Methods and compositions for detecting mycoplasma exposure
Inventors
Campbell, Robert L. • Langer, Melissa E. • Pollard, Jasper N. • Cheek, Robert L. • Dolan, Kevin G. • Stewart, W. William • Bresolin, Stefano • Sink, John Richard • Heineck, Tobias M. • Black, Laura A. • Pope, Nathan F. • Parker, Lauren K. • Jensen, Erik J. • Correa, Jorge Carlos • Azizoglu, Reha O.
Assignees
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Publication Number
US-10222383-B2
Publication Date
2019-03-05
Expiration Date
Abstract
The present invention provides methods and compositions for detecting Mycoplasma exposure in a subject.
Core Innovation
The invention relates to detecting Mycoplasma exposure in a subject using an aqueous sample comprising a biological fluid from the subject. The method contacts the aqueous sample to at least one solid support bearing immobilized mycoplasma antigen so that an antigen/antibody complex forms if the aqueous sample contains an antibody to Mycoplasma. The approach includes detecting the presence or absence of a first detectable group and a second detectable group on the solid support as indicators of exposure.
The method specifically addresses detection using anti-IgG and anti-IgM antibodies. An anti-IgG antibody coupled to a first detectable group is contacted with the aqueous sample on the solid support, and the presence or absence of the first detectable group is detected. In parallel, an anti-IgM antibody coupled to a second detectable group is contacted, and the presence or absence of the second detectable group is detected, where detection of the presence of the first and/or second detectable group detects Mycoplasma exposure.
A further aspect quantifies exposure by determining anti-Mycoplasma IgG and anti-Mycoplasma IgM amounts and using imaging-based relative intensity values. The method calculates a value X from a relative intensity value assigned to the anti-Mycoplasma IgG signal and a value Y from a relative intensity value assigned to the anti-Mycoplasma IgM signal, and then calculates a value Z by adding X and Y. The subject is identified as having Zone 1, Zone 2, or Zone 3 status using high, medium, or low relative intensity values of Z that correspond to active infection or recent exposure, prior exposure, or no prior exposure.
The disclosed system also includes an apparatus configured to image the sample and calculate zone status. The apparatus includes an imaging reader configured to image a sample having anti-Mycoplasma IgG and anti-Mycoplasma IgM antibody intensity signals and a controller configured to receive the signals, calculate X and Y by assigning relative intensity values, calculate Z by adding X and Y, and identify Zone 1, Zone 2, or Zone 3 status. In one refinement, the high, medium, and low relative intensity values for the zones are identified empirically based on clinical symptoms.
Claims Coverage
The provided set contains three independent claims (clm-00001, clm-00016, clm-00018) with inventive features centered on an immunoassay workflow using a solid support with immobilized Mycoplasma antigen and separate anti-IgG/anti-IgM detectable groups, quantitative calculation of X, Y, and Z followed by Zone 1/2/3 classification based on Z intensity, and an apparatus that images IgG/IgM intensity signals and computes the same Zone classification.
Solid-support antigen/antibody complex detection for Mycoplasma exposure
Providing an aqueous sample comprising a biological fluid from the subject; contacting the aqueous sample to at least one solid support having a mycoplasma antigen immobilized thereon to form an antigen/antibody complex if the sample contains an antibody to Mycoplasma; contacting an anti-IgG antibody coupled to a first detectable group and detecting presence or absence of the first detectable group; contacting an anti-IgM antibody coupled to a second detectable group and detecting presence or absence of the second detectable group; wherein detection of the presence of the first and/or second detectable group on the solid support detects Mycoplasma exposure.
Quantitative IgG/IgM intensity calculation and Zone 1/Zone 2/Zone 3 classification using Z
Quantitatively determining the amount of anti-Mycoplasma IgG antibodies and the amount of anti-Mycoplasma IgM antibodies in the biological fluid; 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; calculating a value Z by adding X and Y; and identifying the subject as having Zone 1, Zone 2 or Zone 3 status regarding Mycoplasma exposure, where Zone 1 corresponds to a high relative intensity value indicating an active infection or recent exposure, Zone 2 corresponds to a medium relative intensity value indicating prior exposure, and Zone 3 corresponds to a low relative intensity value indicating no prior exposure.
Imaging-reader apparatus calculating X, Y, and Z to identify Zone status
An 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 receive the anti-Mycoplasma IgG antibody intensity signal and the anti-Mycoplasma IgM antibody intensity signal, 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, calculate a value Z by adding X and Y, and identify the subject as having a Zone 1, Zone 2 or Zone 3 status regarding Mycoplasma exposure based on whether Z is a high, medium, or low relative intensity value.
Across the independent claims, the core coverage is an immunoassay using immobilized Mycoplasma antigen and separate anti-IgG and anti-IgM detectable groups to detect Mycoplasma exposure, combined with a quantitative imaging-based intensity framework that computes Z from IgG and IgM relative intensity values and classifies exposure into Zone 1/2/3. An apparatus claim covers performing the same X/Y/Z calculation from imaging signals and reporting the corresponding zone status.
Stated Advantages
Detects Mycoplasma exposure in a subject using detection of anti-Mycoplasma IgG and/or anti-Mycoplasma IgM detectable groups on a solid support.
Provides quantitative determination via assigning relative intensity values to anti-Mycoplasma IgG and anti-Mycoplasma IgM signals and calculating a value Z.
Identifies exposure status using Zone 1, Zone 2, and Zone 3 corresponding to active infection or recent exposure, prior exposure, and no prior exposure.
Documented Applications
Detecting Mycoplasma exposure in a subject using an aqueous sample comprising a biological fluid, with a solid support bearing immobilized Mycoplasma antigen and detection of anti-Mycoplasma IgG and anti-Mycoplasma IgM signals.
Quantitative classification of Mycoplasma exposure into Zone 1 (active infection or recent exposure), Zone 2 (prior exposure), and Zone 3 (no prior exposure) by calculating Z from anti-Mycoplasma IgG and anti-Mycoplasma IgM relative intensity values.
Using an apparatus with an imaging reader and controller to image anti-Mycoplasma IgG and anti-Mycoplasma IgM antibody intensity signals and identify Zone 1, Zone 2, or Zone 3 status.
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