Methods and compositions for detecting mycoplasma exposure

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, 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 is identified when Z is a medium relative intensity value and indicates a prior exposure to mycoplasma, and

Zone 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, wherein

Zone 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, and

Zone 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; and

a 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.