WO1999056128A1 - Dosage immunitaire avec peptides mixtes - Google Patents

Dosage immunitaire avec peptides mixtes Download PDF

Info

Publication number
WO1999056128A1
WO1999056128A1 PCT/US1999/009331 US9909331W WO9956128A1 WO 1999056128 A1 WO1999056128 A1 WO 1999056128A1 US 9909331 W US9909331 W US 9909331W WO 9956128 A1 WO9956128 A1 WO 9956128A1
Authority
WO
WIPO (PCT)
Prior art keywords
filter
test
reagent
reagent layer
test device
Prior art date
Application number
PCT/US1999/009331
Other languages
English (en)
Inventor
Mohammed Afzal Chowdhury
Mary Ann Childs
David Bernstein
Janece Lovchik
William Trainor
Original Assignee
Universal Healthwatch, 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
Application filed by Universal Healthwatch, Inc. filed Critical Universal Healthwatch, Inc.
Priority to AU36709/99A priority Critical patent/AU3670999A/en
Publication of WO1999056128A1 publication Critical patent/WO1999056128A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis

Definitions

  • the present invention relates to diagnostic test devices that utilize one or more sample/reagent fluids to generate a signal that indicates a positive test result for each analyte tested.
  • Embodiments of the invention relate to improvements in fluid movement within such devices, improved resistance to chemical interferences, and in testing for multiple analytes using mixed peptides.
  • Diagnostic assays have become an indispensable means for detecting analytes in test fluids, and for the most part the medical profession has used highly automated clinical laboratories and sophisticated equipment for these determinations. There is, however, an expanding need for having analytical capabilities in doctors' offices, in the home, and in the field where electricity and other semblances of a laboratory are unavailable. Together with the diagnosis of disease or physiological conditions or disorders, there is a growing need to monitor the effects of drug therapy and chronic illness, to detect the use of drugs of abuse and to detect the presence of contaminants. The generation of test results can be followed by appropriate treatment or prophylaxis to prevent further public health problems.
  • Assay methods accordingly, have been developed which utilize one or more binding reactions that lead to color development in response to the presence of a test antigen.
  • Exemplary of these methods are antibody-based assays that utilize immobilized antibodies on a reagent material within a test device.
  • a test fluid is added to the device and wicks into a first absorbent material and then is transferred to another absorbent material.
  • one or more test reagents, necessary for one or more binding reactions and a color development reaction become wetted or dissolved within one or more materials in the device. Chemical reactions take place and cause a visual response to the presence or absence of analyte in the test fluid.
  • diagnostic devices that provide an accurate readout and also for devices that provide multiple analyte detection signals. This is particularly true for tests of infectious disease organisms such as hepatitis and HIV, where multiple epitope reactivity information is needed to confirm infection.
  • test devices results from their limitation for use with certain types of samples and test analytes.
  • a truly convenient and simple device for accurate testing of, for example, antibodies that indicate past exposure to infectious disease agents does not exist.
  • a unitized device is needed that contains all mechanical parts and one or more diagnostic reagents, such as sample pre-treatment components (eg. cell filters), binding members (eg. antibodies) and signal development reagents (eg. colloidal gold) and which can render a sufficiently accurate and fast result.
  • diagnostic reagents such as sample pre-treatment components (eg. cell filters), binding members (eg. antibodies) and signal development reagents (eg. colloidal gold) and which can render a sufficiently accurate and fast result.
  • test samples sometimes contain particulate matter which clogs up and creates inconsistent flows within a test device.
  • chemical interferences in test samples may non-specifically bind to physical parts of a test device, or react with chemical reagents such as antibodies or enzymes within the test device to cause false positive and false negative test results. Problems in each of these categories lead to uneven test signal development and concomitant mistakes in test development.
  • the first category of problems relating to wicking and seeping within test devices, frequently arises from the dual motions of test and reagent fluids both downward and in lateral directions. Lateral reagent flow contributes to a higher incidence of inaccurate results due to the tendency of spent reagents to accumulate at the periphery of a reaction zone, for example between an antibody and an antigen. Down directed (transverse) motion perpendicular to a surface and crossing from one surface to another between adjacent parts is affected by contours and contact vagaries between the surfaces. In both cases the reagents tend to interact and produce a test signal that can be mistaken for a true positive or negative result.
  • reagent layer such as a membrane
  • reagent layer the reaction portion
  • absorbent pad another absorbent
  • the sandwich method of holding these two absorbents together may provide the test fluid, reaction, or wash fluid an additional and undesirable path to move.
  • a portion of fluid that is applied to the device may go around the reaction portion or penetrate the reaction portion incompletely.
  • test fluid(s) application of test fluid(s) to a dry reagent in a device often leads to uneven dissolution of the reagent and inhomogeneity of test signal development.
  • the unevenness of dissolution that occurs during the wetting process itself, and differences between samples may play a role in this problem, leading to inaccurate test results.
  • the second category of problems is uneven fluid flow due to interference by particulate matter and by viscosity of the test sample. Viscosity and/or particulate matter often limit the ability of a test device to give a correct result from blood or blood derived samples that may contain red blood cells, white blood cells, chylomicrons, debris, precipitates or other "cellular" elements. These elements may choke the flow of fluid within the device and affect reaction of test reagents such as an immobilized binding pair member. In fact, one of the most important challenges in the diagnostic field is the need to test blood which may comprise one-half by volume such particles. Any improvement to this art would have important consequences in the field of public health by allowing earlier diagnosis from samples such as blood that do not require processing at remote locations.
  • the third category of problems relates to chemical interferants that often are encountered in undiluted blood and blood derived products.
  • a chemical interferant in an individual blood sample may give an erroneous positive or negative result for that sample.
  • the biochemical origins of interferents and of their reactions generally are not known. However, attempts have been made to limit the effects of chemical interferents by adding protein such as gelatin, serum albumin and casein to assay buffer or to absorbents used in test devices.
  • a strategy to overcome the first problem of uneven fluid transfer within a test device is to eliminate the transfer of fluid from one absorbent to another by using a single absorbent.
  • US patent No. 5,212,065, issued on May 18, 1993 to Pegg et al. describes a device that employs a single porous membrane that serves as both a reagent support and spent reagent reservoir. This device directs the flow of test fluid and reagents and alleviates lateral diffusion and reagent backflow.
  • this device is limited to the use of a single absorbent.
  • different absorbents should be used within the same device, to optimize parameters such as porosity, hydrophobicity, ability to separately impregnate with reagents, etc. as needed for each step of an assay procedure.
  • housing multiple parts such as a filter, spreading layer and absorbent within a test device creates the problem of reproducible fluid transfer between these parts.
  • the control of fluid flow from one absorbent to another is exacerbated by this strategy.
  • RNA viruses such as HTV and hepatitis C
  • Improvements in such binding reagents should lead directly to more reliable tests and more efficacious therapies that compensate for viral strain specificity.
  • a diagnostic test should incorporate binding reagents that address this problem as well.
  • the inventors have explored both rapid assay devices and peptides used in those devices, particularly for HIV testing. During this work, a rapid and accurate device for diagnostics was discovered and a method of selecting peptides for diagnostic and therapeutic uses was discovered. Finally, specific peptides relating to HIV testing and therapy were designed by the method and used in the device for testing HTV infection. The device and pepides useful for the device are described separately below.
  • the present inventors have discovered that certain advantageous assays can be prepared by orienting components within a test device used to generate a signal in response to a test analyte so that the components contact each other more evenly, and without bridging, leading to more homogeneous fluid flow.
  • this objective is achieved by mounting two parts that form a mutual surface junction such that primarily the two surfaces, and not a mount or holder, participate in fluid contact with the junction.
  • Another embodiment employs materials that expand upon wetting and which are friction-held, leading to a more even contact between adjacent parts during test performance.
  • Another embodiment employs a filter to remove particulate interferences.
  • Another embodiment provides a choice of components and configurations that limits transverse flow of reagent and sample.
  • Yet another embodiment includes a glycosaminoglycan in at least one test component or fluid to prevent effects of chemical interference, particularly for an HIV test.
  • one or more of the above embodiments is combined in a multi-well test device suitable for testing more than one sample simultaneously.
  • Such embodiments serve to: (1) control fluid flow between absorbent materials in test devices; (2) control the problem of particulates and viscosity of samples; and/or (3) limit the effects of chemical interferences. More specific details for how embodiments of the present invention improve the reproducibility and accuracy of test results in the three enumerated problem areas are provided below.
  • a test device comprising a housing; an absorbent pad held by the housing; a reagent layer that comprises an immobilized test reagent, the layer being in intimate contact with the absorbent pad; a filter in contact with the reagent layer; an opening in the housing adjacent to the filter for receiving an aqueous sample into the filter; and a sleeve operatively holding the filter in physical contact with the reagent layer, wherein the filter protrudes from the sleeve such that the sleeve does not contact the reagent layer.
  • the test device further comprises a cover for closing the opening.
  • the cover may be a plug adapted to be attached to the opening by friction.
  • the sleeve is reversibly attached to the housing via a bayonet mount and in another embodiment the sleeve prevents or delays the escape of moisture from the device by covering the reagent layer.
  • the reagent layer of the test device further comprises a binder that immobilizes human antibody as a control to monitor the presence of an adequate sample.
  • at least the filter or reagent layer comprises a glycosaminoglycan.
  • the filter of the test device comprises an upper portion for filtering blood and a lower portion for dispersing filtrate to the reagent layer.
  • the lower portion of the filter in the test device comprises a material that passes fluid at a slower rate compared to the material of the upper portion.
  • the upper portion of the filter comprises two separate filters that are held together and which act together to block the flow of particulates (including cells) in a sample.
  • the filter of the test device has a depth at least one tenth its diameter and in another embodiment the immobilized test reagent comprises a molecule for binding a sample analyte.
  • the sample analyte is an antigen of an infectious disease agent, which may be a human immunodeficiency virus.
  • the sleeve holds the filter by friction and in further embodiments the filter, at least one of its portions, or the sleeve itself is adapted to swell upon wetting.
  • the filter protrudes at least about 0.02 to about 0.25 mm or more from the sleeve. In an acceptable embodiment, the filter protrudes about 0.5 mm (e.g., 0.5 mm) from the sleeve. In other embodiments the filter protrudes more than 0.5 mm from the sleeve.
  • a test device comprising a housing having an opening; an absorbent pad within the housing; a reagent layer having an immobilized test reagent, the layer being positioned on top of the absorbent pad; a filter on top of the reagent layer and aligned with the opening so that the filter is adapted to receive a test sample; an opening in the housing above the filter that can admit an aqueous sample; and a sleeve operatively holding the filter in physical contact with the reagent layer, the sleeve being connected to the housing, wherein the filter protrudes from the sleeve so that the sleeve does not contact the reagent layer.
  • a test device comprising: a housing having an opening; an absorbent pad held by the housing; a reagent layer having immobilized antigens which may include one or more epitopes from HIV-1, HIV-2 and/or HIV-1 subtype o, the layer being positioned in contact with the absorbent pad; at least one filter in contact with the reagent layer and aligned with the opening to receive an aqueous sample into the filter.
  • at least the filter or reagent layer comprises a glycosaminoglycan.
  • a blood HIV diagnostic assay kit contains a test device as described in alternate embodiments above.
  • the test device further comprises a cover for sealing the device and a glycosaminoglycan.
  • the glycosaminoglycan is selected from the group consisting of chondroitin sulfate, dermatan sulfate, heparin sulfate, keratan sulfate, hyaluronic acid and heparin.
  • the kit includes printed instructions for carrying out the assay method. The printed instructions may be present in the form of a booklet.
  • a process for detecting HIV antibody from whole blood comprising the steps of: (a) providing a test device that contains a housing having an opening, an absorbent pad held by the housing, a reagent layer having immobilized antigens that may include HIV-1, HIV-2 and/or HIV-1 subtype o, the layer being positioned in contact with the absorbent pad, at least one filter in contact with the reagent layer and aligned with the opening to receive an aqueous sample into the filter; (b) applying a blood sample to the opening of the test device; (c) applying at least one wash fluid to the test device; (d) allowing one or more reactions to proceed in the reagent layer of the device; and (e) detecting an optical change in the reagent layer in response to the presence of anti-HIV antibody.
  • the wash fluid of the process comprises a glycosaminoglycan and in yet a further embodiment the glycosaminoglycan is selected from the group consisting of chondroitin sulfate, dermatan sulfate, heparin sulfate, keratan sulfate, hyaluronic acid and heparin.
  • the glycosaminoglycan is heparin and is present in the wash fluid in a concentration from about 5 international units per milliliter to about 25 international units per milliliter (e.g., from 5 usp/ml to 25 usp/ml).
  • each test well comprises a housing; an absorbent pad held by the housing; a reagent layer that comprises an immobilized test reagent, the layer being in fluid communication with the absorbent pad; a filter in fluid communication with the reagent layer; an opening in the housing adjacent to the filter for receiving an aqueous sample into the filter; and a sleeve operatively holding the filter in physical contact with the reagent layer, wherein the filter protrudes from the sleeve such that the filter exerts a greater pressure onto the reagent layer than does the sleeve.
  • test device that more accurately confirms exposure to virus by detecting antibodies that react with at least two separate antigens at a plurality of sites.
  • the device detects exposure to more than one virus from the same sample.
  • the invention provides a test device comprising a reagent layer having at least two immobilized test sites, each having an immobilized test reagent that becomes visible upon detecting a target substance, and a control indicia for indicating whether a test is properly carried out.
  • the control indicia is placed in relation to the test sites so that, regardless how the test device is held or positioned, the test sites readily can be identified.
  • a test device is provided wherein the reagent layer has at three immobilized test sites, one of which contains a p24 antigen, another a gp41 antigen, and the other a gpl20 antigen for detecting HIV-1.
  • a test device is provided wherein the reagent layer has at three immobilized test sites, one of which contains an HIV antigen, another an HCV antigen, and the other a syphilis antigen for detecting multiple viruses.
  • the invention provides an HIV-1 diagnostic device comprising a reagent layer positioned in the housing and in contact with the filter, the reagent layer having three test sites, one of which contains an immobilized p24 antigen, another one of which contains an immobilized gp41 antigen, and the other of which contains an immobilized gpl20 antigen, and an indicia line containing an anti -human IgG; a control agent for visually marking the indicia line upon properly applying a test sample; and a control agent for visually marking the three test sites upon detecting HIV-1 antibodies complementary to the immobilized antigens.
  • the control line is placed in relation to the test sites so that, regardless how the test device is held or positioned, the test sites can be readily identified.
  • the invention further provides a multiple virus diagnostic device comprising a reagent layer positioned in the housing and in contact with the filter, the reagent layer having three test sites, one of which contains an immobilized HIV antigen, another one of which contains an immobilized HCV antigen, and the other of which contains an immobilized syphilis antigen, and an indicia line containing an anti-human IgG control agent for visually marking the indicia line upon properly applying a test sample; and a control agent for visually marking the three test sites upon detecting antibodies complementary to the immobilized antigens.
  • the control line is placed in relation to the test sites so that, regardless how the test device is held or positioned, the test sites can be readily identified.
  • compositions for early prevention, detection and treatment of viral infection It is another object of the invention to provide compositions for early prevention, detection and treatment of viral infection. It is a further object of the invention to provide peptide sequences that yield improved solubility for detecting and treating infection such as from HIV compared to detection and treatment using natural protein, recombinant protein or peptide. It is another object of the invention to provide methods for finding antigens most suited for early prevention, detection and treatment of viral infection.
  • One embodiment of the claimed invention is a diagnostic test for detecting viral infection that comprises at least two peptides from the immunodominant region of a viral protein, wherein the peptides have a common overlapping epitopic sequence that differs by more than a single amino acid change and wherein the peptides have differing cross-reactivity with viral strains having natural mutated proteins in diagnostic testing and in therapy.
  • Another embodiment is a method of selecting peptides that are useful for immunodiagnostic testing of antibodies specific for a highly mutagenic virus from a blood sample, comprising (a) collecting blood samples from various individuals infected with the virus; (b) testing a blood sample from each individual with alternative peptides corresponding to the same immunodominant region of a viral protein; (c) selecting a first peptide that reacts with a first set of samples but fails to react with or reacts poorly with a second set of samples; (d) selecting a second peptide that does not react or that reacts poorly with the first set of samples but reacts with the second set of samples; and (e) combining the first and second peptides in a test for the detection of virus exposure in blood samples.
  • Another embodiment of the claimed invention is a combination of at least two peptide antigens in a diagnostic test, the peptide antigens corresponding to an immunodominant region of a viral protein, wherein the peptides have a common overlapping epitopic sequence that differs by at least a single amino acid change and wherein the peptides have differing cross-reactivity with viral strains of mutated protein. It is yet another object of the invention to provide diagnostic test devices and kits for detecting HIV infection and hepatitis C infection having at least two peptide antigens as described herein. Further objects of the invention readily will be apparent from the disclosure herein.
  • FIG. 1 is a perspective view of a test device in accordance with this invention.
  • FIG. 2 is a side view of the test device shown in FIG. 1.
  • FIG. 3 is a cross sectional view taken along the line of 3-3 shown in FIG. 2.
  • FIG. 4 is a perspective view of another test device in accordance with this invention.
  • FIG. 5 is a cross sectional view of another test device in accordance with this invention.
  • FIG. 6 is a cross sectional view of yet another test device in accordance with this invention.
  • FIG. 7 is an exploded view of a 96 multiple test device in accordance with this invention.
  • FIG. 8 shows a top view and a side view of an 8 well test device in accordance with this invention.
  • FIG. 9 is a schematic top view of a test device according to the invention, illustrating three control test sites and an indicia.
  • FIG. 10A-10G schematically illustrate one example used for a HIV-1 Confirmatory test.
  • Test devices of the present invention have a housing comprised of a water impermeable material in which other test components such as an absorbent pad with a reagent layer, filter and a reagent used to obtain a test result are held.
  • the housing has at least one opening to admit a fluid sample although additional openings may be desired.
  • a wick may exist at least partly outside the housing, or the filter may exist at least partly outside the housing.
  • all mechanical parts are completely held within the housing.
  • a housing that comes apart during use so that the user can remove the filter to expose the reagent layer for application of a reagent and/or wash fluid.
  • a sleeve that holds the filter is removably attached to the housing such that contact of the filter is favored over contact of the sleeve with the surface of the reagent layer.
  • the sleeve is attached to the housing by a bayonet mount. After a sample is applied, and an optional wash solution added, the sleeve is removed and further optional reagent solution and a wash solution are added directly to the reagent layer. In some applications a sample is added to the device and further processing is carried out at a separate location or after storage of the device for a few hours. In these situations, the sleeve remains attached to the housing to prevent or delay the release of moisture from the device until the later processing steps are carried out.
  • the housing also may contain a cover to protect the opening and further guard against the release of moisture.
  • Multiple housings can be incorporated into a multi-test unit to allow high volume testing.
  • the latter embodiment is acceptable for infectious disease testing of blood samples at blood banks.
  • a 32 well multiple-test device having overall dimensions of 3.5 inches by 6.75 inches
  • a 48 well multiple-test device having overall
  • the 32 well device is particularly advantageous and is desirably configured as a single array of 4 eight member rows. In one embodiment 4 (or 8) test devices that correspond in size to a column (or row) of a microtiter plate are used in applications where intermediate numbers of samples are processed.
  • the invention has particular advantages when used in a multi-test format over other methods such as microtiter plate enzyme linked immunosorbent assays. For example, the invention obviates waste disposal required by these other methods and allows optical perception of several test spots, (including one or more calibrators) in one well. Furthermore, the invention overcomes problems associated with icteric samples, samples that contain debris, or cellular material. In addition, unlike some other methods, the present invention provides detection signals that are stable.
  • the housing and other parts of the test device may be constructed from well-known materials in accordance with well-known methods of the prior art.
  • Material suitable for the invention should not interfere with the production of a detectable signal and should have a reasonable inherent strength, or strength can be provided by means of a supplemental support, such as, for example, by forming a nitrocellulose layer onto an absorbent pad, by means of a suspension of nitrocellulose.
  • Natural, synthetic, or naturally occurring materials that are synthetically modified can be used including, but not limited to: cellulose materials such as paper, cellulose, and cellulose derivatives such as cellulose acetate and nitrocellulose; fiberglass; cloth, both naturally occurring (e.g., cotton) and synthetic (e.g., nylon); porous gels such as silica gel, agarose, dextran, and gelatin; porous fibrous matrixes; starch based materials, such as Sephadex(r) brand cross-linked dextran chains; ceramic materials; films of polyvinyl chloride and combinations of polyvinyl chloride-silica; and the like. See, for example, U.S. Patent Nos. 5,075,078, 5,552,276, 4,912,034 and 5,212,065 which are incorporated herein by reference in their entireties. Constructions other than those described in these patents readily will be apparent to the skilled artisan.
  • the test device most preferably incorporates the features of (1) positioning parts with a positioning "sleeve” to allow even fluid flow between the parts without interference by the sleeve itself, (2) arranging parts to minimize transverse flow, (3) using friction-held parts and water swellable parts to allow fluid to more evenly flow through junctions between the parts, and (4) using a dispersing layer downstream of the filter to help disperse fluid more evenly to the reagent layer, where, advantageously, the reagent layer is integrated with absorbent
  • a "blood sample” as termed herein means whole blood obtained by, for example, a finger prick or venipuncture either with or without an anti-coagulant added; a plasma sample; serum sample; or a partly processed or diluted derivative of any of these such as centrifuged blood.
  • a “whole blood” sample is a blood sample that contains red blood cells, white blood cells and other components such as chylomicrons and lipoprotein.
  • a “filter” removes more than half of particles that are suspended within a test sample and which are larger than the rated sieving size of the filter material.
  • the "rated sieving size" of a material is determined by and provided from a manufacturer of the material and typically is stated in microns. For example, a glass fiber having a porosity of ten microns should remove red blood cells from a blood sample because such cells have a dimension that is greater than 10 microns. In some cases the rated sieving size of the material may be close to or even larger than one or more dimensions of the particle that is to be held back by the cell filter. Depending on the filter thickness, retardation of particle movement and not simply holding back the particles will suffice for a given test device. How well a given filter material works is best determined empirically, by applying test samples to devices made using the material.
  • the filter When used for test samples that contain red cells the filter preferably removes at least 90% of the red cells from fluid which then contacts the reagent layer.
  • the filter may comprise one or more materials in physical contact. In practice, the filter should have significant depth to allow entrapment of cells that typically comprise about one half of whole blood volume.
  • the filter When used for testing of saliva samples, the filter preferably retards cells and other debris that may exist in such test specimens.
  • the filter comprises two glass fiber pads mechanically held together.
  • the filter also may comprise a first portion that traps or slows the movement of cells and a second portion downstream from the first portion. In this case the second portion serves to disperse filtrate to an adjacent material such as a reagent layer.
  • a "filtrate" of a blood sample or whole blood sample is a portion of the sample that is passed through a filter or component of a filter.
  • the filter may remove blood cells or blood molecules by virtue of binding between the filter surface(s) and the blood cells or blood molecules.
  • the filtrate from a filter will lack more than 90% of the originally applied red cells by virtue of the sieving action of one or more portions of the filter.
  • An "absorbent pad” imbibes water generally and serves as a final repository for much or most of the liquid that enters the housing of a test device.
  • Examples of an absorbent pad include paper, ethyl cellulose, porous plastic, sintered glass, and other polymers that accept water such as polyvinyl pyrrolidone and acrylamide copolymers.
  • An absorbent pad may consist of more than one piece.
  • An acceptable absorbent pad is comprised of ethyl cellulose and is held in place by the device housing, wherein a surface of the pad contacts a reagent layer.
  • an absorbent pad comprising nitrocellulose and cellulose (sold by Schleicher and Schuell (Keene N.H.) as part number 77385 and described as BAC-T-KOTE 0.6um 35 X 35 cm size) can be used.
  • an “analyte” is a molecule, cell or cell component that is detected by the test device.
  • the analyte is a polypeptide such as, for example, a regular polypeptide, a lipopolypeptide or glycopolypeptide produced by a disease causing organism such as HIV virus, but may be a naturally occurring molecule found in the body, or a molecule produced by the body in response to a disease state.
  • An “analyte” includes any antigenic substance or "antigen”, hapten, antibody, and combination thereof.
  • the analyte can be a peptide, an amino acid, a nucleic acid, a carbohydrate, a hormone, a steroid, a vitamin, a pathogenic microorganism for which polyclonal and/or monoclonal antibodies can be produced, a natural or synthetic chemical substance, a contaminant, a drug including those administered for therapeutic purposes as well as those administered for illicit purposes, and a metabolite of or an antibody to any of the above substances.
  • the hormones which are suitable as analytes for this invention are the following: thyroid stimulating hormone (TSH), human chorionic gonadotropin (hCG), luteinizing hormone (LH) and follicle stimulating hormone (FSH).
  • TSH thyroid stimulating hormone
  • hCG human chorionic gonadotropin
  • LH luteinizing hormone
  • FSH follicle stimulating hormone
  • an "antigen” is a molecule that reacts with an antibody.
  • the test antigen preferably contains one or more epitopes that are similar or the same as epitopes of the infectious disease agent.
  • Acceptable peptides for use as a confirmatory HIV test and for other tests are described below under the heading "PEPTIDES USEFUL FOR THE DEVICE.”
  • Acceptable antigens for use in a device for syphilis testing are the 15.5 kDa, 17 kDa, 44.5 kDa (TmpA), and 47 kDa polypeptides, or portions thereof, made by the Treponema pallidum virus as described by Byrne et al. in J. Clin. Microbiol. 30: 115-122 (1992).
  • a positive confirmed syphilis test is when at least three of the four antigens react with a blood sample and produces three colored spots.
  • a confirmed negative syphilis test is when less than two of the four syphilis antigens react.
  • Acceptable antigens for use as a device for hepatitis C testing include, for example, four different "HCV regions" known as core, NS3, NS4 and NS5, as discussed by Feucht et al. in J. Clin. Microbiol. 33:620-624 (1995). A confirmed positive test result can be determined by reactivity against two of these four protein antigens.
  • Antigens useful for testing of exposure to other pathogens such as those responsible for lyme disease, toxoplasmosis, and other microorganisms such as rubella, mycoplasma, cytomegalo virus, herpes, HTLVI, HTLVII, Hepatitis B, and chlamydia are known to the skilled artisan. Antigens also can be determined by cloning genes of a pathogen and testing each expressed gene or portions thereof for reactivity, as demonstrated by Feucht (Id.) using available reagents and methods. Such antigens are too numerous to list here but are contemplated by the inventors in the context of their invention. Chemically synthesized peptides and recombinant proteins can be immobilized within devices as claimed by routine methods, such as spotting a water solution of the antigen onto a nitrocellulose membrane or membrane layer.
  • an "antibody” is IgG, IgE, IgM, IgA and the like and may be obtained from blood, a blood product or saliva.
  • saliva samples may be obtained from blood, a blood product or saliva.
  • the invention is used for the detection of analyte from saliva as well as from blood.
  • Advantages of the invention such as the operation of the filter, extend to saliva samples as well as to blood samples.
  • an “opening in the housing” may be a hole, aperture or simply a region that accepts a fluid sample.
  • the housing itself may be partly water permeable and partly water impermeable. In these instances the water permeable portion(s) act as an "opening" in context of the invention. More than one opening may be used in the test device.
  • passing fluid has its regular meaning that is well known to the skilled artisan.
  • the relative rate of passing fluid can be determined by a number of techniques known to the artisan, including for example, measuring how much time is required for the material to become completely wetted upon contact with water.
  • An advantageous material for trapping cells in the filter is glass fiber and an advantageous material for a dispersant layer
  • this glass fiber 16 in contact with this glass fiber typically is cellulose because cellulose can pass water at a lower rate compared to glass fiber.
  • the second portion or "dispersing" portion of a filter when used, may provide a lower flow rate of sample and of wash fluid compared to the cell trapping material in the filter.
  • the low flow rate provided by the dispersing portion allows more time for chemical substances to react before these substances contact the reagent layer. Increasing the reaction time, particularly for binding reactions, allows greater test sensitivity.
  • Acceptable materials for use in the filter to trap cells include Gelman Cytosep glass fiber filter membrane and Pall corporation blood separation membrane.
  • Acceptable for a "dispersing" portion of a filter if one is used, are cellulose- based materials such as filter paper.
  • a “reagent layer” is an absorbent or group of absorbents that contains at least one reagent, contacts the filter and receives sample from the filter by virtue of this contact.
  • An advantageous reagent layer is a membrane that comprises an immobilized test reactant such as antibody or antigen.
  • an absorbent pad that contacts the reagent layer accepts and draws in fluid from the reagent layer.
  • the reagent layer preferably comprises an immobilized test reactant, the test reactant may become immobilized during the test itself by for example, precipitation or by binding to the reagent layer during the test procedure.
  • the reagent layer may assume any of a variety of shapes.
  • a most advantageous shape is a thin layer between the filter and the absorbent pad, preferably formed on or in the absorbent material itself, although another shape such as a plug may be used, depending on the features of the other components.
  • Fluid preferably moves traverse to the major surfaces of a reagent layer, although some device configurations may utilize lateral flow along the longest axis of a reagent layer.
  • the reagent layer need not be a separate layer but may be part of the absorbent, e.g., it may consist of a pad, rod or other shaped absorbent adhered or otherwise fixed to the upper portion of the absorbent pad, or a surface on or within a portion of such a material, or the reagent simply may be deposited onto the surface of the absorbent pad, in which case the reagent layer is that portion of the absorbent pad that comprises the reagent.
  • a “sleeve” is a water impermeable solid surface that holds the filter.
  • the sleeve may be a portion of the test device housing or may be a separate part that is separately attached to the housing. In the latter case, the sleeve preferably is reversibly attached to the housing by a bayonet mount.
  • An advantageous construction in this context is to place the filter into the sleeve and to assemble the housing from two portions, an upper housing portion and a lower
  • the filter preferably has a depth that is at least one tenth its diameter.
  • the filter particularly the dispersant portion, if included), or, (in some cases), the sleeve surface, to swell upon wetting.
  • swell in the context of a part or part surface used in the test device means that the part increases its size or that the surface increases its thickness upon wetting. Acceptable in this context is cellulose, although the use of many alternate materials is readily appreciated by a skilled artisan.
  • An "optical change" in the context of determining the presence or absence of a test analyte means an absorbance, reflectance, fluorescence, phosphorescence, or chemiluminescence change produced within the test device. This change may be determined by eye or by an instrument.
  • One acceptable embodiment employs gold particles to produce a reflectance change that is visually perceived.
  • the filter is separated from the reagent layer by removal of the sleeve, which exposes a portion of the reagent layer where an additional reagent may be added and a signal is developed from the presence of gold particles.
  • a "bayonet mount” has the usual meaning in the art and refers to a fastening means whereby a first part firmly attaches to a second part by overlapping indentations along a circular surface that fits into a depression of the second part. After inserting one part into the other, either part is rotated to firmly seal the two together.
  • a "chemical interferant" in a sample is a chemical that interferes with one or more chemical reactions of the test assay such as a binding reaction or enzymatic reaction.
  • a chemical interferant is a rheumatoid factor that can enhance certain agglutination reactions.
  • Another example is an auto-antibody that affects some binding interactions. Chemical interferants may cause false positive assay results and false negative assay results.
  • a "glycosaminoglycan” is a linear heteropolysaccharide possessing a characteristic disaccharide repeat sequence (as reviewed by Jackson et al., Physiological Reviews 71: 481- 539 (1991)) which is herein incorporated in its entirety by reference.
  • One monosaccharide of the disaccharide is an amino sugar with D-glucosamine or galactosamine, and the other unit is typically, but not always, a uronic acid residue of either D-glycuronic acid or iduronic acid. Both units are variably N- and O-sulfated, which adds to the heterogeneity of these complex macromolecules.
  • glycosaminoglycans examples include chondroitin sulfate, dermatan sulfate, heparin sulfate, keratan sulfate, hyaluronic acid and heparin. Some of these molecules are covalently attached, typically at their reducing ends through an O-glycosidic linkage to a serine residue or N-linked to asparagine in a core protein to form a proteoglycan.
  • a given glycosaminoglycan can vary by size or degree of sulfation and various subtypes can be prepared by synthesis or by purification of naturally occurring glycosaminoglycan.
  • heparin of 11,000 molecular weight and a sulfated pentasaccharide derivative of heparin as described by Jackson et al. (Id.) also are include within the ambit of the invention.
  • the inventors have discovered that adding a glycosaminoglycan to assays that contain certain HIV antigen polypeptides will improve resistance to false positive interferences.
  • glycosaminoglycan prevents false positives when the assay uses a polypeptide antigen that contains four or five basic amino acids within a 10 amino acid long (decapeptide) segment. Further, the improvement by glycosaminoglycan may occur when these basic residues exist in two pairs of basic amino acids that are separated by between 2 and 6 amino acids.
  • a "basic amino acid” in this context is arginine or lysine.
  • glycosaminoglycan to improve diagnostic assay performance is advantageous in combination with use of a recombinant polypeptide that has been designed with four or more positive charged amino acids within a short region of twenty amino acids.
  • the glycosaminoglycan advantageously is used with an antigen having four or more amino acids within a short region of ten amino acids.
  • the glycosaminoglycan is advantageously used with a recombinant polypeptide that has been designed by deleting a portion of a preexisting sequence, yielding a "new" segment which forms a tertiary structure that has not been determined by natural selection. Such a new structure often is unstable and tends to bind other substances.
  • glycosaminoglycans stabilize basic amino acid clusters of such proteins and prevent undesirable false positive assay results with such assays. This discovery solves a general problem of using engineered polypeptides as antigens in diagnostic assays.
  • An advantageous device configuration comprises an absorbent pad with reagent provided on its upper surface, and a two-part filter within a single housing.
  • Acceptable materials for the housing include water impermeable plastics such as polystyrene, polypropylene, polyvinyl chloride and the like.
  • Acceptable materials for the filtering portion of the filter, the dispersing portion of the filter, the reagent layer and absorbent pad are glass fiber, ethyl cellulose, nitrocellulose and ethyl cellulose respectively.
  • the material of the filter should be chosen for its ability to premix the test sample and any test reagent that may be present in the filter.
  • Two or more materials can be physically combined to make up a filter, reagent layer or absorbent pad.
  • Two materials are acceptable for the filter, an upper filtering material and a lower dispersal material. Most acceptable is a dispersal layer that passes fluid at a slower flow rate compared to passage of fluid through an upper filtering material.
  • the applicants have found that the use of a dispersal layer is surprisingly superior over the use of a single part filter.
  • the layer may remove irregularities in the flow that arise from irregular collection of particles in the upper regions of the filter.
  • a second advantage of using a two- part filter is that the dispersal layer, by virtue of its slow fluid flow rate, can prolong one or more reaction times for substances that react before entering the reaction layer, thereby increasing sensitivity.
  • the separate reagent layer and absorbent pad can be replaced by an absorbent, the top surface of which comprises the reagent(s).
  • the reagent layer comprises the portion of the absorbent pad that contains the reagent(s).
  • absorbent, porous or capillary possessing materials through which a solution containing the analyte can be transported by a wicking action are suitable for construction of the test device and are known to the skilled artisan.
  • the inventors extensively have studied natural sequence variation of the immunodominant region of gp41 (HIV-1) and gp36 (HIV-2) envelope peptides and have searched Africa for "problem" HIV strains that do not cross-react well with known sequences of this region.
  • New peptide antigens were designed and prepared and successfully tested in a device as described above that contains the immunodominant region of gp41 and gp36 envelope proteins.
  • the inventors discovered methods of finding alternative peptides of the same epitope that (I) have improved solubility and stability and (ii) can be used together to detect infection from a broad range of virus strains found in nature. Using these methods, the inventors designed, prepared and tested peptides that can detect HIV infection at an earlier stage compared to other screening assays.
  • the method of optimizing peptide sequences for a given immunoreactive site of a viral protein is exemplified with the HIV-1 gp41 antigen but works with other viral proteins such as the N3, N4 or env70 proteins of hepatitis C virus.
  • the invention is useful particularly for detection and therapy of infection by RNA viruses such as HIV and HCV although non- RNA viruses and even cancer cell antigens that may undergo fast mutation are contemplated as well.
  • the method begins with the selection of an antigen that can stimulate a specific antibody response which occurs early in the course of a viral infection.
  • a specific antigen is chosen that stimulates a response such that circulating antibodies to the antigen can be detected in the blood of an infected mammal long after initial infection.
  • a most desirable immunoreactive site is amino acid numbers 594 through 609 of the gp41 protein because antibodies that recognize epitopes from this site are found early in infection and their titer remains high during infection.
  • alternative peptides are made that are similar to or identical to known naturally occurring sequence(s) from the site. These peptides should be between 16 and 100 amino acids long and preferably between 24 and 50 amino acids long.
  • a sequence of the immunodominant region of gp41, from the O group such as MVP5180 is known to act immunologically different from an M sequence.
  • an M sequence peptide can be selected along with an O sequence.
  • proteins of the D subtype of H ⁇ V-1 (group M) appear to have different epitopes from many other proteins and is particularly advantageous to combine a peptide from this subtype with another type.
  • the peptide(s) that have been selected are then tested with samples that have been exposed to the virus. Most advantageously, these samples should comprise a wide range of serological virus types. Test results are then compared to see which serological virus type samples fail to react, or react less well with the selected peptides. If, after comparison, it seems that some samples are not detectable using any of the selected peptides, then advantageously, another peptide can be further included to provide further reactivity with one or more samples that do not cross-react (or react poorly) with the originally chosen peptides.
  • a peptide sequence need not be identical to a naturally occurring protein sequence from the virus, and preferably will differ, as described in the above-cited co-pending patent applications. The process of designing/selecting peptide sequences and testing with multiple blood samples is repeated as needed until substantially all of the known positive (from known infected individuals) samples will react with at least one of the peptides.
  • the peptides are included in a test for detecting infection by the virus.
  • the test will employ binding reaction(s) between the peptides and antibodies in a sample such as blood from a mammal, to determine whether the mammal has been infected with the virus.
  • the peptides may be used together in the test or may be used separately.
  • two or more peptides are dissolved in the same water solution and the water solution is spotted onto a solid phase such as a nitrocellulose membrane or polystyrene plastic
  • peptides are in separate solutions or in separate locations in the test and separate results are obtained either simultaneously or sequentially from the same sample.
  • the first strategy is that the peptide sequences preferably are chosen so that, when used together, the peptides allow broad reactivity against early anti-HIV antibodies.
  • the second strategy is that the peptide sequences preferably are modified to make the peptides more reactive and more soluble.
  • the peptides advantageously have a size between 16 and 100 amino acids, and most advantageously between 25 and 50 amino acids long.
  • the claimed invention provides minimum-sized antigens that contain early epitopes of HIV infection. This feature allows a very high number of antigens to contact a blood sample in a test and directly leads to faster tests of greater sensitivity that can detect infection at an earlier stage compared to other tests.
  • the invention makes possible a "peptide only" test that lacks the above-mentioned disadvantages of using large proteins.
  • the second strategy of modifying a peptide sequence also directly leads to faster tests of greater sensitivity by increasing solubility of the peptide, thus allowing more copies of stronger reacting epitopes in a test to detect HIV from a sample.
  • the embodiment of altering a hydrophobic amino to a less hydrophobic amino acid alleviates the problem of gp41 pep tide/protein aggregation caused by hydrophobic residues in the sequence.
  • the first step is to determine an immunodominant region of a protein from the target virus.
  • a first peptide then is chosen that comprises at least a portion that corresponds to the chosen immunodominant region. It is most advantageous to pick a peptide that cross reacts broadly with many types of strains, such as taught in the co-pending applications cited above. Particularly advantageous in this context for HIV are Group O reactive peptides, such as those that comprise one or more of the alternative sequences listed
  • an immunodominant region of the N3 peptide may be advantageously chosen, or another well known immunodominant region of a hepatitis C protein. Although not sufficiently described elsewhere, an even more advantageous immunodominant region would be from the gp70 envelope protein region that has a high rate of mutation.
  • a second peptide that comprises at least a portion corresponding to the chosen immunodominant region also is contemplated as peptide of the invention.
  • the first peptide then is challenged with a diverse set of strains in binding assays to select one or more strains that do not react as well with the peptide.
  • the selected strain(s) constitute a second group.
  • a second peptide that corresponds to the same immunodominant but which cross-reacts with the second group better than the first peptide then is chosen based on known characteristics of the group and optionally on experimental results.
  • the inventors discovered that Subtype D peptides cross-react well with the very rare HIV-1 samples that fell into the second group. Accordingly, one embodiment of the claimed invention with respect to HIV-1 tests is a combination of Group O reactive peptide with Subtype D reactive peptide.
  • Subtype D peptide sequences are found in co-pending application entitled "HIV-1 Subtype D Peptides" filed October 16, 1998 (Attorney docket No. 073294/0193).
  • the invention as practiced with HIV testing and therapy is particularly advantageous with Group O and Subtype D peptides as described in the two above referenced applications.
  • Secondary structure in this context refers to polypeptide helix or pleated sheet that forms primarily by multiple hydrogen bonding between peptide bond hydrogen and oxygen. Most advantageous is alpha helix structure that forms within a stretch of the peptide.
  • the degree of stabilization has a great influence on performance of a peptide used in diagnosis or therapy.
  • the inventors learned that the 25 amino acid peptide of sequence ALETLIQNQQRLNLWGCKGKLICYT fails to detect some HJN-1 infected samples in an immunoassay.
  • a longer peptide having an extra 5 amino acids that form a more extended alpha helix at the amino terminus: RARLQALETLIQ ⁇ QQRL- ⁇ LWGCKGKLICYTSVKW ⁇ T successfully detected all HIV-1 samples tested.
  • the extra 5 amino acids, "RARLQ” provide a more stable peptide by virtue of extending the alpha helix at the amino terminal side of the immunodominant region.
  • hydrophobic to hydrophilic amino acid residue shift can be that the new peptide may have greater solubility in water.
  • An increase in water solubility can lead directly to improved diagnostic assay or vaccine performance by allowing a greater amount of peptide to be used.
  • This attribute also facilitates the use of more than one peptide together in the same solution without causing a precipitate at higher concentrations of one or more of the peptides.
  • a peptide antigen according to the invention is greater than 16 amino acid residues long but smaller than 100 amino acid residues long. This size range is termed "intermediate size.” The upper size limit reflects the fact that an intermediate size peptide according to the
  • 25 invention is shorter than most proteins, which have tertiary structure due to folding of the peptide sequence.
  • the polypeptide chain folds upon itself (forms tertiary structure) to, among other things, allow mutual association of hydrophobic residues in order to maximize entropy of a water solution that contains the polypeptide.
  • Intermediate sized peptides in accordance with the invention on the other hand, generally are smaller, generally fold less and have less tertiary structure than an intact protein but have secondary structure.
  • Their minimum size limit of 16 amino acids reflects the fact that peptides smaller than 16 residues long generally have little structure outside the primary structure of amino acid sequence and are less improved by making an alteration according to the claimed embodiment.
  • intermediate sized peptides were synthesized having additional substitutions of hydrophilic amino acid residues for hydrophobic residues. These peptides have sequences that correspond to (i.e., at least half of the amino acids correspond in identity with) naturally-occurring sequences.
  • the synthesized peptides showed greater specificity for HIV-1 O Group specimens compared to peptides that have sequences that are identical to sequences from naturally occurring proteins. That is, the peptide sequences of the claimed invention exhibit different immunological characteristics than the corresponding sequences of naturally occurring proteins. The different characteristics can include a loss of one or more immunological properties, exemplified by the loss of HIV-2 reactivity for peptides obtained from a naturally-occurring HIV-1 envelope protein sequence.
  • the inventors theorize that altering a hydrophobic amino acid such as leucine, valine and isoleucine etc. to a hydrophilic amino acid such as glutamine, asparagine, serine, threonine etc., particularly in the immunodominant region, helps prevent structural instability when present in an intermediate sized (16-100 residue-long) peptide that lacks complex protein (i.e. tertiary structure).
  • the inventors theorize that hydrophobic amino acid residues in a large protein come together to form an interior oily pocket that excludes water and stabilize the structure of the complete large protein.
  • a peptide antigen less than about 100 amino acids (e.g.
  • less than 100 amino acids particularly less than about 75 amino acids (e.g. less than 75 amino acids) and more particularly less than about 50 amino acids (e.g. less than 50 amino acids) is prepared to mimic antigenically this same protein, individual hydrophobic residues no longer can avoid water by optimally coming together and instead randomly are exposed to water and increase disorder of the peptide in water.
  • the disorder contributes to less stable and unrecognizable epitopic structures which react less well or react less specifically with
  • the embodiment of replacing one or more hydrophobic amino acids with one or more hydrophilic amino acids particularly relates to intermediate sized peptides from 16 amino acids to 100 amino acids in length, and more particularly to peptides between 25 to 50 amino acids, 36 to 50 amino acids and 41 to 50 amino acids.
  • the improved effect is seen particularly with intermediate sized peptides because, at very small sizes of less than about 16 (e.g., 16), and particularly less than 10 amino acids, the epitope recognized by an antibody more closely resembles the primary structure of the short segment, namely, the individual amino acid residues themselves. That is, antibody reactivity (if any) to such a short peptide arises primarily from chemical characteristics of the amino acid residues themselves.
  • peptides between about 25 to 100 amino acid residues long, and particularly 25-50 amino acids long advantageously are used. These intermediate sized antigens are larger than short pieces studied by Horal, Aleanzi and others, and have more advantageous secondary structure in water solution. In this case, altering a hydrophobic amino acid to a hydrophilic amino acid provides an advantage to the peptide.
  • peptide antigens of most interest for diagnostics and therapy generally have more advantageous secondary and tertiary structures which are more sensitive to disruption by a hydrophobic residue, yet the hydrophobic residue(s) present in these peptides need a large protein for proper orientation.
  • the claimed invention is exemplified by, for example, altering a leucine to a glutamine but works well with shifts of other hydrophobic amino acids such as I, V, M, F and W to hydrophilic amino acids, and even to hydrophilic charged amino acids.
  • Most advantageous in this aspect is to replace a leucine, which has a three carbon long residue with a methyl group attached, with a glutamine, which also has a three carbon long residue with an additional amine group attached.
  • a peptide between 25 and 50 amino acids long is used for diagnostic tests that has only one hydrophobic residue within an 8 residue long portion. Altering this hydrophobic residue to a hydrophilic residue improves reactivity (sensitivity and/or selectivity). In yet another embodiment, 2 hydrophobic residues within an 8 amino acid long portion exist and at least one of these is altered to a hydrophilic amino acid to provide the benefit. Altering 2 or more residues within a short region can provide great improvement to solubility and the ability to incorporate the peptide, alone or with other peptide(s) in a diagnostic test reagent or therapeutic agent.
  • an isoleucine, leucine, valine, or methionine is replaced with glutamine.
  • any of these hydrophobic amino acids is replaced with asparagine.
  • any of these hydrophobic amino acids is replaced with threonine, serine, alanine or glycine.
  • any of these hydrophobic amino acids is replaced with histidine or proline.
  • any of these hydrophobic amino acids is replaced with aspartic acid, glutamic acid, arginine or lysine.
  • a phenyl alanine can be converted to a glutamine.
  • a phenyl alanine can be converted to any of the other hydrophilic amino acids.
  • a computer modeling software program such as "Peptide Companion” advantageously is used and a specific alteration is chosen, using the program, to maintain the predicted pre-existing secondary or tertiary structure of the protein.
  • At least one additional amino acid is added to at least one terminus of a peptide of the claimed invention.
  • Such added amino acid(s) facilitates linking the peptide to another peptide, coupling to a carrier, or coupling to a support.
  • 28 amino acid(s) also can be chosen to alter the physical, chemical or biological properties of the peptide, such as, for example adding another epitope for T-cell stimulation.
  • Suitable amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, and the like, can be introduced at the C- or N-terminus of the peptide.
  • a peptide of the invention can differ from the natural sequence by being modified by terminal-NH sub 2 acylation, e.g., acetylation, or thioglycolic acid amidation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule, thereby providing a linker function.
  • terminal-NH sub 2 acylation e.g., acetylation, or thioglycolic acid amidation
  • terminal-carboxyl amidation e.g., ammonia, methylamine, etc.
  • these modifications may provide sites for linking to a support or other molecule, thereby providing a linker function.
  • the peptides of the claimed invention or analogs or homologs thereof may be further modified beyond the sequence considerations given above, as necessary to provide certain other desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially the biological activity of the unmodified peptide.
  • the peptides can be modified by extending, decreasing or substituting amino acids in the peptide sequence by, for example, the addition or deletion of suitable amino acids on either the amino terminal or carboxyl terminal end, or both, of peptides derived from the sequences disclosed herein.
  • substitutions for HIV-1 testing are described by, for example, SEQ ID Nos. 1-20, further conservative substitutions are possible and sometimes desirable for HIV-1 testing.
  • conservative substitutions is meant replacing an amino acid residue with another that is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another.
  • the substitutions include combinations such as Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • Other amino acid substitutions are provided as groups within individual claims.
  • the portion of the peptide sequence that is intended to mimic an antigen of HIV will not differ by more than about 30% from any of the sequences provided herein, except where additional amino acids may be added at either terminus for the purpose of modifying the physical or chemical properties of the peptide for, for example, ease of linking or coupling, and the like.
  • additional amino acids may be added at either terminus for the purpose of modifying the physical or chemical properties of the peptide for, for example, ease of linking or coupling, and the like.
  • regions of the peptide sequences are highly variable, it may be desirable to vary one or more particular amino acids to mimic more effectively differing epitopes of different HIV strains.
  • the contributions made by the side chains of the residues can be probed via a systematic replacement of individual residues with a suitable amino acid, such as Gly or Ala.
  • a suitable amino acid such as Gly or Ala.
  • 29 peptide are required for binding to a specific MHC protein, (or other component of the immune system) are known. See, for instance, Allen et. al., Nature, 327, 713-717; Sette et. al., Nature, 328, 395-399; Takahashi et. al, J. Exp. Med., 170, 2023-2035 (1989); and Maryanski et. al, Cell, 60, 63-72 (1990).
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art as cited in U.S. No. 5,703,057 (citing Kyte and Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant peptide which in turn defines the interaction of the peptide with other molecules, for example, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a peptide with similar biological activity, i.e., still obtain a biological functionally equivalent peptide.
  • substitution of amino acids whose hydropathic indices are within +- 2 is preferred, those which are within +- 1 are particularly preferred, and those within +- 0.5 are even more particularly preferred.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 +- 1) glutamate (+3.0 +- 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0) threonine (-0.4); proline (-0.5 +- 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0)
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent peptide.
  • substitution of amino acids whose hydrophilicity values are within +- 2 is preferred, those which are within +- 1 are particularly preferred, and those within +- 0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Peptides that tolerate multiple amino acid substitutions generally incorporate small, relatively neutral molecules, e.g., Ala, Gly, Pro, or similar residues.
  • the number and types of residues that can be substituted, added or subtracted will depend on the spacing necessary between the essential epitopic points and certain conformational and functional attributes that are sought.
  • types of residues it is intended, e.g., to distinguish between hydrophobic and hydrophilic residues, among other attributes. If desired, increased binding affinity of peptide analogs to can also be achieved by such alterations.
  • any spacer substitutions, additions or deletions between epitopic and or conformationally important residues will employ amino acids or moieties chosen to avoid stearic and charge interference that might disrupt intramolecular binding of the peptides and intermolecular binding of peptides to other molecules.
  • Peptides that tolerate multiple substitutions while retaining the desired immunological activity also may be synthesized as D-amino acid-containing peptides.
  • Such peptides may be synthesized as "inverso" or “retro-inverso” forms, that is, by replacing L-amino acids of a sequence with D-amino acids, or by reversing the sequence of the amino acids and replacing one or more L-amino acids with D-amino acids.
  • the D-peptides are substantially more resistant to peptidases, and therefore are more stable in serum and tissues compared to their L-peptide counterparts, the stability of D-peptides under physiological conditions may more than compensate for a difference in affinity compared to the corresponding L-peptide.
  • L-amino acid-containing peptides with or without substitutions can be capped with a D-amino acid to inhibit exopeptidase destruction of the antigenic peptide.
  • An advantageous embodiment is to prepare the peptide by chemical synthesis.
  • the peptide is made recombinantly.
  • modifications, including conservative modifications, are best carried out by changing a DNA sequence that codes for the peptide.
  • the following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule.
  • the amino acid changes may be achieved by changing the codons of the DNA sequence, according to the following codon table:
  • Biologically functional universal peptides can be prepared through specific mutagenesis of the underlying DNA.
  • the technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art.
  • Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.
  • Site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained.
  • recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • the peptide combinations are particularly useful in dot blot immunoassays and a test device as described above is particularly preferred.
  • FIG. 1 depicts device housing 10 that consists of sleeve assembly 20 and container 30.
  • Container 30 consists of top half 40 and bottom half 50.
  • Sleeve assembly 20 consists of top surface 60 and opening 70 made by sleeve 80.
  • Sleeve assembly 20 is reversibly mounted onto container 30 and can be removed after application of a sample for further processing of the sample by adding wash and/or reagent solution to the middle of the top surface of the container.
  • FIG. 2 is a side perspective drawing of device housing 10.
  • FIG. 3 shows a cross section of the device shown in FIG. 1 and FIG. 2.
  • Top half 40 and bottom half 50 of container 30 snap together to form a solid structure.
  • Nestled within bottom half 50 is absorbent pad 90.
  • reagent layer 100 On the top side of absorbent pad 90 is reagent layer 100.
  • circular washer 110 On top of reagent layer 100 is circular washer 110.
  • Circular washer 110 is comprised of a water impermeable plastic and forms a tight seal with container top half 130, leaving space 200 at the bottom of top half 130.
  • Sleeve assembly 20 consists of lip region 120 that forms funnel 130. Assembly 20 is a single body that forms sleeve 140 underneath funnel 130. Within sleeve 140 are first filter portion 150, second filter portion 160 and dispersant layer 170. First filter portion 150, second filter portion 160 and dispersant layer 170 form a "filter” having an upper surface 180 and a lower surface 190. Lower surface 190 protrudes out of sleeve 140 to contact the upper surface of reagent layer 100, leaving space 200 between the side edge of dispersant layer 170 and circular washer 110. Absorbent pad 90 and reagent layer 100 are depicted as two separate portions of a common absorbent in this figure.
  • FIG. 4 depicts an embodiment of the device shown in FIG. 3 that further contains cover 210 attached at location 220 of sleeve assembly 20.
  • FIG. 5 is a cross section view of an alternate device that differs from the device of FIG. 1 - FIG. 4 by having reagent layer 100 inside sleeve 140.
  • This figure depicts an embodiment wherein reagent layer 100 is not compressed by sleeve 140 because it is held inside sleeve 140 along with filter portions 150 and 160.
  • the reagent layer is in fluid contact with both the filter and absorbent pad 90 (i.e. it may contact both directly or via an intervening layer(s)).
  • the reagent layer protrudes beyond the sleeve to the absorbent pad as shown in this figure.
  • the filter, one of the filter portions, or the sleeve be removable to allow direct optical detection of a signal that forms below the filter.
  • a reagent layer comprised of a clear polycarbonate porous membrane in which antigen immobilized on polystyrene microparticles have been spotted.
  • an optical signal develops at the bottom of the polycarbonate filter and can be seen by inspecting the bottom of the polycarbonate filter after removal of the filter.
  • a colloidal gold detection reagent in the filter in a dried form so that the detection reagent may become resuspended during application of a test sample.
  • an optional wick may be used to bring a fluid sample into the container.
  • a portion of the wick that is exposed to the outside of the container is contacted with the sample, which then wicks into the container.
  • the wick may itself have filtering properties and can replace the filter.
  • FIG. 6 shows an alternate embodiment that uses lateral flow within a reagent pad and two openings.
  • Housing 200 is made up of top half 202 and bottom half 204 which snap together.
  • the housing has openings 210 and 220. Opening 210 is formed by cylinder 230 which holds filter 240. Filter 240 contacts reagent layer 250. End 260 of reagent layer 250 contains immobilized HIV antigen. Above end 260 is opening 220. Opening 220 holds dispersant layer 270 which contacts end 260 of the reagent layer. End 260 is situated above and in contact with absorbent pad 280. Dispersant layer 270 is held in contact with reagent layer 260 by virtue of friction between the dispersant layer and sleeve 290. Spaces 300 are optional and may be used to help direct the flow of fluid into absorbent 280.
  • a sample is applied to opening 210, enters filter 240 and then enters reagent layer 250. Fluid moves across reagent layer 250 to end 260 above absorbent pad 280. An optional reagent and wash fluid are applied to opening 220 and an optical signal is developed at the reagent layer surface below this opening.
  • the reagent may be present within the filter or the reagent layer and dissolves or becomes suspended upon application of fluid to the device.
  • FIG. 7 shows the use of the invention in a 96 well multiple test format.
  • a 96 well cassette 310 consists of upper housing 320 and lower housing 330. Each well position of upper housing 320 contains a filter and dispersant layer. Each well position of lower housing 330 contains reagent layer and absorbent pad. In an advantageous embodiment the lower housing contains a continuous sheet of absorbent layer and a continuous absorbent pad.
  • Optional multiple test device assembly 340 contains 8 individual tests and can be assembled into 96 well cassette 310 for test sequences in which up to 8 tests are performed at once.
  • cassette 310 can be assembled with 96 tests as a single unit.
  • FIG. 8 shows a top view and a side view of optional multiple test device assembly 340.
  • the top view shows tab 360 for grasping a section of 8 tests that can be moved together.
  • the side view shows individual sleeves 370 attached directly and indirectly to tab 360.
  • Filter 380 Inside each sleeve and exposed to that sleeve's opening is filter 380. Filter 380 contacts, at its lower surface, dispersant layer 390. Dispersant layer 390 protrudes from sleeve 370 to form protrusion 400.
  • assembly 340 is inserted into a row of a 96 well plate that contains reagent layer and absorbent pad. Up to 8 samples can be processed at the same time using an 8 position pipetter.
  • sample and wash fluid are added to the 8 well sections of multiple device assembly 340, followed by removal of this assembly. Removal exposes the reagent layer for direct addition of fluid and also direct optical detection of a test signal from the reagent layer.
  • individual absorbent pads are replaced with a large surface continuous absorbent sheet inside lower housing 420.
  • FIG. 9 shows an embodiment according to the present invention that contains three test sites A, B, C and an indicia in a form of a control line CL formed on the top surface of the absorbent pad.
  • a detecting substance for example, polypeptide antigen, antibody, or another substance (that reacts with the biological substance to be detected) can be immobilized at any portion of the reagent layer 100 by dissolving the detecting substance in an aqueous solution and then spotting this solution at the desired sites A, B, C.
  • the spotted sites are then dried, for example, by storing the absorbent pad, (e.g., nitrocellulose or a fused nitrocellulose/cellulose absorbent), in an atmosphere having a low relative humidity.
  • the absorbent pad e.g., nitrocellulose or a fused nitrocellulose/cellulose absorbent
  • the detecting substance to be immobilized is dissolved to a high concentration to keep the spotted site small, and to minimize drying requirements.
  • polypeptide antigen is dissolved in water only, and about 0.25 microliters of the solution are spotted onto the surface of the reagent layer 100 to form a dot.
  • the control line can be formed by applying about 0.375 microliters of, for example, a solution of anti-human IgG in a streaking motion on the top surface of the absorbent pad.
  • control line One of the uses of the control line is for a "positive control", which indicates that the user has applied human antibody to the device and the reaction between the immobilized antigen and the antibody in the sample has been carried out properly.
  • the positive control line gives a test for indicating whether a test sample and additional liquids, such as wash liquid, have been applied properly.
  • control line is a positive control, which includes a molecule that binds to human antibody.
  • the molecule is immobilized to the reagent layer in the form of a line and preferably comprises an anti-human antibody.
  • the molecule used for making the control line is goat antibody directed against human IgG.
  • a test user adds human blood, which contains human antibody. A portion of the human antibody reacts with the goat antibody in the control line.
  • a signal developing reagent such anti-human IgG with gold particle additive, is used to develop both the test spot signals (if anti-HIV antibody is present) and the control line signal (if blood was applied and processed).
  • This example concerns a test for HIV exposure based on binding to anti-HIV antibody in a human blood test sample, but specific reagents suitable for other analytes and tests are readily determined by the skilled artisan.
  • another of the advantageous features of a test device in accordance with this invention is that the surface of a filter that protrudes beyond a sleeve within the test device indirectly mates in a flush manner with a surface of an absorbent pad. More particularly, a diffuser (lower portion of filter that disperses filtrate) mates with the reagent layer such that the total surface of the diffuser contacts a surface of the reagent layer. Most acceptably, the diffuser protrudes beyond the sleeve so that a space is maintained when the surface of the diffuser contacts the surface of the reagent layer.
  • a single test device is obtained and sample and wash fluids are added directly to either the filter or the reagent layer of the device.
  • An additional wick also can be used to bring a fluid sample into the device, if desired.
  • a sample can be introduced into the device by leaving a portion of the filter exposed to the outside, where it can be used to directly absorb sample fluid and then bring this fluid, or a filtrate into the housing.
  • the wash solution preferably contains a non-ionic detergent such as Tween 20 (r) and may also contain a protein such as bovine serum albumin.
  • the filter is removed by for example, twisting off the sleeve assembly 20 (in a bayonet mount) from the container. Then a reagent
  • An advantageous reagent solution in this context contains a detection agent such as colloidal gold coated with protein A.
  • the detection agent is optionally washed with a wash solution.
  • the sleeve be attached to the device reversibly by, for example a bayonet mount.
  • contact between the filter and a reagent layer is established upon attaching the sleeve that holds the filter to the device housing. This contact is broken when the sleeve is removed after washing a sample into the reagent layer.
  • the sleeve remains in place and the opening at the top of the sleeve is covered after addition of sample (and optional wash). The cover (and optionally the sleeve) can be removed later for further processing if needed.
  • Reagents needed for operation of the test device may be placed in the filter, reagent layer, dispersant (if used), and/or in an aqueous fluid that is applied to the device.
  • a reagent such as gold sol can be prepared and used as a liquid phase reagent and added during operation of the device.
  • the lower portion of the filter i.e. the "dispersant layer” extends beyond the sleeve that holds the filter such that when the filter is attached to the container, the dispersant layer portion exerts a greater pressure onto the reagent layer than does the sleeve.
  • "Greater pressure” in this context means that the mechanical force per unit area exerted by the filter onto the reagent layer exceeds the mechanical force per unit area exerted by the sleeve onto the reagent layer.
  • the filter exerts at least twice as much mechanical pressure onto the reagent layer compared to the sleeve. More preferably, the sleeve does not contact the reagent layer. By relying principally on the filter itself to contact the reagent layer, the sleeve does not deform the reagent layer. This allows a more even and reproducible transfer of fluid from the filter to the reagent layer.
  • a detection agent such as colloidal gold is placed in an optional lower portion ("dispersant") of the filter during manufacture of the device.
  • a user would apply a blood sample to the device, followed by a wash, removal of the filter, and then another optional wash.
  • Adding sample and wash solution starts sequential and automatic reactions in the test device.
  • These reactions include one or more binding reaction(s), an optional separation step and an optional enzymatic reaction step according to the test format employed.
  • Many types of chemistry formats are useful for the test device and are contemplated for the invention.
  • One acceptable format is a binding assay in which an immobilized antigen in the reagent layer binds to an analyte molecule.
  • the analyte has at least one epitope or binding site for which there' exists a naturally occurring, complementary specific binding member or for which a specific binding member can be prepared.
  • two molecules that bind specifically i.e.
  • a signal producing substance form an optical signal.
  • An advantageous signal producing substance is colloidal gold.
  • Molecules that act as "binding partners" with an analyte in a test sample typically are antibodies specific to the analyte, but, as the skilled artisan will readily appreciate, other molecules that bind specifically to the analyte molecule also can be used.
  • the test device may contain a chemical label to generate the signal or the user may add this label.
  • the label generally is any substance which is attached to a specific binding member and which is capable of producing a signal
  • the above- summarized procedure can be modified by the use of an aqueous "pre- treatment" solution that is applied to the device before test fluid application.
  • the pre- treatment solution comprises a protein and a detergent.
  • the protein may be, for example, bovine serum albumin, human serum albumin, casein, non-fat milk product or gelatin.
  • the protein is preferably at a concentration of from 0.05% to 10% and more preferably between 0.2% and 2% (wgt/vol). Most acceptable is a 1% solution of bovine serum albumin.
  • the detergent is an amphoteric molecule or composition such as triton X-100, Tween 20, Tween 80, NP-40, zwitterionic detergents and the like.
  • the detergent is preferably at a concentration above its critical micelle concentration and typically should be at a concentration of between 0.05% and 3% (wgt/vol).
  • the use of Tween-20 detergent is most acceptable at a concentration of between 0.2% and 2%.
  • the pretreatment solution should have a pH in the range of 5.0 to 10.0 and preferably contains a buffering component such as sodium phosphate or Tris-Cl at a concentration of between lmM and 1M and more preferably between lOmM and 200mM. Most acceptable is a 50mM concentration of Tris-Cl at pH 8.0.
  • a suitable absorbent pad comprises nitrocellulose/cellulose material with three test sites A, B, C and a control line CL.
  • the sites A, B, C are respectively spotted with p24, gp41, and gpl20 antigens.
  • the control line is formed with an anti-human IgG in a streaking motion on the reagent layer 100.
  • a blood sample is introduced or dropped into the filter portion 150, along with appropriate wash liquid so that the filtrant contacts the three test sites A, B, C.
  • the filter is then removed and wash fluid is added to the top of the absorbent pad, i.e., where the reagents are located.
  • colloidal gold labelled protein A and wash buffer solutions are added.
  • FIG. 10A shows a positive control, indicating that the user has applied human antibody to the device and the reaction between the immobilized antigen and the antibody in the sample has been carried out properly.
  • the positive control line gives a test for indicating whether a test sample and additional liquids, such as wash liquid, have been applied properly. If the test sample is properly applied, the control line will become visible. Otherwise, the control line CL will not be visible.
  • FIGs. 10B-G show that the test sample has been properly applied.
  • FIG. 10B shows presence of p24 antigen
  • FIG. 10C shows presence of gp41
  • FIG. 10D shows presence of
  • Figs 10E-G indicate the detection of at least two of the three types of epitopes of HIV-1 antibody in the sample.
  • the detection is visual.
  • the control line determines whether the test was conducted properly. This control line also is used as a guide for identifying the test sites regardless how the device is positioned or held, or is placed during manufacture of the test device.
  • the embodiment shown in FIG. 9, can detect three different viruses.
  • a suitable absorbent pad of nitrocellulose/cellulose is provided with three test sites A, B, C and a control line CL.
  • the sites A, B, C are respectively spotted with HIV, HCV, and syphilis antigens.
  • the control line again is formed with an anti-human IgG in a streaking motion on the reagent layer 100.
  • a blood sample is introduced or dropped into the filter portion 150, along with appropriate wash liquid so that the filtrant contacts the three test sites A, B, C.
  • the filter is then removed to permit addition of wash fluid.
  • Wash buffer is added, followed by colloidal gold labelled protein A, followed by wash buffer.
  • glycosaminoglycan typically may be chondroitin sulfate, dermatan sulfate, heparin sulfate, keratan sulfate, hyaluronic acid or a heparin, although other glycosaminoglycan compounds not recited here can be used.
  • the glycosaminoglycan compound typically may be at a concentration of from about lug/ml to about lOmg/ml (e.g., lug/ml to lOmg/ml) and more acceptably at a concentration of from about O.lmg/ml to about lmg/ml (e.g., O.lmg/ml to lmg/ml) upon dissolution in the assay or in the pre-treatment solution.
  • the compound may be a heparin salt such as sodium heparin, ammonium heparin or lithium heparin. If used, the heparin advantageously is at a concentration of from about 1 usp/ml to about 50 usp/ml (e.g., 1 usp/ml to 50 usp/ml) and more advantageously from about
  • the glycosaminoglycan compound does not work when present at a high concentration. Heparin, for example, should not be used at a final dissolved concentration that exceeds 25 usp/ml although chondroitin sulfate was found to remain active at 10 mg/ml final concentration.
  • Optimal levels of the glycosaminoglycan compound are determined by adding the compound to an assay at various concentrations and determining test results from different test samples.
  • the acceptable embodiment whereby all solutions are added to a single horizontal location at the top of the device (either before or after separating the top and bottom halves) is particularly suited for automated instrumentation.
  • the inventors realized that a filter and a reagent-layer/absorbent-pad can be sized to allow their manufacture into a 96 well microtiter plate.
  • 8 (or 12) devices can be manufactured into a vertical (or horizontal) strip of multiple test devices that can be assembled to become part of a microtiter plate.
  • Such microtiter plate embodiments can be processed by existing microtiter plate instruments. In these embodiments the user can add sample, wash and/or reagent solutions by hand or by automated instrumentation.
  • a particularly advantageous embodiment is a 32 well plate comprising 4 rows of 8 wells each.
  • each well presents an opening of about 0.3 to 0.8 inch diameter- to a single device in the group of 32.
  • a device of the present invention can be packaged with other components such as an instruction pamphlet, a wash reagent that may be either a dried material that is redissolved in water or buffer, or already present as a fluid, and optionally one or more reagents to add to the device such as gold particles treated with a binding partner for a binding reaction.
  • the invention allows the production and use of kits for infectious disease testing from blood.
  • kits for infectious disease testing from blood.
  • One advantage of these kits, according to an advantageous embodiment is that they can contain a glycosaminoglycan to eliminate false measurements.
  • a test fluid preparation filter holder was constructed with a filter composed of the following components in sequential order from top to bottom: a blood separation filter; a backup blood separation filter; and a dispersant layer.
  • the dispersant layer was situated in direct contact with a reagent layer in which a member of a binding pair, namely HIV antigen, was immobilized.
  • the blood separation filters and the dispersant layer were chemically treated by exposure to a solution of buffer, detergent, protein and anticoagulant.
  • test fluid whole blood, EDTA treated blood, heparinized blood, acid citrate dextran treated blood, plasma or serum
  • test fluid whole blood, EDTA treated blood, heparinized blood, acid citrate dextran treated blood, plasma or serum
  • test fluid preparation filter holder is then removed from the detection device. After removing the filter holder, the reagent layer of the test device is exposed. Exposing the reagent layer allows for the addition of wash fluid. Two drops of wash buffer are added to the reagent layer on which HIV antigens have been immobilized. Then two drops of labelled colloidal gold labelled protein A are added, followed by two drops of wash buffer. The presence of anti-HTV antibody in the sample is detected as color formation from the deposition of colloidal gold. More than 100 whole blood samples that were obtained from patients suspected of carrying HIV were tested for antibodies to HIV. The test results correlated 100% with results obtained by a western blot method.
  • Example one a device is constructed as described in example one.
  • the device is used in accordance with the procedure of Example One except that after sample application and wash, the test device is maintained at room temperature for 4 hours. The remaining steps of the assay are then carried out. Blood samples tested in accordance with this device and method and which contain anti-HIV antibody accurately formed color from the deposition of
  • peptides having the sequences Q-A-R-L-L- A-W-E-K-T-L-K-D-Q-Q-L-L-G-I-W-G-C-S-G-K-H-I-C-T-T-T-T-V-P-W-N-S (a preferred subtype D sequence) and R-A-R-L-Q-A-L-E-T-L-I-Q- N-Q-Q-R-L-N-I-W-G-C-K-G-K-L-V-C-Y-T-S-V-K-W-N-R (a preferred Group O sequence) were combined in a test device as described above for HIV-1 infection testing.

Abstract

L'invention concerne un dispositif de tests diagnostiques qui comprend un filtre et au moins deux peptides qui correspondent au même épitope d'analysat. Le dispositif de tests manifeste un transfert amélioré du mouvement des fluides entre les éléments d'un dosage et sert à effectuer des dosages simultanés de plusieurs analysats. Le filtre fait partie intégrante d'une bandelette et peut être utilisé pour des tests avec bandelettes du sang entier et, en général, d'autres solutions comportant des particules. Les surfaces des parties à l'intérieur du dispositif sont combinées de différentes manières spécifiques pour améliorer le mouvement des fluides d'échantillon et de réactif ; un éventuel additif chimique améliore la qualité des tests. En guise d'exemple, on cite des cas de tests du sang entier pour le VIH, y compris les tests de confirmation, qui sont faciles à effectuer, manifestent une résistance chimique améliorée aux faux résultats positifs et une plus grande capacité de détection d'une large gamme de souches virales.
PCT/US1999/009331 1998-04-30 1999-04-30 Dosage immunitaire avec peptides mixtes WO1999056128A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU36709/99A AU3670999A (en) 1998-04-30 1999-04-30 Immunoassay with mixed peptides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US6993598A 1998-04-30 1998-04-30
US09/069,935 1998-04-30

Publications (1)

Publication Number Publication Date
WO1999056128A1 true WO1999056128A1 (fr) 1999-11-04

Family

ID=22092115

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/009331 WO1999056128A1 (fr) 1998-04-30 1999-04-30 Dosage immunitaire avec peptides mixtes

Country Status (2)

Country Link
AU (1) AU3670999A (fr)
WO (1) WO1999056128A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1347292A1 (fr) * 2000-12-27 2003-09-24 Matsushita Electric Industrial Co., Ltd. Biocapteur
WO2004040306A1 (fr) * 2002-10-31 2004-05-13 Labrea Holding S.A. Dispositif de diagnostic et kit
CN104215786A (zh) * 2014-09-30 2014-12-17 博奥赛斯（天津）生物科技有限公司 一种化学发光免疫分析设备外加机械手
CN104237545A (zh) * 2014-09-30 2014-12-24 博奥赛斯（天津）生物科技有限公司 一种化学发光免疫分析混匀装置
WO2016035099A1 (fr) * 2014-09-05 2016-03-10 Meril Diagnostics Private Limited Dispositif à flux traversant pour la détection de substances biologiques à analyser et procédé associé
US20210333262A1 (en) * 2020-04-28 2021-10-28 Brandon Heeger Rapid testing mechanism and method for respiratory viral pathogens

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0401913A1 (fr) * 1989-06-05 1990-12-12 Janssen Pharmaceutica N.V. Dosage en phase solide à utiliser avec un développeur physique
WO1996012809A2 (fr) * 1994-10-20 1996-05-02 Institut Pasteur Sequences nucleotidiques d'antigenes retroviraux vih-1 groupe (ou sous-groupe) o
WO1996038474A2 (fr) * 1995-05-31 1996-12-05 Bionova Corporation Diagnostic de et vaccination contre un virus a arn double brin positif a l'aide d'un polypeptide isole et non traite
DE19536166C1 (de) * 1995-09-29 1997-03-06 Siegfried Dr Krell Verfahren zur Bestimmung von Antikörpern gegen Treponema pallidum (Syphilis)
WO1998013519A1 (fr) * 1996-09-25 1998-04-02 Universal Healthwatch, Inc. Dispositif pour epreuve diagnostique ameliorant la circulation des fluides et la resistance aux interactions chimiques

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0401913A1 (fr) * 1989-06-05 1990-12-12 Janssen Pharmaceutica N.V. Dosage en phase solide à utiliser avec un développeur physique
WO1996012809A2 (fr) * 1994-10-20 1996-05-02 Institut Pasteur Sequences nucleotidiques d'antigenes retroviraux vih-1 groupe (ou sous-groupe) o
WO1996038474A2 (fr) * 1995-05-31 1996-12-05 Bionova Corporation Diagnostic de et vaccination contre un virus a arn double brin positif a l'aide d'un polypeptide isole et non traite
DE19536166C1 (de) * 1995-09-29 1997-03-06 Siegfried Dr Krell Verfahren zur Bestimmung von Antikörpern gegen Treponema pallidum (Syphilis)
WO1998013519A1 (fr) * 1996-09-25 1998-04-02 Universal Healthwatch, Inc. Dispositif pour epreuve diagnostique ameliorant la circulation des fluides et la resistance aux interactions chimiques

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1347292A1 (fr) * 2000-12-27 2003-09-24 Matsushita Electric Industrial Co., Ltd. Biocapteur
EP1347292A4 (fr) * 2000-12-27 2005-03-02 Matsushita Electric Ind Co Ltd Biocapteur
WO2004040306A1 (fr) * 2002-10-31 2004-05-13 Labrea Holding S.A. Dispositif de diagnostic et kit
WO2016035099A1 (fr) * 2014-09-05 2016-03-10 Meril Diagnostics Private Limited Dispositif à flux traversant pour la détection de substances biologiques à analyser et procédé associé
CN104215786A (zh) * 2014-09-30 2014-12-17 博奥赛斯（天津）生物科技有限公司 一种化学发光免疫分析设备外加机械手
CN104237545A (zh) * 2014-09-30 2014-12-24 博奥赛斯（天津）生物科技有限公司 一种化学发光免疫分析混匀装置
US20210333262A1 (en) * 2020-04-28 2021-10-28 Brandon Heeger Rapid testing mechanism and method for respiratory viral pathogens
US11644456B2 (en) * 2020-04-28 2023-05-09 Brandon Heeger Rapid testing mechanism and method for respiratory viral pathogens

Also Published As

Publication number Publication date
AU3670999A (en) 1999-11-16

Similar Documents

Publication Publication Date Title
EP0351248B1 (fr) Détection d'un anticorps pathogène-specifique félin et d'un antigène pathogène félin dans un immunoessai
AU748387B2 (en) Neutralization of polycations in a chromatographic device for whole blood use
JP2626721B2 (ja) Ｈｔｌｖ―３の蛋白質の分離、エイズおよび前エイズ条件を持つ患者の血清中ｈｔｌｖ―３に対する抗体の血清学的検出、およびｈｔｌｖ―３およびその蛋白質を使用した免疫分析によるｈｔｌｖ―３感染の検出
ES2207757T3 (es) Deteccion de igg especifica para antigenos.
Kelen et al. Rapid detection of Australia/SH antigen and antibody by a simple and sensitive technique of immunoelectronmicroscopy
KR20200037846A (ko) 개선된 분석물 검출을 위한 분석 방법
KR20000071894A (ko) 단백질칩 및 이를 이용한 다목적 자동화 진단시스템
Araujo et al. Use of monoclonal antibodies to detect antigens of Toxoplasma gondii in serum and other body fluids
EP0644950A1 (fr) Dosage pour la detection de l'antigene du vih et de l'anticorps du vih
JPH10197534A (ja) 凝集により試験液中の分析物を検出するための方法及び試薬キット、固定リガンド分子を担持する合成粒子並びにその使用
JPH04502325A (ja) Ｈｉｖ−１又はｈｉｖ−２抗体の存在又は量の確定方法、免疫特異的試薬、合成ペプチド、診断用キット、ｈｉｖ−１又はｈｉｖ−２抗体の調製方法、免疫原及び抗体
JP4130505B2 (ja) Ｄ−アミノ酸からなるペプチドによる診断方法の干渉の排除
CA2126247A1 (fr) Anticorps monoclonal contre le vih-2 et ses utilisations
CA2266747A1 (fr) Dispositif pour epreuve diagnostique ameliorant la circulation des fluides et la resistance aux interactions chimiques
WO1999056128A1 (fr) Dosage immunitaire avec peptides mixtes
EP0698214A1 (fr) Dosage immunologique vih a l'aide de proteines recombinantes et de reactifs peptidiques de synthese
AU754309B2 (en) Diagnosis of feline immunodeficiency virus infection using env/gag polypeptide markers
US4879211A (en) Rapid immunoagglutination test for presence of HIV in body fluids
EP0993615A1 (fr) Test de confirmation rapide d'infections microbiennes
ES2220982T3 (es) Peptidos destinados a la deteccion del vih-1.
JP3421336B2 (ja) 合成ペプチドを用いてのｈｔｌｖ−▲ｉ▼とｈｔｌｖ−▲ｉｉ▼との区別
WO1995032293A1 (fr) Dosage du groupe o du vih-1 a l'aide d'au moins un peptide gp41
CA1339006C (fr) Systeme de diagnostic autonome pour immuno-essais multiples
JPH06265548A (ja) 抗原と抗体の同時測定法
JPH09171019A (ja) アデノウイルス抗原検出キット

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase