Abstract:
The present invention provides an assay for detection of  M. bovis -infected animals. A tracer, comprising a peptide of  M. bovis  protein MPB70 conjugated to a fluorophore, is added to a serum sample from an animal to form a mixture. The fluorescence polarization of the mixture in then measured and compared to the fluorescence polarization of a control. The present invention further provides a tracer for use in fluorescence polarization assay to detect antibodies specific for  M. bovis.  The tracer comprises a peptide of  M. bovis  protein MPB70 conjugated to a fluorophore, such that the tracer is able to bind to antibodies specific for  M. bovis  to produce a detectable change in fluorescence polarization.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application claims priority from U.S. Patent App. No. 60/335,368, filed 31 Oct. 2001. All patents, patent applications, as well as all other scientific and/or technical writings referred to herein are incorporated by reference to the extent that they are not contradictory. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    1. Field of the Invention  
           [0003]    This invention relates to the field of diagnostic assays. More particularly, this invention relates to a peptide-based assay that uses changes in fluorescence polarization to detect serum antibodies to  Mycobacterium bovis.    
           [0004]    2. Description of Related Art  
           [0005]    Fluorescence polarization is a well-known technique that has been used for a number of applications, including animal disease diagnostics, detection of food-borne pathogens, and grain mycotoxin determination. See M. S. Nasir, M. E. Jolley (1999) “Fluorescence Polarization: An analytical tool for Immunoassay and Drug Discovery.”  Combinatorial Chemistry  &amp;  High Throughput Screening,  vol. 2, pp. 177-190. The overall strengths of fluorescence polarization tests lie in the simplicity, ease, rapidity, and cost-effectiveness of the tests protocols. For example, fluorescence polarization tests typically do not require washing steps.  
           [0006]    In general, a fluorescence polarization test for disease detection is run as follows. A small quantity of sample is added into a tube containing a buffer solution. A blank is read in the instrument. A fluorescent tracer specific for the disease is added in the same tube and the resultant polarization value is noted within seconds to minutes.  
           [0007]    U.S. Pat. No. 6,110,750, which is fully incorporated herein by reference, disclosed a fluorescence polarization technique for detection of animals infected with  Mycobacterium bovis.  The technique was based on the MPB70 protein secreted by  M. bovis.  The MPB70 protein was considered to be a highly species-specific immunodominant antigen containing at least three separate  M. bovis -specific epitopes. The fluorescence polarization technique used a tracer comprising MPB70 protein conjugated to a fluorophore to detect antibodies to  M. bovis  in sera from animals, such as cattle, bison, llama, and elk.  
           [0008]    However, in order to increase the potential sensitivity of fluorescence polarization assays for  M. bovis  infection, it is desirable to develop fluorescence tracers based on molecules smaller than MPB70.  
         SUMMARY OF THE INVENTION  
         [0009]    In a first principal aspect, the present invention provides an assay for detection of  M. bovis -infected animals. A tracer, comprising a peptide of  M. bovis  protein MPB70 conjugated to a fluorophore, is added to a serum sample from an animal to form a mixture. The fluorescence polarization of the mixture is then measured. The presence of  M. bovis  antibodies in the animal is detected from the measured fluorescence polarization of the mixture.  
           [0010]    In a second principal aspect, the present invention provides a tracer for use in a fluorescence polarization assay for antibodies specific for  M. bovis.  The tracer comprises a peptide of  M. bovis  protein MPB70 conjugated to a fluorophore, such that the tracer is able to bind to antibodies specific for  M. bovis  to produce a detectable change in fluorescence polarization. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0011]    A number of different peptides of MPB70 were screened for their suitability to detect serum antibodies to  M. bovis  using fluorescence polarization. Specific examples are described herein.  
       EXAMPLE 1  
       [0012]    A peptide with the following amino acid sequence was manufactured: PTNAAFSKLPASTIDELKTN (Pro Thr Asn Ala Ala Phe Ser Lys Leu Pro Ala Ser Thr Ile Asp Glu Leu Lys Thr Asn) (Ref. No.: 554; SEQ. ID NO.: 1).  
         [0013]    The peptide was then labeled in the following manner. The peptide was dissolved (2 mg/ml) in 1M sodium bicarbonate pH 8.3 at room temperature. A dye, 5-carboxyfluorescein succinimidyl ester (5-FAM, SE, Molecular Probes, Oregon) was then dissolved (10 mg/ml) in dimethyl sulfoxide (DMSO, Sigma-Aldrich Canada, Oakville, Ontario, Canada) and a sufficient quantity was added to the peptide solution to yield a dye:peptide molar ratio of 1:1. The mixture was stirred for 1 hour at room temperature in the dark. Hydroxylamine buffered to pH 8.5 (Molecular Probes) was added to the mixture and the stirring was continued for an additional 30 minutes. The mixture was then added to a Sephadex G-25 fine column (1×50 cm), which was pre-equilibrated with phosphate buffered saline (PBS, 0.01 M sodium phosphate+0.85% sodium chloride, pH 7.2) supplemented with 0.02% sodium azide. The labeled peptide was separated from the free dye by elution with PBS. Fractions (1 ml) were collected and the absorbance monitored at 492 nm. The elution profile showed 2 well separated peaks, the first of which contained the fluorescently-labeled peptide. The fractions containing the first peak were then pooled and concentrated using an Amicon Ultrafiltration Cell (Millipore, Corporation, Nepean, Ontario, Canada) fitted with an Amicon YM-1 membrane (Millipore).  
         [0014]    This labeled peptide was then tested for its suitability used as a fluorescent tracer for detecting serum antibodies to  M. bovis.  Specifically, the labeled peptide was tested by adding it to serum samples from cattle, elk, and deer, some of which were known positives for  M. bovis  infection and some of which were known negatives for  M. bovis  infection. It was found that the positive and negative sera gave different fluorescence polarization measurements after labeled peptide was added. Thus, it was determined that this labeled peptide can be used to detect serum antibodies to  M. bovis.    
       EXAMPLE 2  
       [0015]    A peptide with the following amino acid sequence was manufactured: SVQGMSQDPVAVAASNNPEL (Ser Val Gln Gly Met Ser Gln Asp Pro Val Ala Val Ala Ala Ser Asn Asn Pro Glu Leu) (Ref. No.: 555; SEQ. ID NO.: 2).  
         [0016]    This peptide was labeled and purified using the procedures described in Example 1, except that a different dye, fluorescein-5-EX, succinimidyl ester (Molecular Probes) was used, and the dye:peptide molar ratio was 20:1. This dye compound has a succinimidyl reactive group that is separated from the fluorophore by a seven atom spacer. This spacer serves to minimize interaction between the fluorophore and the amino acids to which the dye is conjugated.  
         [0017]    This labeled peptide was then tested as described above for Example 1. It was found that this labeled peptide can also be used to detect serum antibodies to  M. bovis,  i.e., that positive and negative sera gave different fluorescence polarization measurements upon addition of the labeled peptide.  
       EXAMPLE 3  
       [0018]    The MPB70 protein was epitope scanned to make small fluorescent peptides. Epitope scanning was accomplished using a Multipin™ Peptide Synthesis block (cleavable DKP, Catalog No. KT-96-0-DKP, batch no. 1252-2A) consisting of 96 gears, which was purchased from Mimotypes Pty Ltd., Calyton, Victoria, Australia. Specifically, this kit was used, in accordance with the Multipin™ instructions, to synthesize 96 peptides of 15 amino acids each, each corresponding to a peptide of MPB70. After synthesis, each peptide was covalently attached with 6-carboxyfluorescein (isomer 2). The gears were then washed with excess DMF and methanol, and the resultant block was immersed overnight in a 96 deep well (1 ml capacity, with each well containing 800 microliters of 0.1 M phosphate:CH 3 CN (60:40) solution) to obtain solutions of free labeled peptide.  
         [0019]    The free labeled peptide solutions were then diluted appropriately for conducting fluorescence polarization assays. Each labeled peptide was tested by adding it to cattle sera, both positive and negative for  M. bovis,  and measuring the resulting fluorescence polarization. In this way, it was found that two labeled peptides could be used to detect serum antibodies to  M. bovis,  i.e., that positive and negative sera gave different fluorescence polarization measurements upon addition of the labeled peptide. Two promising peptides had the following amino acid sequences: AVAASNNPELTTLTA (Ala Val Ala Ala Ser Asn Asn Pro Glu Leu Thr Thr Leu Thr Ala) (SEQ. ID NO.: 3) and PTNAAFSKLPASTID (Pro Thr Asn Ala Ala Phe Ser Lys Leu Pro Ala Ser Thr Ile Asp) (SEQ. ID NO.: 4).  
       EXAMPLE 4  
       [0020]    Additional peptides were generated and labeled essentially as described above. The peptides were labeled with 6-carboxyfluorescein (6-FAM or 6-fam; Sigma-Aldrich # 54115; Molecular Probes #C-1360 or #C-6164 (6-FAM, succinimidyl ester)). The sequences of the peptides were:  
                               6-fam-GMSQDPVAVAASNNPELTTLTAALS;   (Ref. No.: 708; SEQ. ID NO.: 5)                   and               6-fam-SVQGMSQDPVAVAASNNPELTTLTAALS.   (Ref. No.: 799, SEQ. ID NO.: 6)          
 
         [0021]    The peptides, as well as the full-length MPB70 protein, were used in fluorescence polarization assays as described above to screen sera from  Mycobacterium bovis -infected animals as well as non-infected animals. The fluorescence polarization assays were positive for  Mycobacterium bovis  infection when the measured fluorescence polarization exceeded a predetermined threshold value. The charts below summarize the results from these assays:  
                                                                                                                                                                       SERUM   # of       MPB70   Peptide 555   Peptide 708   Peptide 799   Peptide 554            SOURCE   SERA   Classification   Sens. %   Spec. %   Sens. %   Spec. %   Sens. %   Spec. %   Sens. %   Spec. %   Sens. %   Spec. %                    North   5666   Presumed       98.3                                       America       negative       (5570/5666)       Canada   28   Culture positive   92.9                   (26/28)       #1   50   Skin test positive   36   N.D.   76   N.D.                   (18/50)       (38/50)       #2   90   17 skin test   64.7   98.6   82.4   97.3               positive               73 skin test   (11/17)   (72/73)   (14/17)   (71/73)               negative       #3   49   Lesion positive   55.1   N.D.   61.2   N.D.           67.3       51.0                   (27/49)       (30/49)               (33/49)       (25/49)                                           71.4       71.4                                           (35/49)       (35/49)                                           (peptides       (peptides                                           555 +       555 +                                           799)       554 +                                                   799)       North   557   Presumed               98.75                       98.75       America       negative               (550/557)                       (550/557)       Ontario 02   69   65 skin test                   55.4   25               positive               4 skin test                   (36/65)   (1/4)           5/63               negative                                   (1 unique)                                                   58                                                   (40/69)                                                   (peptides                                                   708 +                                                   554)           48   Lesion positive   56.3       71.4       64.4       68.8       46.5                   (27/48)       (30/42)       (29/45)       (33/48)       (25/48)                                           72.9                                           (35/48)                                           (peptides                                           708 +                                           799)                  
 
         [0022]    The foregoing description of the invention is presented for purposes of illustration and description, and is not intended, nor should be construed, to be exhaustive or to limit the invention to the precise forms disclosed. The description was selected to best explain the principles of the invention and practical application of these principles to enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention not be limited by the specification, but defined by the claims.  
     
       
       
         1 
         
           
             6  
           
           
             1  
             20  
             PRT  
             Mycobacterium bovis  
           
            1 

Pro Thr Asn Ala Ala Phe Ser Lys Leu Pro Ala Ser Thr Ile Asp Glu 
1               5                   10                  15 

Leu Lys Thr Asn 
            20 

 
           
             2  
             20  
             PRT  
             Mycobacterium bovis  
           
            2 

Ser Val Gln Gly Met Ser Gln Asp Pro Val Ala Val Ala Ala Ser Asn 
1               5                   10                  15 

Asn Pro Glu Leu 
            20 

 
           
             3  
             15  
             PRT  
             Mycobacterium bovis  
           
            3 

Ala Val Ala Ala Ser Asn Asn Pro Glu Leu Thr Thr Leu Thr Ala 
1               5                   10                  15 

 
           
             4  
             15  
             PRT  
             Mycobacterium bovis  
           
            4 

Pro Thr Asn Ala Ala Phe Ser Lys Leu Pro Ala Ser Thr Ile Asp 
1               5                   10                  15 

 
           
             5  
             25  
             PRT  
             Mycobacterium bovis  
             
               misc_feature  
               (1)..(1)  
               labeled at the amino terminus with 6-FAM  
             
           
            5 

Gly Met Ser Gln Asp Pro Val Ala Val Ala Ala Ser Asn Asn Pro Glu 
1               5                   10                  15 

Leu Thr Thr Leu Thr Ala Ala Leu Ser 
            20                  25 

 
           
             6  
             28  
             PRT  
             Mycobacterium bovis  
             
               misc_feature  
               (1)..(1)  
               labeled at the amino terminus with 6-FAM  
             
           
            6 

Ser Val Gln Gly Met Ser Gln Asp Pro Val Ala Val Ala Ala Ser Asn 
1               5                   10                  15 

Asn Pro Glu Leu Thr Thr Leu Thr Ala Ala Leu Ser 
            20                  25