WO2000019202A1 - DOSAGE DIAGNOSTIQUE A POLARISATION DE FLUORESCENCE DESTINE A DES SEROVARS DE $i(LEPTOSPIRA) - Google Patents

DOSAGE DIAGNOSTIQUE A POLARISATION DE FLUORESCENCE DESTINE A DES SEROVARS DE $i(LEPTOSPIRA) Download PDF

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WO2000019202A1
WO2000019202A1 PCT/US1999/022210 US9922210W WO0019202A1 WO 2000019202 A1 WO2000019202 A1 WO 2000019202A1 US 9922210 W US9922210 W US 9922210W WO 0019202 A1 WO0019202 A1 WO 0019202A1
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sequence
protein
amino acids
flab
seq
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PCT/US1999/022210
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English (en)
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Nasreen I. Bughio
Min Lin
Om P. Surujballi
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Diachemix Corporation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6445—Measuring fluorescence polarisation
    • 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
    • G01N33/56905—Protozoa

Definitions

  • This invention relates to a homogeneous immunoassay for detection of antibodies to a range of leptospira serovars.
  • a fluorescent antigen probe is provided as a diagnostic reagent that, when combined with the target antibodies, shows an increase in fluorescence polarization, thereby quantitating the antibody present.
  • Leptospirosis is a zoonotic infection, which exhibits a broad spectrum of clinical manifestations, ranging in severity from acute to chronic with multi-organ syndrome, to fatal. Leptospirosis affects wild rodents and domestic animals such as cattle, swine, horses, sheep, goats and dogs. Infections with leptospires also represent an occupational hazard to farmers, butchers and veterinarians. The pathogenic leptospires were formerly classified as Leptospira interrogans. but the genus has recently been reorganized and pathogenic leptospires are now identified in seven species of Leptospira.
  • MAT microscopic agglutination test
  • Fluorescence polarization is a measure of the time-averaged rotational motion of fluorescent molecules.
  • FPA fluorescence polarization assay
  • Leptospires have a characteristic corkscrew-like movement, which is mediated by two periplasmic flagella (PF) inserted subterminally into the protoplasmic cylinder.
  • PF periplasmic flagella
  • Sequence analysis of flagellar genes and proteins from several spirochetes show that there are two distinct classes of proteins, FlaA and FlaB in the filament. FlaA proteins are associated with the sheath surrounding a core that is composed of FlaB proteins.
  • Electrophoresis has revealed that the PF is composed of three prominent proteins with molecular weights of 31, 37 and a 33 - 34 kDa doublet (See Kelson, J. Med. Microbiol, 26:47 (1987)). Additionally, several proteins with molecular weights between 30-67 kDa have been identified from L. interrogans serovar hardjo (See Nunes-Edwards, et al., Infect. Immun., 48:492-497 (1985)). However, the significance of these proteins in diagnosis, pathogenesis or immunity to infection is not yet clear.
  • the invention provides a fluorescent antigen probe comprising a fluorophore conjugated to a purified and isolated protein comprising a sequence of amino acids that is at least 98.6%> homologous with the sequence of amino acids set forth in SEQ ID NO:2.
  • the fluorescent antigen probe binds to serum antibodies to Leptospira serovars to produce a detectable change in fluorescence polarization.
  • the invention provides an assay for serum antibodies reactive with an antigen common to a range of Leptospira serovars.
  • a serum specimen suspected of containing antibodies reactive with an antigen of Leptospira is diluted with a buffer solution, to provide a buffered specimen.
  • a fluorescent antigen probe is added to this buffered specimen.
  • the fluorescent antigen probe comprises a fluorophore conjugated to a purified and isolated protein comprising a sequence of amino acids that is at least 98.6%o homologous with the sequence of amino acids set forth in SEQ ID NO:2.
  • the buffered specimen with added probe is incubated for a time sufficient to permit binding in solution of the antibodies to the antigen probe to provide a reaction product.
  • the invention provides a diagnostic assay kit for detecting serum antibodies to a range of Leptospira serovars.
  • the kit includes a fluorescent antigen probe in an amount suitable for at least one assay and suitable packaging.
  • the fluorescent antigen probe comprises a fluorophore conjugated to a purified and isolated protein comprising a sequence of amino acids that is at least 98.6%o homologous with the sequence of amino acids set forth in SEQ ID NO:2.
  • the fluorescence polarization-based diagnostic assay utilizing a fluorescent antigen probe, is rapid, easy to use, and has a high sensitivity to and specificity for a range of Leptospira serovars.
  • Figure 1 is a photograph showing the results of SDS-PAGE applied to samples from five steps in the purification process of the FlaB protein used to make the fluorescent antigen probe in accordance with a preferred embodiment of the present invention.
  • Figure 2A is a photograph of the gel under UV illumination showing the results of SDS-PAGE analysis of labeled and unlabeled recombinant FlaB protein.
  • Figure 2B is a photograph of the gel of Figure 2B after Coomassie blue staining.
  • Figure 3A is a photograph showing the results of a Western blot analysis of both labeled FlaB and unlabeled FlaB probed with bovine sera containing antibodies against serovar pomona.
  • Figure 3B is a photograph showing the results of a Western blot analysis of both labeled FlaB and unlabeled FlaB probed with bovine sera containing antibodies against serovar hard]o.
  • Figure 3C is a photograph showing the results of a Western blot analysis of both labeled FlaB and unlabeled FlaB probed with bovine sera containing antibodies against serovar sejroe.
  • Figure 4 is a graph showing the receiver operating characteristic (ROC) curve for the fluorescence polarization assay, in accordance with a preferred embodiment of the present invention, applied to 208 MAT-positive and 208 MAT-negative bovine serum samples.
  • ROC receiver operating characteristic
  • Figure 5 is a bar graph showing the results of the fluorescence polarization assay, in accordance with a preferred embodiment of the present invention, as applied to 208 MAT-positive and 208 MAT-negative bovine serum samples.
  • the 35-kDa protein was highly purified from an outer sheath antigen preparation (see Auran et al, Infect. Immun. 5:968 (1972)) through a high-performance liquid chromatography size exclusion column (TSK G2000 SWG; 21.5 by 600 mm; Phenomenex, Torrance, Calif.) with 50 n M ammonium acetate as the elution buffer.
  • the purified serovar pomona protein antigens were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto a polyvinylidene difluoride membrane, and excised for the determination of the N-terminal amino acid sequence.
  • Amino acid sequencing was performed on a model 470A gas phase sequencer equipped with an on-line model 120A PTH analyzer (Applied Biosystems, Foster City, Calif).
  • the N-terminal amino acid sequence found for this protein is provided herein as SEQ ID NO: 1.
  • N-terminal amino acid sequence of the 35-kDa protein was performed against the sequence databases through the National Center for Biotechnology Information (NCBI) BLAST E-mail server.
  • NCBI National Center for Biotechnology Information
  • a striking homology (61.5 to 92.2%) identity) between the N-terminal amino acid sequence (13 amino acids) of the serovar pomona protein and those of the flagellins from other bacteria was found.
  • This considerably high level of homology suggested that the 35-kDa protein is a constituent polypeptide of PF.
  • the PF from several different spirochetes has been found to be complex structures consisting of several different polypeptide subunits.
  • the PF filaments consist of a core structure surrounded either by one outer sheath layer as in Spirochaeta aurantia and Treponema pallidum or by two outer layers as in L. interrogans. Sequence analyses of the PF genes and proteins from several spirochetal sources have shown that there are two distinct classes of proteins, namely FlaA and FlaB, which form the outer sheath and core layers of the PF filament, respectively.
  • FlaA and FlaB which form the outer sheath and core layers of the PF filament, respectively.
  • the 35-kDa protein described here is a FlaB protein based upon its N-terminal amino acid sequence, and its gene is therefore referred to as flaB.
  • PCR primers which contain the Sacl and Bam ⁇ l cognition sequences at their 5' ends, were derived, respectively, from the N-terminal amino acid sequence of the serovar pomona 35-kDa protein and the conserved C-terminal amino acid sequence of a leptospiral periplasmic flagellar subunit (see Mitchison, et al, J. Gen. Microbiol, 137:1529 (1991)). PCR was performed with Taq DNA polymerase (Life Technologies, Burlington, Ontario, Canada).
  • the highly conserved FlaB in leptospires may explain the observation that Ml 38 cross-reacted with various pathogenic serovars, and the cross-reactivity of periplasmic flagellar proteins among various strains of Leptospira has been previously described.
  • the S ⁇ cl and if ⁇ /wHI-digested 850-bp amplified gene fragment from the serovar pomona genomic DNA was ligated into pUC 118 at Sacl and BamHl sites and cloned in Escherichia coli TGI .
  • Recombinant plasmids pUC118pomFla-l and pUC118pomFla-2 prepared from the cultures of two selected white colonies, were restriction analyzed with Sacl and Bam ⁇ I.
  • Both recombinant plasmids contain an insert with an apparent molecular size corresponding to that of the 850-bp amplified gene.
  • DNA sequencing was performed on an ABI model 373 automatic sequencer with an ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer, Foster City, Calif.) according to the manufacturer's instructions.
  • the recombinant plasmid DNA was used as sequencing templates. Both strands of the DNA insert were initially sequenced with pUC/M13 forward and reverse primers, 5'GTAAAACGACGGCCAGT3' and 5'CAGGAAACAGCTATGAC3 ⁇ respectively, and completed with an additional pair of internal primers, 5'TTATAATAAGCTCCCATATC3' and
  • 5'TACTGAAGACGGAATGAGTT3' synthesized according to the initial nucleotide sequence obtained with the pUC/M 13 primers .
  • the inserted DNA fragment contains an open reading frame of 849 bp with a G + C content of 46.88%> which codes for a 283-amino-acid (aa) protein.
  • the encoded protein contains 31 (10.95%) acidic (D and E), 34 (12.0%) basic (K and R) , and 102 (36.0%) hydrophobic (A, I, L, F, W, and V) amino acids.
  • the deduced protein sequence contains no cysteine residue and only one tryptophan residue.
  • the calculated molecular mass and predicted pi value of the encoded protein were 31.297 kDa and 9.065, respectively.
  • the G + C content of the serovar pomona flaB gene is significantly lower than the 54.7%> found for serovar hardjo FlaB but higher than the 39% G + C content of the leptospiral genome reported in LeFebvre, et al., J. Clin. Microbiol. 25:2094-2097 (1987).
  • the deduced protein sequence is provided herein as SEQ ID NO:2.
  • the nucleotide sequence for the flaB gene is provided herein as SEQ ID NO:3.
  • the calculated molecular mass of the serovar pomona FlaB is remarkably similar to that reported for the serovar hardjo FlaB (see Mitchison, et al., J. Gen. Microbiol, 5 137:1529-1530 (1991)), but it is smaller than the size of the native protein (35 kDa) as determined by SDS-PAGE. At least two possibilities may account for this difference.
  • the serovar pomona flagellin shows a close relationship with the serovar hardjo protein, with a homology (identity) of 98.6%. In comparison with other bacterial species, the serovar pomona protein has an overall 42.0% identity with the Bacillus sp.
  • strain C-125 flagellin 20 39.6%o identity with the flagellins from two Borrelia species (B. hermsii and B. burgdorfe ⁇ ), and 55.5% to 57.2% identity with the T. pallidum flagellins (FlaBl, FlaB2, and FlaB3).
  • PCR amplification of pUCl 18pomFla-l gave a product of 870 bp, comprising the flaB structural gene and a restriction endonuclease recognition sequence at each end to allow cloning into the vector, pProEx HT.
  • the pProEx HT expression vector was obtained from Life Technologies (Burlington, Ontario, Canada). This plasmid contains
  • IPTG IPTG
  • trc promoter a gene from the trc promoter.
  • an expression vector designated pHTFlaB
  • the pHTFlaB construct enables production of a FlaB fusion protein with a polyhistidine tag.
  • the polyhistidine tag facilitates affinity purification of the desired protein and may be removed by proteolysis with TEV protease.
  • the recombinant FlaB protein was purified by affinity chromatography on a nickel-nitriloacetic acid (Ni-NTA)-agarose column followed by electroelution. Samples 5 from each step in the purification process of recombinant FlaB protein were subjected to SDS-PAGE, and the results are shown in Figure 1. Lane 1 shows the results for soluble lysate of uninduced E. coli; lane 2 the results for soluble lysate of induced E. coli; lane 3 the results for the crude extract of induced cells prior to Ni-NTA affinity chromatography; lane 4 the results for the proteins eluted from the Ni-NTA column with
  • Lanes 1 and 2 contain total cell proteins from 1 ml of culture with an A 59 o of 0.2.
  • the SDS-PAGE was performed by the method of Laemmli in 12% (wt/vol)
  • the NC was reacted with diluted bovine antiserum (1 :100) for 3 hours at room temperature and then with anti-bovine immunoglobulin (IgG) conjugated with a horseradish peroxidase (1 :5000).
  • the NC was washed (five times) between each reaction step with TBS-T. Bound conjugate was visualized with a horseradish peroxidase substrate kit (Bio-Rad). The molecular weights of the separated proteins were determined by comparison with prestained protein standards (Bio-Rad).
  • the first step in the purification process is obtaining the lysate from E. coli. Briefly, IPTG- induced E. coli cells harboring pHTFlaB were resuspended in 10 ml of buffer A (8 M urea, 0.1 M Na 2 HPO 4 , 0.01 M Tris-HCl, pH 8.0), lysed for one hour at
  • the recombinant protein FlaB was partially purified by Ni-NTA affinity chromatography (See Hochuli, ⁇ ., p. 87-98, in Setlow, J.K. (ed.), Genetic engineering, principle and method, Plenum, New York (1990); and Holzinger, et al., News Qiagen, 4:14-15 (1996)) according to the manufacturer's instructions (Qiagen).
  • the supernatant was applied to an Ni-NTA column (1 x 7.5 cm) pre-equilibrated with buffer A.
  • coli proteins were eluted by sequential washing with progressively acidic buffers, namely, 50 ml each of buffer B (8 M urea, 0.1 M Na 2 HPO 4 , 0.01 M Tris-HCl, 1 M NaCl, pH 6.3), buffer C (same composition as buffer B except pH 5 9) Selective elution of FlaB protein was achieved with buffer D (same composition as buffer C except containing 80 mM imidazole) The fractions were collected and the absorbance was measured at 280 nm m a spectrophotometer (Pharmacia LKB Ultrospec plus) All eluted fractions were subjected to SDS-PAGE to confirm the presence of FlaB protein The peak fractions containing the FlaB protein were pooled and stored at -20°C
  • the FlaB was further purified by electroelution Following Ni-NTA affinity chromatography, the partially pu ⁇ fied FlaB preparation was subjected to SDS-PAG ⁇ as described above Individual protein bands were visualized by staining with Tns- Coomassie buffer (0 125 M Tris-HCl, pH 6 8) containing 0 2% Coomassie brilliant blue, and destammg with 0 125 M Tris-HCl (pH 6 8) containing 0 1% SDS and 1 mM ⁇ DTA The FlaB protein band was excised and electroeluted with a Bio-Rad ⁇ lectroelutor model 422 at a constant current of 10 mA/tube for 4 to 6 hours at room temperature using elution buffer (25 mM T ⁇ s-HCl, 192 mM glycine, 0 1% SDS, pH 8 5) The pu ⁇ fied FlaB was concentrated m an Ultrafiltration Stirred Cell (Amicon) fitted with a PM-10 (25
  • the reaction mixture was applied to a Sephadex G-25 column (1 x 23 cm)(Pharmacia), pre-equilibrated with 0.1 M sodium phosphate buffer (pH 7.0). The absorbance of the fractions was monitored at 492 nm (Pharmacia LKB Ultrospec plus). Eluted fractions were pooled and analyzed by SDS-PAGE. The protein bands were visualized by UV light (Transilluminator, VWR Scientific) followed by staining with Coomassie brilliant blue.
  • FIG. 2 A is a photograph of the gel taken under UV illumination, and Figure 2B shows the same gel after Coomassie blue staining.
  • lane 1 shows the results for the reaction mixture comprising FlaB and FITC;
  • lane 2 shows the results for unlabeled FlaB;
  • lane 3 shows the results after gel filtration chromatography.
  • An arrowhead indicates the recombinant FlaB protein in Figures 2 A and 2B.
  • the antigenicity of labeled and unlabeled FlaB was confirmed by Western blotting using Bovine sera containing antibodies against L. interrogans serovar pomona, and L. borgpetersenii serovars hardjo and sejroe. Sera from uninfected animals were used as negative controls. Western blot analysis revealed that both labeled and unlabeled FlaB were recognized by anti-Leptospira antibodies against various serovars, thus indicating the broad antigenic cross-reactivity of this recombinant protein and indicating that the labeling did not effect the antigenicity.
  • Figures 3A, 3B, and 3C show the Western blot results, wherein unlabeled FlaB (lane 1) and labeled FlaB (lane 2) were electrophoresed on 12% SDS-polyacrylamide gel, transblotted onto NC and probed with bovine sera containing containing antibodies against serovar pomona (Figure 3 A), serovar hardjo (Figure 3B), and serovar sejroe (Figure 3C).
  • Figures 3A-3C the recombinant protein FlaB is indicated by an arrowhead.
  • the FPA was performed using FITC-labeled FlaB as a tracer antigen.
  • the FPA was carried out on a FPM-1 Fluorescence Polarization Analyzer (Jolley Consulting and
  • the baseline for the tracer antigen alone was established with 0.1 M PBS (pH 7.2). Serum samples were diluted (1:50) in 0.01 M PBS containing 0.1% sodium azide and 0.05% lithium dodecyl sulfate. Two milliliters of diluent were dispensed into a glass tube (12 x 75 cm). Test serum (0.04 ml) was then added to the diluent tube and mixed thoroughly. Each sample was blanked in the fluorescence polarization analyzer. The predetermined amount of the tracer antigen (0.02 ml) was then added to the serum sample and mixed thoroughly. The mixture was allowed to equilibrate for 10 to 30 minutes at room temperature. Following the equilibration period, the fluorescence polarization was measured as millipolarization units (mP) in a fluorescence polarization analyzer.
  • mP millipolarization units
  • mP fluorescence millipolarization
  • five MAT-positive and four MAT-negative bovine serum samples (1 :50) were analyzed by FPA.
  • MAT procedure used herein was a modification of the method previous described in Cole, et al, "Improved microtechnique for the leptospiral microscopic agglutination test," Appl. Microbiol, 25:976-980 (1973).
  • Live 4-day-old Leptospira cultures (Leptospira serovars sejroe, hardjo, pomona, canicola, icterohaemorrhagiae, and grippotyphosa) were used as antigens.
  • the endpoint titer was the highest dilution of serum that agglutinated at least 50%) of the leptospires. Agglutination at a serum dilution of 1 TOO was considered positive.
  • ROC receiver operating characteristic
  • the ROC curve plots the relative sensitivity (true-positive ratio) versus relative specificity (false-positive ratio) while the cut-off value for a positive or negative result is varied, and is completely independent of disease prevalence.
  • the area under the ROC curve provides a quantitative assessment of a test's diagnostic performance, in this case, the ability of FPA to discriminate the sera with or without antibodies. The larger the area under the ROC curve the better the test discriminates.
  • a perfect test has an area of 1.0, whereas a non-informative test has an area of 0.5 or less.
  • the 95%> confidence intervals are calculated as indicators of the precision of the relative sensitivity and relative specificity estimates.
  • the fluorescent antigen probe of the present invention is preferably made available in kit form.
  • the kit preferably includes a quantity of buffer solution for diluting serum specimens suspected of containing antibodies to Leptospira, the fluorescent antigen probe in amount suitable for at least one assay, along with suitable packaging and instructions for use.
  • the fluorescent antigen probe may be provided in solution, as a liquid dispersion, or as a substantially dry powder (e.g., in lyophilized form).
  • the suitable packaging can be any solid matrix or material, such as glass, plastic, paper, foil, and the like, capable of separately holding within fixed limits the buffer and the synthetic fluorescent antigen probe.
  • the buffer solution and the fluorescent antigen probe may be provided in separate labeled bottles or vials made of glass or plastic.
  • the fluorescent antigen probe comprises a fluorophore conjugated to a purified and isolated protein that preferably corresponds to the FlaB flagellar protein of Leptospira interrogans serovar pomona described herein.
  • This protein may be prepared recombinantly, using the nucleotide sequence set forth in SEQ ID NO:3, and purified using Ni-NTA affinity chromatography, followed by electroelution, as described herein.
  • the deduced amino acid sequence for this flagellar FlaB protein is set forth in SEQ ID NO:2.
  • a fluorophore is conjugated, i.e., covalently attached, to this FlaB flagellar protein in the fluorescent antigen probe.
  • the fluorophore is preferably fluorescein isothiocyanate.
  • other fluorophores such as rhodamine, BODIPYTM, Texas RedTM and Lucifer yellow, could also be used.
  • the fluorescent antigen probe is preferably prepared by mixing about 0.3 ml of FITC (1 mg/ml) in a 0.15 M sodium
  • the kit preferably provides the fluorescent antigen probe prepared in this way in an amount suitable for at least one assay, in suitable packaging.
  • the buffer solution provided in the kit is preferably 0.01 M PBS that also contains 0.1% sodium azide and 0.05% lithium dodecyl sulfate.
  • other buffers could also be used.
  • the diagnostic assay kit is intended to be used in the following way, as should be described in the instructions for use.
  • a serum specimen suspected of containing antibodies to Leptospira is diluted with a quantity of the buffer solution provided in the kit to provide a buffered specimen.
  • a dilution of about 1 :50 in a total volume of about 2.0 ml is preferred.
  • the buffered specimen is then blanked in the reader, after which about 0.02 ml of the fluorescent antigen probe is added, the fluorescent antigen probe having been prepared as described above.
  • the buffered specimen with added probe is then incubated for a time sufficient to permit binding in solution of Leptospira antibodies with the antigen probe to provide a reaction product.
  • the fluorescence polarization of the reaction product is then compared to a negative serum control, i.e., compared to a buffered solution of serum obtained from an animal known to be free of Leptospira, to which the fluorescent antigen probe is added at about the same concentration.

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Abstract

L'invention concerne un dosage immunologique homogène de leptospirose dans lequel des anticorps propres à des antigènes présents dans la protéine FlaB de sérovars de Leptospira se fixent à un réactif de diagnostic comprenant une protéine FlaB recombinante marquée avec de l'isothiocyanate de fluorescéine, provoquant une augmentation détectable de la polarisation de fluorescence. Ce dosage est facile, rapide à réaliser et sûr.
PCT/US1999/022210 1998-09-25 1999-09-24 DOSAGE DIAGNOSTIQUE A POLARISATION DE FLUORESCENCE DESTINE A DES SEROVARS DE $i(LEPTOSPIRA) WO2000019202A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6482601B1 (en) 2000-09-11 2002-11-19 Diachemix Llc Fluorescence polarization-based homogeneous assay for fumonisin determination in grains
US6812036B2 (en) 2000-09-11 2004-11-02 Diachemix Llc Fluorescence polarization-based homogeneous assay for deoxynivalenol determination in grains

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE HCAPLUS, WOODWARD ET AL.: "Deoxynucleotide sequence conservation of the endoflagellin subunit protein gene, F1aB within the genus Leptospira" *
LIN ET AL.: "Identification of a 35-kilodalton serovar-cross-reactive flagellar protein, F1aB, from leptospira interrogans by N-terminal sequencing, gene cloning and sequence analysis", INFECTION AND IMMUNITY,, vol. 65, no. 10, October 1997 (1997-10-01), pages 4355 - 4359, XP002923643 *
VET. MICROBIOLOGY,, vol. 40, no. 3-4, 1994, pages 239 - 251 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6482601B1 (en) 2000-09-11 2002-11-19 Diachemix Llc Fluorescence polarization-based homogeneous assay for fumonisin determination in grains
US6812036B2 (en) 2000-09-11 2004-11-02 Diachemix Llc Fluorescence polarization-based homogeneous assay for deoxynivalenol determination in grains

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