.
  • Other DNA sequences complementary to the LTR may be used, alternatively or in addition to this oligonucleotide.
  • the length of the oligonucleotide is optimized for efficiency in capturing HIV RNA having a complementary LTR region.
  • a fluorescent oligonucleotide (siGLO from Thermo Scientific Dharmacon, CO) was added directly to the membrane of the ULTraPID cassette, and the binding was insufficient to prevent the fluorescent marker from being washed off the membrane surface with 0.3 M NaCl, which is normally included in the gold nanoparticle staining buffer.
  • a fluorescent oligonucleotide was first conjugated with chitosan nanoparticles, and the conjugate was applied to the surface of the membrane.
  • Figure 24 illustrates that the conjugate was stably immobilized on the membrane in this example.
  • Figure 24 has a green fluorescence 607 of the fluorescent oligonucleotide (converted to white in this image for purposes of illustration only), which contrasts with the uniformly dark (no fluorescence) background 606 of a sample prepared without first conjugating the fluorescent oligonucleotide with chitosan nanoparticles.
  • the fluorescent oligonucleotide is rinsed from the cellulose filter paper in the background 606 image, while the oligonucleotide conjugated with chitosan and chitosan derivatives immobilizes the fluorescent oligonucleotide on a portion of the cellulose filter paper.
  • the chitosan forms a cationic polymer that binds to the matrix of the membrane when conjugated with an oligonucleotide.
  • the chitosan helps to immobilize the complimentary oligonucleotides to the membrane used for testing for viral RNA or other RNA or DNA to be detected.
  • the one or more complementary oligonucleotides functionalizing nanotubes or nanoparticles may be hybridized on the target RNA or DNA to be detetected, providing contrast between the genetic probe test spots (G) and the background of the cellulose filter paper.
  • the genetic probe may be used to detect the presence of a specific RNA or DNA sequence that binds to the complementary oligonucleotides immobilized on the filter paper and coupled with the nanotubes and/or nanoparticles, and may be used to distinguish viral DNA from the mere presence of antibodies for viral DNA, for example.
  • bovine serum albumin (BSA) or streptavidin are used either alone or in combination with conjugation with chitosan nanoparticles to enhance the binding of the 33-nt oligonucleotide on a cellulose filter paper membrane, such as the membranes used in the examples of antibody test kits,
  • a portion of the membrane is coatedwith streptavidin, and a biotinylated 33-nt oligonucleotide is applied to the streptavidin coated portion of the membrane.
  • FIG. 25 a schematic of a detector 2500 is sketched that is capable of measuring light emitted by a detection region 2510 or transmitted through a detection region 2510 of a slide 2511.
  • a light source 2501 is provided in a housing -2502, and a charge coupled device or other photodetector or spectral analyzer 2505 is provided with a shield 2515 or collimator to capture and analyze the light.
  • Filters and optics may be provided as is known in the art for such detectors,
  • Table IB shows flow rate data for nitrocellulose mixed ester membranes with various pore sizes.
  • Table 4 Assay comparison for commercial kits and example test kits.

Abstract

La présente invention concerne un kit de test rapide qui peut être doté d’une sonde génétique, et une sonde de détection d’anticorps ou une combinaison d’une sonde génétique et d’une sonde de détection d’anticorps disposées dans une ou plusieurs fenêtres de test du kit de test. Une membrane de papier-filtre en cellulose avec un débit sélectionné dans une plage allant d’environ 0,04 à environ 0,4 ml/min/cm2 est utilisée dans un exemple. Le kit de test permet le criblage rapide d’ADN, d’ARN ou de fragments d’ADN ou d’ARN dans un fluide corporel ou des anticorps indiquant une exposition à cet ADN ou à cet ARN. La sonde génétique peut inclure un ADN monocaténaire ou un fragment d’ADN monocaténaire, tel qu’une amorce, immobilisé sur le papier-filtre, et un ADN monocaténaire, tel que la même amorce ou une amorce différente, conjugué à un marqueur, tel qu’un nanotube ou une nanoparticule. Par exemple, une nanoparticule d’or ou un nanotube de carbone peut être utilisé en tant qu’agent de coloration par conjugaison de la nanoparticule d’or ou du nanotube de carbone à une sonde génétique, telle qu’une amorce d’ADN capable de se lier à un ADN complémentaire ou à un ARN viral ou à un fragment de l’un de ceux-ci. La comparaison du contraste ou de l’intensité d’un point test à une norme permet de signaler une charge virale. En comparant une région test utilisant un marqueur génétique et une région test utilisant un antigène pour détecter des anticorps, on peut réaliser un test sensible et spécifique lors de l’utilisation d’un vaccin afin de déterminer l’efficacité du vaccin, par exemple.
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Cited By (1)

* Cited by examiner, † Cited by third party
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RU2557951C1 (ru) * 2014-05-27 2015-07-27 федеральное государственное бюджетное учреждение "Федеральный научно-исследовательский центр эпидемиологии и микробиологии имени почетного академика Н.Ф. Гамалеи" Министерства здравоохранения Российской Федерации (ФГБУ "ФНИЦЭМ им. Н.Ф. Гамалеи" Минздрава России) Способ получения мультиплексного риккетсиального диагностикума

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CN101978072A (zh) 2011-02-16
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WO2009128960A3 (fr) 2010-03-11
WO2009128960A2 (fr) 2009-10-22

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