Method and devices for rapid diagnosis of foot-and-mouth disease

Abstract

A rapid immunoassay method and apparatus for detecting foot and mouth disease virus are disclosed. The method and test device permit pen-side testing of animals and provide test results within a relatively short time period. In a preferred embodiment, the method and apparatus provide a means for differentiating between FMDV-infected and FMDV-vaccinated animals.

Claims

1. A method for simultaneously detecting and differentiating between antibodies of interest comprising antibodies to FMDV structural proteins and antibodies to FMDV non-structural proteins in a sample, comprising: a. obtaining a fluid test sample to be assayed for said antibodies of interest; b. supplying a rapid measurement device comprising a membrane strip with a proximal end containing a labeled binding partner and a labeled control reagent, and a distal end containing a zone having at least one immobilized capture reagent specific for an FMDV structural protein comprising at least one of VP1, VP2, VP3 or VP4, and a second zone having at least one immobilized capture reagent specific for an FMDV non-structural protein comprising at least one of Lb, 2B, 2C, 3A, 3AB, or 3ABC, and a third band of immobilized control capture reagent wherein said fluid test sample moves from said proximal end to said distal end by capillary action; c. contacting said membrane strip with said fluid test sample; d. maintaining said membrane strip under conditions which are sufficient to allow said antibodies of interest to bind to said labeled binding partner, forming a specific binding complex, and to allow fluid to transport said specific binding complex and said labeled control reagent in said fluid by capillary action through said membrane strip to said immobilized capture reagent specific for an FMDV structural protein and said adjacent immobilized capture reagent specific for an FMDV non-structural protein and to said immobilized control capture reagent; e. maintaining said membrane strip under conditions which are sufficient to allow said specific binding complex in said fluid to bind to at least one of said immobilized capture reagent specific for an FMDV structural protein and/or to bind to at least one of said immobilized capture reagent specific for an FMDV non-structural protein and said labeled control reagent to bind to said immobilized control capture reagent; f. detecting an amount of said antibodies of interest in said fluid test sample and an amount of internal control signal, as indicated by an intensity of a signal producing system; and g. differentiating between said antibodies of interest.

2. The method of claim 1 wherein said labeled binding partner is at least one of VP1, 3D, 2C or 3ABC and at least one of VP1, VP2, VP3 and VP4.

3. The method of claim 1 wherein said labeled binding partner is protein G and/or protein A.

4. The method of claim 1 wherein said membrane strip comprises an application point comprising a filter pad containing at least one of said labeled binding partner and said labeled control reagent.

5. The method of claim 1 wherein aid labeled binding partner is protein G.

6. The method of claim 1 wherein aid labeled binding partner is protein A.

7. The method of claim 1 wherein aid labeled binding partner is protein G and protein A.

8. The method of claim 1 wherein said membrane strip comprises an application point comprising a filter pad containing said labeled binding partner and said labeled control reagent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram of the genetic map of FMDV RNA and proteins.

(2) FIG. 2 is an elevation view of the strip configuration of the assay device.

(3) FIG. 3 is a schematic diagram of an assay for the detection of antibody in the sample.

(4) FIGS. 4 and 4A are the nucleotide and amino acid sequence of the VP-1 protein.

(5) FIG. 5 is the plasmid map of pBM-VP1Tw97F.

(6) FIGS. 6, 6A and 6B are the nucleotide and amino acid sequence of the 2C protein.

(7) FIG. 7 is the plasmid map of pBM-2CTw97F.

(8) FIGS. 8, 8A, 8B and 8C are the nucleotide and amino acid sequence of the 3ABC protein.

(9) FIG. 9 is the plasmid map of pBM-3ABCTw97F.

(10) FIGS. 10, 10A, 10B and 10C are the nucleotide and amino acid sequence of the 3D protein.

(11) FIG. 11 is the plasmid map of pBM-3DTw97F.

(12) FIG. 12 is a schematic diagram of the test kit formulation.

(13) FIG. 13 is a schematic diagram of the two-line test kit configuration.

(14) FIG. 14 is a chart comparing test results over time of a 3ABC ELISA and the test of the presenting invention for an FMDV-infected cow.

(15) FIG. 15 is a chart comparing test results over time of a 3ABC ELISA and the test of the presenting invention for a second FMDV-infected cow.

(16) FIG. 16 is a chart comparing test results over time of a 3ABC ELISA and the test of the presenting invention for an FMDV-infected pig.

(17) FIG. 17 is a chart comparing test results over time of a 3ABC ELISA and the test of the presenting invention for a second FMDV-infected pig.

DETAILED DESCRIPTION OF THE INVENTION

(18) The Test Device

(19) The present invention relates to a device for the detection of an analyte in a sample of biological fluid through the use of immunochemical ligand-receptor reactions and specially selected, treated, and arranged filter materials (FIG. 2; see also U.S. Pat. No. 5,559,041 which is hereby incorporated by reference). The present invention includes a non-reactive cover (also referred to as an enclosure or casing) around the device. The cover encloses at least the strip to avoid contact with, and contamination of, the capture sites. In one embodiment, the cover also includes a raised area adjacent to the application pad to facilitate receiving and/or containing a certain volume of the test sample and/or wicking solution. In a further embodiment of the invention, the cover includes a cut out area or areas in the form of a letter, number, icon, or symbol or any combination thereof. In this embodiment, the cut out area or areas form the design for a particular capture site(s) once the strip is completely enclosed. In another embodiment of the invention, a sufficient portion of the strip is encased to prevent the applied test sample from contacting the capture sites without first passing through a portion of the strip.

(20) The Application Pad

(21) It is contemplated and within the scope of the present invention that the solid phase can be any suitable porous material with sufficient porosity to allow access by detection antibodies and/or antigens and a suitable surface affinity to bind antigens and/or antibodies. Microporous structures, in general, are preferred, but materials with gel structure in the hydrated state may be used as well. Such useful solid supports include, but are not limited to, nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), porous polyethylene fit or pads, and glass fiber filter paper; either used by themselves or in conjunction with other materials.

(22) The surface of such supports may be activated by chemical processes that cause covalent linkage of the antigen and/or antibody to the support. Alternatively, the irreversible binding of the antigen and/or antibody is obtained by adsorption on the porous material by poorly understood hydrophobic forces. Other suitable solid supports are well known to those of skill in the art. It is also well known in the art that the material chosen for the solid support(s) is one that is compatible with the analyte and assay reagents used.

(23) 2 Pad System/3 Pad System

(24) The filter pad 11 is separate and distinct from said reservoir pad 10, and wicking membrane 15, and interposed between and contiguous with the wicking membrane 15, and the reservoir pad 10′(3 Pad system). Or one filter pad 11′ functions as both reservoir 10′ and filter zone (2 Pad system). The filter zone has impregnated therein a labelled immunochemical component capable of binding to an analyte of interest in sample to form an immuno-complex. The filter zone is operable to permit passage of any specific immuno-complex to the wicking membrane 15, while impeding passage of larger components then contained in the sample; and at least one immobilized substance disposed in at least one assay indicia zone 4 of the wicking membrane 15 downstream of the filter zone and defining assay indicia 13, the immobilized substance being operable to bind a specific immuno-complex contained in the sample to form the assay indicia 13.

(25) Reagents are Incorporated within the Test Device

(26) In addition, the application pad typically contains one or more assay reagents either diffusively or non-diffusively attached thereto. Reagents that are contained in the application pad include, but are not limited to, labeled reagents, ancillary specific binding members, and/or signal producing system components needed to produce a detectable signal. The isolation of assay reagents in the application pad also separates the interactive reagents, thereby facilitating the manufacturing process.

(27) In an embodiment of the present invention, predetermined amounts of signal producing components and ancillary reagents are incorporated within the device, thereby avoiding the need for additional protocol steps or reagent additions. Thus, it also is within the scope of this invention to provide more than one reagent to be immobilized within the application pad and/or the strip material.

(28) This invention covers detection method for FMDV antibody, PRRSV(Porcine Respiratory and Reproductive Symptom Virus) antibody and antigen, FeLV(Feline Leukemia Virus) antigen, FIV(Feline Immunodeficiency Virus) antibody, Mad Cow disease marker, CSF(Classical Swine Fever) antibody and antigen, B. canis(Brucellocis canis) antibody and antigen, Johnes disease antibody and BVDV(Bovine Viral Diarhrea Virus). Antigen. Also this invention covers detection of the cancer markers, hormones, enzyme and drugs, and antigens that may be applied as disease or biological makers.

(29) Chromatographic Material Transports Liquids

(30) The invention disclosed herein provides assay devices and methods wherein strips of chromatographic material capable of transporting liquids are used in the assay. In one embodiment of the present invention, the assay device includes test sample application pads that are in fluid flow contact with the strip which functions to regulate the flow of the test sample to the chromatographic material, to filter the test samples and to deliver and/or mix assay reagents. For example, not meant to limit the invention in any way, during a binding assay the labeled reagent is contained in the application pad and is released from the pad to the strip containing the applied test sample, thereby eliminating the need to combine the test sample and labeled reagent prior to using the device (FIG. 3).

(31) In a further embodiment of the invention the assay reagents are incorporated within the chromatographic material, as well as in the application pad. By varying the configuration of reagent-containing sites on the device, qualitative and quantitative displays of assay results are obtained. The reagents are situated in the devices in such a way as to make the assay substantially self-performing and to facilitate the detection and quantitation of the assay results. Any signal resulting from the reaction(s) at the reagent-containing site(s) is detected by instrumentation or by direct visual observation.

(32) Test Device for Diagnosis of FMD

(33) In one embodiment the test device for the assay includes, but is not limited to, a nitrocellulose membrane strip upon which are placed, and allowed to dry in separate distinct capture areas, highly purified recombinant antigens derived from FMDV and/or specific monoclonal antibodies to FMDV (FIG. 2). The test device further includes a filter pad which contains a labeled indicator, such as gold colloid conjugated with protein G and/or A, suspended in a fluid containing nitrocellulose blocking proteins, which have been dried prior to assembly and affixed to the distal end of the nitrocellulose membrane (FIGS. 2 and 3). The entire device is held permanently in place by a top clear laminating material which bears an adhesive surface in contact with the top surface of the nitrocellulose membrane and attached to the conjugate pad, and a bottom laminating material which bears an adhesive surface in contact with the bottom surface of the nitrocellulose membrane. The fluid test sample flows from the distal end to the proximal end. In one embodiment of the device, there is a test sample pad and reactivity zone upon which the test sample is placed. The read out (in the capture areas and/or in the test sample reactivity zone) is either visual, without the aid of laboratory equipment, or automated. In a further embodiment of the invention, the test device is enclosed in a casing of molded plastic or other suitable material.

(34) Analyte Detection

(35) An exemplary embodiment of the present invention, which is not meant to define or constrain the invention described herein, is performed as follows. A test sample, such as, but not limited to, animal serum is contacted with the labeled indicator, such as protein G and/or protein A-gold conjugate, on the filter pad at the sample application point on the test device. IgG and/or IgM antibodies in the sample are bound by the protein G and/or protein A-gold conjugate and the protein G and/or protein A-gold conjugate—antibody complexes are chromatographed along the length of the absorbent pad (such as, but not limited to, a nitrocellulose membrane). As an internal control for efficacy, a labeled control reagent is also present in the filter pad. As the fluid, containing both the aforementioned complexes and the internal control reagent, flow, they pass over the line where the FMDV recombinant antigens have been applied in test band 13 (FIG. 3). If the complexes contain specific antibody (IgG and/or IgM) to the recombinant antigens in the test band 13, the antigens in the test band will form a complex with the protein G and/or A antibody complex and a detectable signal is generated. Simultaneously, the labeled control reagent present in the fluid will flow, via capillary action, to the control band 14 wherein an immobilized control capture reagent will bind to the labeled control reagent, generating a positive signal. A further embodiment of the invention disclosed herein incorporates the quantitation of the antibody and/or antigen in the test sample, as determined by the intensity of the signal generated relative to an intensity of signals generated in a standard curve.

EXAMPLES

(36) Oligonucleotides for gene construction and sequencing were synthesized at ResGen (Huntsville, Ala.). Unless otherwise indicated, DNA sequencing was also performed at ResGen.

(37) Polymerase Chain Reaction (PCR)

(38) For PCR, Vent DNA polymerase and buffer were purchased from New England Biolabs, Inc. (Beverly, Mass.) and a mixture of dNTPs was purchased from Amersham-Pharmacia (Piscataway, N.J.) and used according to the manufacturer's specifications, unless otherwise indicated. PCR amplifications were performed on a GeneAmp 2400 thermal cycler from Perkin-Elmer Corporation (Foster City, Calif.). The PCR product was purified using Qiagen PCR spin column (Qiagen Inc., Chatsworth, Calif.), as recommended by the manufacturer. Unless indicated otherwise, restriction enzymes were purchased from New England BioLabs and DNA fragments were initially isolated on agarose (Sigma-Aldrich) gels prior to the restriction digestion for their cloning.

(39) Isolation of the Desired Clone(s)

(40) The desired fragment was excised and the DNA was extracted with a QIAEX II gel extraction kit, as recommended by the manufacturer. DNA was resuspended in H.sub.2O or IE. Ligation of the isolated fragment into the vector was performed using DNA ligase (Boehringer Mannheim Corporation, Indianapolis, Ind.), as recommended by the manufacturer. The ligation reaction was incubated at 16° C. overnight. Bacterial transformations were performed using E. coli XL1-Blue competent cells. Unless indicated otherwise, transformations and bacterial re-streaks were plated on LB agar (Lennox) plates supplemented with 100 ug/ml ampicillin. All bacterial incubations (plates and liquid cultures) were conducted overnight (16 hours) at 37° C.

(41) Screening of transformants to identify desired clones was accomplished by restriction enzyme digestion of mini-prep DNA and/or by colony PCR. Mini-prep DNA was prepared according to Molecular Cloning: A Laboratory Manual, unless otherwise indicated. Colonies containing desired clones were propagated from the transfer plate or stocked in glycerol at −70° C.

Example 1

Antigen Production

Preparation of Recombinant FMDV VP1 Antigen

(42) A. Construction of FMDV VP1 Expression Vectors

(43) (i) Construction of Synthetic VP1 Gene

(44) VP1 protein FMDV Taiwan Type O 97 sequence was retrieved from NCBI GenBank database (genbank accession number: G15921457) and oligonucleotides for synthesis of the gene were synthesized at ResGen (Huntsville, Ala.). In the synthetic oligonucleotides, the native FMDV codons were altered to conform to E. coli codon bias in an effort to increase expression levels of the recombinant protein in E. coli. (see, for example, M. Gouy and C. Gautier, Nucleic Acids Research 10:7055 (1982); H. Grosjean and W. Fiers, Gene 18:199 (1982); J. Watson et al. (eds.), Molecular Biology of the Gene, 4th Ed., Benjamin Kumming Publishing Co., pp. 440 (1987)). The recursive PCR method was used to assemble the oligonucleotides into a full VP1 gene. The gene construction strategy involved synthesis of a series of overlapping oligonucleotides with complementary ends. When annealed, the ends served as primers for the extension of the complementary strand. The fragments were then amplified by outside primers.

(45) The oligonucleotide was designed to contain a BamHI restriction site for cloning into the expression vector pGEX-4T-1. The anti-sense oligonucleotide contains a translational termination codon (TAA) and an EcoRI restriction site. When outside primers TW97-1 (SEQ B) NO: 1) and TW97-16 (SEQ ID NO: 16) were used, a full-length VP1 (213 amino acids) gene was synthesized (FIG. 4).

(46) Recursive PCR (100 ul volume) was set up as follows: Vent DNA polymerase (1U) and 1× buffer, along with 25 uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 50 pmol each of oligonucleotides TW97-1 (SEQ ID NO: 1) and TW97-16 (SEQ ID NO: 16), and 0.25 pmol each of oligonucleotides TW97-2 through TW97-15 (SEQ ID NO: 2 through SEQ ID NO: 15, respectifully). The reaction was incubated at 95° C. for 5 minutes, and then amplified with 30 cycles of 95° C. for 15 seconds, 58° C. for 15 seconds and 72° C. for 60 seconds, followed by incubation at 72° C. for 5 minutes. The PCR-derived product was purified using Qiagen PCR spin column.

(47) (ii) Cloning of the PCR Product.

(48) The PCR product amplified as described herein was digested with the restriction endonucleases Bam HI and Eco RI and ligated into the gel-purified vector pGEX-4T-1 that had been digested with Bam HI and Eco RI. The ligation product was used to transform XL-1 Blue competent cells. The transformed cells were plated on LB plates supplemented with 100 ug/ml ampicillin. Mini-prep DNAs were prepared from overnight cultures of colonies and digested with Bam HI and Eco RI to screen the desired clones. The clone with right insert was designated as pBM-VP1Tw97F (FIG. 5).

(49) The pBM-VP1Tw97F clone was sequenced with the oligonucleotide primers pGEX5 (SEQ ID NO: 116) and pGEX3 (SEQ ID NO: 117).

(50) B. Growth and Induction of E. coli Strains with VP] Plasmids

(51) Overnight seed cultures of each E. coli colonies were prepared in 500 ml sterile LB supplemented with 100 μg/ml ampicillin, and placed in a shaking orbital incubator at 37° C. Fifty milliliter inoculums from seed cultures were transferred to flasks containing 0.5 liter sterile LB supplemented with 100 μg/ml ampicillin. Cultures were incubated at 37° C. until the cultures reached mid-logarithmic growth and then induced with 1 mM IPTG for 3 hours at 37° C. After the induction period, cells were pelleted by centrifugation and harvested following standard procedures currently used in the art. Pelleted cells were stored at −70° C. until further processed.

(52) C. Preparation of VP] Antigen

(53) Frozen cells obtained above were resuspended in PBS with 1 mM PMSF. The cells were disrupted by ultrasonication (Branson). Inclusion bodies were separated from soluble proteins by centrifugation. These pelleted inclusion bodies were washed and pelleted sequentially in PBS followed by water. The washed inclusion bodies were resuspended in PBS and 5 M urea with a brief sonication. The inclusion bodies were then separated from the solubilized proteins by centrifugation. The pelleted inclusion bodies were fully solubilized in 7M guanidine-HCl. The solubilized recombinant antigens were clarified by centrifugation and passed through a 0.2 μm filter.

(54) The guanidine-HCl solubilized fusion protein was denatured by diluting in water and precipitated by centrifugation. The pellet was washed with water and then resuspended in water. A 2M NaOH solution was added to solubilize the denatured protein completely and 2N HCl was added to neutralize the pH of the protein solution.

Example 2

Preparation of Recombinant FMDV 2C Antigen

(55) A. Construction of FMDV 2C Expression Vector

(56) The genome sequence of FMDV 2C protein was retrieved from NCBI GenBankdatabase (GI: 5921457, 0 strain Chu-Pei) and oligonucleotides for the synthesis of the entire 2C gene were synthesized, and confirmed by sequencing, at ResGen (Huntsville, Ala.). The coding DNA sequence is 954 nucleotides long, which encodes 318 amino acids (FIG. 7).

(57) (i) Construction of Synthetic Full-Length 2C Gene

(58) To obtain the 2C gene of FMDV, 24 oligonucleotide primers were synthesized, each with complementary ends, at Resgen. The recursive PCR method was used to assemble the oligonucleotides into a full-length 2C gene. The gene construction strategy involved synthesis of a series of overlapping oligonucleotides with complementary ends. When annealed, the ends served as primers for the extension of the complementary strand. The fragments were then amplified by excessive outside primers.

(59) Due to the large size of the 2C gene that was to be synthesized, the oligonucleotides were divided into three groups and the respective recursive PCRs were performed. The three DNA products were designated as the A, B and C fragments. The B and C fragments were joined with PCR and then the B-C fragment was joined with A fragment to produce a full-length 2C gene. One of the oligonucleotides on the end of the full-length gene was designed to contain a BamHI restriction site for cloning into the expression vector pGEX-4T-1. The reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 53° C. for 30 seconds and 73° C. for 100 seconds, followed by incubation at 73° C. for 5 minutes. An aliquot of the reaction mixture was analyzed by electrophoresis on agarose mini-gel.

(60) (ii) Cloning of the PCR Product.

(61) The amplified PCR product as described herein was digested with the restriction endonucleases Bam HI and Hind III and ligated into the vector pGEX-4T-1 that had been digested with Bam HI and Hind III. The ligation product was used to transform E. coli XL-1 Blue competent cells. The transformed cells were plated on LB plates supplemented with 100 μg/ml ampicillin. Mini-prep DNAs were prepared from overnight cultures of transformed colonies using Q1Aprep plasmid DNA mini-preparation kit and digested with Bam HI and Hind III to screen the desired clones. The clone with right insert was designated as pBM-2CTw97F (FIG. 8).

(62) The pBM-2CTw97F clone was sequenced with the oligonucleotide primers pGEX5 (SEQ ID NO: 116), pGEX3 (SEQ ID NO: 117), 2C-25 (SEQ ID NO: 41) and 2C-26 (SEQ ID NO: 42).

(63) B. Growth and Induction of E. coli Strains with FMDV 2C Plasmid

(64) Overnight seed cultures of pGEX-2CTw97F were prepared in 500 ml sterile LB supplemented with 100 μg/ml ampicillin, and placed in a shaking orbital incubator at 37° C. A 50 ml inoculum from seed cultures was transferred to flask containing 0.5 liter sterile LB supplemented with 100 μg/ml ampicillin. Cultures were incubated at 37° C. until it reached mid-logarithmic growth and then induced with 1 mM IPTG for 3 hours at 37° C. After the induction period, cells were pelleted by centrifugation and harvested following standard procedures. The pelleted cells were stored at −70° C. until further process.

(65) C. Preparation of FMDV 2C Antigen

(66) Frozen cells obtained above were resuspended in PBS with 1 mM PMSF and Triton X-100 detergent, and then disrupted by ultrasonication (Branson). Inclusion bodies were separated from soluble proteins by centrifugation. The protein fraction enriched with 2C was obtained through 3-4 rounds of washing off the contaminants and solubilization of the cell lysate pellet in urea or guanidin-HCl. Recombinant 2C was purified through size exclusion chromatography (FPLC, Sephacryl S 200 HR) under denaturing conditions (5N GuHCl, PBS (pH7.4)) and the eluted fraction containing 2C was identified by SDS-PAGE and then dialyzed against 20 mM phosphate buffer (pH 9.0). Sodium azide (0.05%) was added to the protein solution, which was stored at 4° C. For longer storage (over 1 month), the protein solution was aliquoted and frozen at −70° C.

Example 3

Preparation of Recombinant FMDV 3ABC Antigen

(67) A. Construction of FMDV 3ABC expression vector

(68) The genome sequence of FMDV 3ABC protein was retrieved from NCBI GenBank data (GI: 5921457, 0 strain Chu-Pei) and oligonucleotides for the synthesis of whole 3ABC gene and sequencing were synthesized at ResGen (Huntsville, Ala.). The coding DNA sequence is 1281 nucleotides long, which encodes 427 amino acids (FIG. 9).

(69) (i) Construction of Synthetic Full-Length 3ABC Genes

(70) To obtain the 3ABC gene of FMDV, 33 oligonucleotide primers were synthesized, each with complementary ends, at ResGen. The recursive PCR method was used to assemble the oligonucleotides into a full-length 3ABC gene. The gene construction strategy involved synthesis of a series of overlapping oligonucleotides with complementary ends. When annealed, the ends served as primers for the extension of the complementary strand. The fragments then were amplified using outside primers. Due to the large size of 3ABC gene to be synthesized, the oligonucleotides were divided into four groups and respective recursive PCRs were performed. The four DNAs were designated as the A, B, C or D fragment. The A and B fragments were joined and the C and D fragments were joined through PCR. Then the A-B fragment was joined with the C-D fragment to produce a full-length 3ABC gene.

(71) One of the end oligonucleotides used in the recursive PCR above was designed to contain a Bamill restriction site for cloning into the expression vector pGEX-4T-1. The anti-sense oligonucleotide contains a translational termination codon (TAA) and an EcoRI restriction site. When N- and C-terminal primers, 3ABC-1 (SEQ ID NO: 43) and 3ABC-33 (SEQ ID NO: 75), were used, a full-length 3ABC (427 amino acids) gene was synthesized.

(72) The PCR reaction (100 11.1 volume) was set up as follows: Vent DNA polymerase (1U) and 1× buffer, along with 25 μM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 μl 100 mM MgSO.sub.4 and 100 pmol of each oligonucleotide. The template was mixture of the A-B fragment and the C-D fragment. The reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 60° C. for 30 seconds and 73° C. for 120 seconds, followed by incubation at 73° C. for 5 minutes. The PCR-derived product was run on the agarose gel and the DNA band was excised and eluted from the gel using Quigen gel extraction kit.

(73) (ii) Cloning of the PCR Product.

(74) The PCR product amplified as described above was digested with the restriction endonucleases Bam HI and Hind III and ligated into the vector pGEX-4T-1 that had been digested with Bam HI and Hind III. The ligation product was used to transform E. coli XL-1 Blue competent cells. The transformed cells were plated on LB plates supplemented with 100 μg/ml ampicillin. Mini-prep DNAs were prepared from overnight cultures of transformed colonies using Q1Aprep plasmid DNA mini-preparation kit and digested with Bam HI and Hind III to screen for the desired clones. The clone with right insert was designated as pBM-3ABCTw97F (FIG. 10).

(75) The pBM-3ABCTw97F clone was sequenced with the oligonucleotide primers pGEX5 (SEQ ID NO: 116), pGEX3 (SEQ ID NO: 117), 3ABC-36 (SEQ ID NO: 78) and 3ABC-37 (SEQ ID NO: 79).

(76) B. Growth and Induction of E. coli Strains with 3ABC Plasmid

(77) Overnight seed cultures of pGEX-3ABCTw97F were prepared in 500 ml sterile LB supplemented with 100 μg/ml ampicillin, and placed in a shaking orbital incubator at 37° C. A 50 ml inoculum from seed cultures was transferred to flask containing 0.5 liter sterile LB supplemented with 100 μg/ml ampicillin. Cultures were incubated at 37° C. until it reached mid-logarithmic growth and then induced with 1 mM IPTG for 3 hours at 37° C. After the induction period, cells were pelleted by centrifugation and harvested following standard procedures known in the art. Pelleted cells were stored at −70° C. until further process.

(78) C. Preparation of FMDV 3ABC Antigen

(79) Frozen cells obtained above were resuspended in PBS with 1 mM PMSF and Triton X-100 detergent and disrupted by ultrasonication (Branson). Inclusion bodies were separated from soluble proteins by centrifugation. Protein fraction enriched with 3ABC was obtained through 3-4 rounds of washing off the contaminants and solubilization of cell lysate pellet in urea. Recombinant 3ABC was run through ion-exchange chromatography (FPLC, Q-Sepharose FF) under denaturing condition (8M urea, 10 mM DTT, 20 mM potassium phosphate, pH 7.0) and eluted using a NaCl gradient. The eluted fraction was dialyzed against 20 mM phosphate buffer (pH 9.0). After measuring the protein concentration by the Bradford method and adding sodium azide to 0.05%, the protein solution was stored at 4° C. For longer storage (over 1 month), protein solution was aliquoted and frozen at −70° C.

Example 4

Preparation of Recombinant FMDV 3D Antigen

(80) A. Construction of FMDV 3D Expression Vector

(81) (1) Construction of Synthetic full-length 3D Genes

(82) To obtain the 3D gene of FMDV, 36 oligonucleotides were synthesized, each with complementary ends, at ResGen. We used the recursive PCR method to assemble the oligonucleotides into a full 3D gene (FIG. 11). The gene construction strategy involved synthesis of a series of overlapping oligonucleotides with complementary ends. When annealed, the ends served as primers for the extension of the complementary strand. The fragments were then amplified by excessive outside primers. Because of the large size of 3D gene to be synthesized, the oligonucleotides were divided into three groups and recursive PCRs were performed. The produced DNAs were designated as the A, B and C fragments. The B and C fragments were joined with PCR and then the B-C fragment was joined with the A fragment to produce the full-length 3D gene.

(83) One of the end oligonucleotides was designed to contain a BamHI restriction site for cloning into the expression vector pGEX-4T-1. The anti-sense oligonucleotide contains a translational termination codon (TAA) and an EcoRI restriction site. When N- and C-terminal primers, 3d-1 A (SEQ ID NO: 80) and 3d-36A (SEQ ID NO: 115), were used, a full-length 3D (470 amino acids) gene was synthesized. These steps are detailed herein below.

(84) 1. 3DA Fragment PCR

(85) The PCR reaction (100 μl volume) was set up as follows: Vent DNA polymerase (1U) and 1× buffer, along with 25 μM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 μl 100 mM MgSO.sub.4, 100 pmol each of oligonucleotides 3d-1 A (SEQ ID NO: 80) and 3d-14 (SEQ ID NO: 93). The template was a mixture of 0.83 pmole of each of the oligonucleotides 3d-1 A to 3d-14. The reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 53° C. for 30 seconds and 73° C. for 100 seconds, followed by incubation at 73° C. for 5 minutes. An aliquot of the reaction mixture was analyzed by electrophoresis on agarose mini-gel.

(86) 2. 3DB Fragment PCR

(87) The PCR reaction (100 μl volume) was set up as follows: Vent DNA polymerase (1U) and 1× buffer, along with 25 μM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 μl 100 mM MgSO.sub.4, 100 pmol each of oligonucleotides 3d-13 (SEQ ID NO: 92) and 3d-24 (SEQ ID NO: 103). The template was mixture of 0.83 pmole of each oligonucleotides 3d-13 to 3d-24. The reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 90 seconds, followed by incubation at 72° C. for 5 minutes. An aliquot of the reaction mixture was analyzed by electrophoresis on agarose mini-gel.

(88) 3. 3DC Fragment PCR

(89) The PCR reaction (100 μl volume) was set up as follows: Vent DNA polymerase (1U) and 1× buffer, along with 25 μM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 μl 100 mM MgSO.sub.4, 100 pmol each of oligonucleotides 3d-25 (SEQ ID NO: 104) and 3d-36A (SEQ ID NO: 115). The template was mixture of 0.83 pmole of each oligonucleotides 3d-25 to 3d-36A. The reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 53° C. for 30 seconds and 73° C. for 100 seconds, followed by incubation at 73° C. for 5 minutes. An aliquot of the reaction mixture was analyzed by electrophoresis on agarose mini-gel.

(90) 4. 3DB-C Fragment PCR

(91) The PCR reaction (100 μl volume) was set up as follows: Vent DNA polymerase (1U) and 1× buffer, along with 25 1AM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 μl 100 mM MgSO.sub.4, 100 pmol each of oligonucleotides 3d-13 (SEQ ID NO: 92) and 3d-36A (SEQ ID NO: 115). The template was a mixture of the B and C fragments described above. The reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 55° C. for 30 seconds and 73° C. for 90 seconds, followed by incubation at 73° C. for 5 minutes. An aliquot of the reaction mixture was analyzed by electrophoresis on agarose mini-gel.

(92) 5. Full-Length 3D (ABC) PCR

(93) The PCR reaction (100 μl volume) was set up as follows: Vent DNA polymerase (1U) and 1× buffer, along with 25 μM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 μl 100 mM MgSO.sub.4, 100 pmol each of oligonucleotides 3d-1A (SEQ ID NO: 80) and 3d-36A (SEQ ID NO: 115). The template was a mixture of A, B and C fragments. The reaction was incubated at 95° C. for 5 minutes, and then amplified with 35 cycles of 95° C. for 30 seconds, 60° C. for 30 seconds and 73° C. for 120 seconds, followed by incubation at 73° C. for 5 minutes. The PCR-derived product was run on the agarose gel and the DNA band was cut from the gel and then eluted using Quigen gel extraction kit.

(94) (ii) Cloning of the PCR Product.

(95) The PCR product amplified as described herein was digested with the restriction endonucleases Bam HI and Eco RI and ligated into the gel purified vector pGEX-4T-1 that had been digested with Bam HI and Eco RI. The ligation product was used to transform XL-1 Blue competent cells. The transformed cells were plated on LB plates supplemented with 100 μg/ml ampicillin. Mini-prep DNAs were prepared from overnight cultures of colonies and digested with Bam HI and Eco RI to screen the desired clones. The clone with right insert was designated as pBM-3DTw97F (FIG. 12).

(96) B. Growth And Induction of E. coli Strains with pBM-3DTw97F

(97) To express recombinant GST-3D protein, the pBM-3DTw97F plasmid was transformed into E. coli BL21(DE3) and transformants were plated on LB-agar plate supplemented with 100 μg/ml ampicillin. Overnight seed cultures of the pBM-3DTw97F clone were prepared in 500 ml sterile LB supplemented with 100 μg/ml ampicillin, and placed in a shaking orbital incubator at 37° C. Fifty milliliter inoculums from seed cultures were transferred to flasks containing 0.5 liter sterile LB supplemented with 100 μg/ml ampicillin. Cultures were incubated at 37° C. until the cultures reached mid-logarithmic growth and then induced with 1 mM IPTG for 3 hours at 37° C. After the induction period, cells were pelleted by centrifugation and harvested following standard procedures. Pelleted cells were stored at −70° C. until further processed.

(98) C. Preparation of GST-3D Protein

(99) Frozen cells obtained above were resuspended in PBS with 1 mM PMSF. The cells were lysed by sonication (Branson, model S-125). A soluble crude lysate was prepared by centrifugation of the cell-lysate (10,000 rpm, 30 min) and filtered with 0.45 μm syringe filter (Sartorius). Glutathione affinity chromatography was carried out to purify rGST-3D protein. The soluble cell lysate was loaded onto glutathione sepharose 4B (Pharmacia) column equilibrated with PBS. After washing the column with three bed volumes of PBS, GST-3D was eluted with 10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0 buffer solution. The eluted fractions were analyzed on the 8% SDS-PAGE. The fractions that contained the fusion protein were dialyzed in PBS overnight.

Example 5

FMDV Antibody Detection Kit Formulation

(100) A. Preparation of Antigen Printed Membrane

(101) From the stock solution, recombinant 2C and 3ABC were mixed to 0.5 mg/ml each with 20 mM phosphate buffer (pH 9.0) and filtered through 0.22 μm filter unit Millex-GV (Millipore). An avidin solution (0.5 mg/ml) in PBS (pH 7.4) was used as an internal control. The antigen mixture and control solution were applied to the nitrocellulose membrane (S&S, 8 μm in pore size or equivalent) using Bio-Dot equipment (Bio-Dot) and following the manufacturers protocol. After the sample was dried in a low humidity room overnight, the membrane was blocked with 3% BSA in PBS for 20 min and then dried on a fan at least for 2 hours. The processed membrane plates are stored in an enclosed container with desiccant or low humidity room.

(102) B. Preparation of Protein G and/or Protein A-Gold Conjugate

(103) Recombinant protein G and/or protein A that had been engineered to eliminate any non-specific binding to serum albumin was purchased from Sigma and reconstituted with 10 mM sodium carbonate buffer (pH 9.6) to a concentration of 1 mg/ml. A gold particle suspension was adjusted to pH 9.0 with 50 mM potassium carbonate (pH 9.6) and the protein G and/or protein A was then added dropwise to the gold solution while stirring. The protein G and/or protein A was added so that a final concentration of 10 μg/ml was obtained. The solution was further stirred for 15 min. Next, 30 μl of 15% BSA solution was added per ml of gold particle suspension. After stirring for another 15 min, coupled gold solution was centrifuged and the supernatant was discarded, thereby removing any unbound protein G and/or protein A. To the pellet of 200 ml of coupled gold solution, 12 ml of 2% BSA (in deionized water) was added. The pellet solution was then sonicated in a sonic bath (Branson model #2200 or equivalent) to resuspend the pellet. The suspension was centrifuged again and the final pellet was suspended in the same volume of 2% BSA (10 mM Sodium carbonate, pH 9.6) and stored in refrigerator.

(104) C. Preparation of Biotin-BSA-Gold Conjugate: Control Indicator

(105) Biotinylated BSA (Pierce) was purchased and was coupled to the gold. The conjugation procedures were basically the same as described as for protein G and/or protein A above. Ten micrograms of biotinylated BSA per ml of gold particle suspension was added to the gold solution, which had been adjusted to pH 4.4 by adding 40 mM phosphoric acid with vigorous stirring. After about 1 min, 16.6 μl of 40 mM potassium carbonate per ml of coupled gold solution was added and allowed to stir for 10-15 min. At the end of the coupling reaction, 30 μl of 15% BSA solution was added per ml of gold particle suspension. After stirring for another 15 min, the Biotin-BSA coupled gold conjugate suspension was centrifuged and the supernatant was discarded to remove any unbound Biotin-BSA. The pellet from 200 ml of coupled gold solution was washed with 12 ml of 2% BSA (10 mM Sodium phosphate, pH 7.5). The resultant pellet was then resuspended in the same volume of 2% BSA (10 mM Sodium phosphate, pH 7.5) and stored in refrigerator.

(106) D. Preparation of the Dye-Pad

(107) Protein G and/or protein A coupled gold solution was diluted using dye dilution buffer (1% casein, 100 mM sodium phosphate, pH 7.0). Biotin-BSA coupled gold solution was added to generate the control line, which binds to avidin on the membrane (see FIG. 3). A lysate of the same E. coli strain used for production of recombinant FMDV antigens, but without a recombinant plasmid, was added to eliminate any anti-E. coli antibodies that might be present in the sample. The diluted gold solution was spread onto the Lydall pad strip (microglass paper) and dried in a lyophilizer. The Lydall pad was pre-soaked in pretreatment buffer (1% NP-40, 20 mM EDTA, 0.25% L-7600, 1% PVP 10, 10 mM sodium phosphate and 0.1% sodium azide, pH 7.0), excess liquid was blotted off, and the pad was dried on a fan. The pad is stored in a low humidity room until use.

(108) E. Filter Pad Preparation

(109) The cellulose filter paper was pre-soaked in pretreatment buffer (0.5% NP-40, 2% β-lactose, 1% PEG 15K, 100 mM sodium phosphate, and 0.1% sodium azide, pH 7.0) excess liquid was blotted off, and the paper was dried on a fan. The prepared filter pad was stored in a low humidity room.

(110) F. Device Assembly

(111) A protective sheet at the top of the plate was peeled off and the absorbent pad was attached along the long axis of the plate. A protective sheet at the bottom of the plate was peeled off and the dye pad was attached beneath the test membrane area along the long axis of the plate. The dye pad should overlap the bottom of the test membrane about 2-3 mm. Next, the filter pad was attached to the plate to cover the bottom of the dye pad. Finally, the dressed membrane plate was cut into 0.765 cm wide strips (FIG. 2).

Example 6

Kit Assay

(112) A schematic diagram of the test kit is shown in FIG. 13. A test sample containing antibodies to FMDV or infected with FMDV (membrane shown on the left) will display a positive signal when contacted with FMDV antigen or FMDV antibodies, respectively. This is indicated by the color reaction at the band containing the immobilized capture reagent (FMDV antigen or FMDV antibodies; T), whereas a sample that does not contain FMDV (membrane shown on the right) will not display any color at the test band (T). A positive control is incorporated into the test kit by applying an albumin-biotin gold conjugate to the filter pad containing the labeled reagent. The albumin-biotin gold conjugate will bind to the avidin in the control band, thus the control band will be positive in both the test strips.

(113) FIG. 14 is a schematic diagram of a two-line test kit configuration. An animal infected with FMDV, as shown in the figure labeled “infected”, will reveal a positive signal on both the T1 (which contains SPs VP1 or 3D) and T2 (which contains NSPs 2C or 3ABC) test bands. The vaccinated sample will only reveal a positive signal at the T1 test band. No antibodies to NSPs and no NSP antigens will be present in the vaccinated sample. Thus the present invention is able to differentiate, within a very short time period, between the infected animal and one that is immune to infection (i.e., has been vaccinated).

Results

(114) Analysis of Whole Blood and Serum Samples in FMDV-Infected Animals

(115) Both whole blood and serum samples from FMDV-infected sheep (3) and goats (3) were analyzed for the presence of antibodies to the non-structural proteins 2C and 3ABC (Tables 1 and 2).

(116) TABLE-US-00001 TABLE 1 Results Of Pen-Side Test Using Whole Blood And Serum Samples From FMDV-Infected Sheep Ovine #716 Ovine #717 Ovine #718 Whole Whole Whole DPI Blood serum blood serum blood serum 0 − − − − − − 2 − − − − − − 4 − − − − − − 6 − − − − − − 8 + ± − − − − 10 + + − − + + 12 + + ± ± + + 14 ++ ++ ++ ++ ++ ++ Legend: +++ Prominently Visible Line ++ Clearly Visible Line + Detectable Line ± Unclear Line − No Line

(117) TABLE-US-00002 TABLE 2 Results Of Pen-Side Test Using Whole Blood And Serum Samples From FMDV-Infected Goats Caprine #804 Caprine #808 Caprine #809 Whole Whole Whole DPI Blood serum blood serum blood serum 0 − − − − − − 2 − − − − − − 4 − − − − − − 6 − − − − − − 8 + + − − − − 10 + + + + ± + 12 + + ++ ++ ++ ++ 14 ++ ++ ++ ++ ++ ++ Legend: +++ Prominently Visible Line ++ Clearly Visible Line + Detectable Line ± Unclear Line − No Line

(118) A positive signal was first detected at 8 days post infection (DPI) in ovine #716. There was no difference in the results from a whole blood sample or a serum sample.

(119) Antibodies in ovine #717 were also detected, although a positive signal was not detected until 12 DPI. The antibodies in the whole blood and serum samples were detected at the same time after infection.

(120) Antibodies in ovine #718 were detected at 10 DPI. As in ovine #716 and #717, the antibodies in the whole blood and serum samples were detected on the same DPI after infection.

(121) The time frame in which the antibodies to the non-structural proteins in FMDV were detected in the goats (Table 2) was similar to those described for the sheep (Table 1). The anti-non-structural protein antibodies in the three goats were detected in both the whole blood and serum between day 8 and day 10 post infection.

Performance Characteristics of The Assay

(122) A total of 1540 identified clinical samples from cattle, swine, goat and sheep sera, provided by PIADC, were tested at the Plum Island Animal Disease Center, USDA (Greenport, N.Y.) using the assay of the invention and commercially available ELISA.

(123) The samples were negative samples prior to vaccination, vaccinated samples that were not infected, and infected samples. The results, shown in Table 3 below, illustrate the excellent agreement between the assay of the invention and the reference ELISA.

(124) The assay of the invention demonstrated a relative sensitivity of 98.6% (69/70) and relative specificity of 98.6% (1449/1470) when compared with the reference test. The overall accuracy was 98.6% (1518/1540). (see also Table 4)

(125) TABLE-US-00003 TABLE 3 Rapid Immunoassay vs. ELISA Test Results Rapid Immunoassay ELISA (AHIS Plum Island) Positive Negative Total Positive Negative Total Infected (+) 52 0 52 52 0 52 Naive (−) 8 1003 1011 0 1011 1011 Vaccinated (−) 1 109 110 0 110 110 single 6 64 70 0 70 70 Total 67 1176 1243 52 1191 1243

Sensitivity And Specificity of The Test Device

(126) The sensitivity and specificity of the assay device disclosed herein was determined on the basis of samples that had been previously tested using the standard test method (ELISA). Antibodies against the 3ABC protein (one of the non-structural proteins) were measured 10 DPI using the method disclosed herein. The results shown in Table 4 reveal that the assay method of the present invention provides a sensitive, accurate and specific assay system that distinguishes, simultaneously, between an infected animal and one that is protected from infection (i.e., vaccinated) in a single step within 30 minutes.

(127) TABLE-US-00004 TABLE 4 The Sensitivity And Specificity Of The Pen-Side Test For Bovine, Swine, Ovine, And Caprine Relative (%) Bovine Swine Ovine Caprine Sensitivity 95.6 100   100 100   (22/23) (12/12) (13/13) (8/8) Specificity 98.5 99.3 100 96.8  (316/320)) (796/801) (31/31) (30/31) Accuracy 98.8 99.3 100 97.4 (338/343) (808/813) (43/43) (38/39)

Efficacy of the Assay Device

(128) FIGS. 15-18 compare the efficacy of the standard ELISA test to that of the assay presented herein. Antibodies to 3ABC were detected 6-7 DPI in bovine #19 and 21 (FIGS. 15-16), whereas the standard ELISA remained negative until 9 DPI. FIGS. 17-18 show the efficacy of the assay in pigs. Antibodies were detected in swine #183 12 DPI, while seroconversion in swine #186 was detected 10 DPI. While efficacy of the ELISA method was similar, the results of this method are not available for a few days. The results of the assay of the invention are available within 30 minutes, a significant advantage since FMDV is highly contagious and will spread rapidly through a herd.

(129) The foregoing descriptions of specific embodiments of the present invention have been presented for the purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application, to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.