초록

The present invention provides methods and compositions for detecting Mycoplasma exposure in a subject.

대표청구항 ▼

1. A method of detecting Mycoplasma exposure in a subject, comprising: (a) providing an aqueous sample comprising a biological fluid from the subject;(b) contacting said aqueous sample to at least one solid support, each said at least one support having a mycoplasma antigen immobilized thereon, unde

1. A method of detecting Mycoplasma exposure in a subject, comprising: (a) providing an aqueous sample comprising a biological fluid from the subject;(b) contacting said aqueous sample to at least one solid support, each said at least one support having a mycoplasma antigen immobilized thereon, under conditions wherein an antigen/antibody complex will form if said aqueous sample contains an antibody to Mycoplasma; (c) contacting an anti-IgG antibody coupled to a first detectable group with said aqueous sample of step (b) on said solid support, under conditions wherein an antibody/antibody complex will form if said aqueous sample contains an antibody to Mycoplasma; (d) detecting the presence or absence of said first detectable group on said at least one solid support;(e) contacting an anti-IgM antibody coupled to a second detectable group, with said aqueous sample of step (b) on said solid support, under conditions wherein an antibody/antibody complex will form if said aqueous sample contains an antibody to Mycoplasma ; and(f) detecting the presence or absence of said second detectable group on said at least one solid support,wherein detection of the presence of said first detectable group and/or of said second detectable group on said solid support detects Mycoplasma exposure in said subject,wherein said aqueous sample comprises said biological fluid and an aqueous diluent; and wherein said aqueous diluent comprises raw whole milk that contains no detectable anti-Mycoplasma antibodies. 2. The method of claim 1, wherein said at least one solid support comprises two separate supports, and said contacting step (b) comprises contacting a first portion of said sample to a first of said solid supports, and contacting a second portion of said sample to a second of said solid supports. 3. The method of claim 1, wherein said first detectable group and said second detectable group are the same. 4. The method of claim 1, wherein said first detectable group and/or said second detectable group comprises a detectable particle. 5. The method of claim 1, wherein said first detectable group and/or said second detectable group comprises a fluorescence moiety. 6. The method of claim 1, wherein said solid support comprises an absorbent material. 7. The method of claim 1, wherein said anti-IgG antibody comprises an anti-IgG heavy chain antibody. 8. The method of claim 1, wherein said anti-IgM antibody comprises an anti-IgM heavy chain antibody. 9. The method of claim 1, wherein said mycoplasma antigen comprises inactivated whole Mycoplasma. 10. The method of claim 1, wherein said mycoplasma antigen is from Mycoplasma bovis. 11. The method of claim 1, wherein said steps are carried out in a lateral flow, dipstick, or flow through format. 12. The method of claim 1, wherein said biological fluid comprises milk, and said aqueous sample is filtered. 13. The method of claim 1, wherein said biological sample comprises blood or a blood fraction. 14. The method of claim 1, wherein said detecting step (d) and/or said detecting step (f) comprises quantitatively determining the amount of anti-Mycoplasma IgG antibodies and/or the amount of anti-Mycoplasma IgM antibodies in said biological fluid. 15. The method of claim 1, wherein said biological fluid is collected from a mammalian subject. 16. A method of detecting Mycoplasma exposure in a subject, comprising: (a) providing an aqueous sample comprising a biological fluid from the subject;(b) contacting said aqueous sample to at least one solid support, each said at least one support having a mycoplasma antigen immobilized thereon, under conditions wherein an antigen/antibody complex will form if said aqueous sample contains an antibody to Mycoplasma; (c) contacting an anti-IgG antibody coupled to a first detectable group with said aqueous sample of step (b) on said solid support, under conditions wherein an antibody/antibody complex will form if said aqueous sample contains an antibody to Mycoplasma; (d) detecting the presence or absence of said first detectable group on said at least one solid support;(e) contacting an anti-IgM antibody coupled to a second detectable group, with said aqueous sample of step (b) on said solid support, under conditions wherein an antibody/antibody complex will form if said aqueous sample contains an antibody to Mycoplasma ; and(f) detecting the presence or absence of said second detectable group on said at least one solid support,wherein detection of the presence of said first detectable group and/or of said second detectable group on said solid support detects Mycoplasma exposure in said subject, further comprising the steps of:(g) quantitatively determining the amount of anti-Mycoplasma IgG antibodies and the amount of anti-Mycoplasma IgM antibodies in said biological fluid;(h) calculating a value X by assigning a relative intensity value to the amount of anti-Mycoplasma IgG antibodies and a value Y by assigning a relative intensity value to the amount of anti-Mycoplasma IgM antibodies;(i) calculating a value Z by adding X and Y of step (h); and(j) identifying the subject as having Zone 1, Zone 2 of Zone 3 status regarding Mycoplasma exposure, whereinZone 1 status is identified when Z is a high relative intensity value and indicates an active infection or recent exposure to mycoplasma,Zone 2 is identified when Z is a medium relative intensity value and indicates a prior exposure to mycoplasma, andZone 3 is identified when Z is a low relative intensity value and indicates no prior exposure to mycoplasma. 17. The method of claim 1, further comprising the steps of: (g) quantitatively determining the amount of anti-Mycoplasma IgG antibodies and the amount of anti-Mycoplasma IgM antibodies in said biological fluid using an imaging reader instrument;(h) calculating a value X using the imaging reader instrument; and(i) identifying the subject as having Zone 1, Zone 2 or Zone 3 status regarding Mycoplasma exposure, whereinZone 1 status is identified when X is equal to or greater than 16 and indicates an active infection or recent exposure to mycoplasma,Zone 2 is identified when X is between 6 and 16 and indicates a prior exposure to mycoplasma, andZone 3 is identified when X is equal to or less than 6 and indicates no prior exposure to mycoplasma. 18. An apparatus for determining Mycoplasma exposure in a subject, the apparatus comprising: an imaging reader configured to image a sample having an anti-Mycoplasma IgG antibody intensity signal and an anti-Mycoplasma IgM antibody intensity signal; anda controller configured to(a) receive the anti-Mycoplasma IgG antibody intensity signal and the anti-Mycoplasma IgM antibody intensity signal from the image reader;(b) calculate a value X by assigning a relative intensity value to the anti-Mycoplasma IgG antibody intensity signal and a value Y by assigning a relative intensity value to the anti-Mycoplasma IgM antibody intensity signal;(c) calculate a value Z by adding X and Y of step (b); and(d) identify the subject as having a Zone 1, Zone 2 or Zone 3 status regarding Mycoplasma exposure, wherein Zone 1 status is identified when Z is a high relative intensity value and indicates an active infection or recent exposure to mycoplasma;Zone 2 status is identified when Z is a medium relative intensity value and indicates a prior exposure to mycoplasma;Zone 3 is identified when Z is a low relative intensity value and indicates no prior exposure to mycoplasma. 19. The apparatus of claim 18, wherein the high relative intensity value of Zone 1, the medium relative intensity value of Zone 2 and the low relative intensity value of Zone 3 are determined empirically from clinical symptoms. 20. The apparatus of claim 18, wherein the sample comprises a lateral flow assay. 21. The method of claim 16, wherein said at least one solid support comprises two separate supports, and said contacting step (b) comprises contacting a first portion of said sample to a first of said solid supports, and contacting a second portion of said sample to a second of said solid supports. 22. The method of claim 16, wherein said first detectable group and said second detectable group are the same. 23. The method of claim 16, wherein said first detectable group and/or said second detectable group comprises a detectable particle. 24. The method of claim 16, wherein said first detectable group and/or said second detectable group comprises a fluorescence moiety. 25. The method of claim 16, wherein said solid support comprises an absorbent material. 26. The method of claim 16, wherein said anti-IgG antibody comprises an anti-IgG heavy chain antibody. 27. The method of claim 16, wherein said anti-IgM antibody comprises an anti-IgM heavy chain antibody. 28. The method of claim 16, wherein said mycoplasma antigen comprises inactivated whole Mycoplasma. 29. The method of claim 16, wherein said mycoplasma antigen is from Mycoplasma bovis. 30. The method of claim 16, wherein said steps are carried out in a lateral flow, dipstick, or flow through format. 31. The method of claim 16, wherein said biological fluid comprises milk, and said aqueous sample is filtered. 32. The method of claim 16, wherein said biological sample comprises blood or a blood fraction. 33. The method of claim 16, wherein said detecting step (d) and/or said detecting step (f) comprises quantitatively determining the amount of anti-Mycoplasma IgG antibodies and/or the amount of anti-Mycoplasma IgM antibodies in said biological fluid. 34. The method of claim 16, wherein said biological fluid is collected from a mammalian subject.