Assay for detecting tuberculosis in nonhuman primates
Inventors
Lyashchenko, Konstantin • Esfandiari, Javanbakhsh • McCombs, Candace
Assignees
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Publication Number
US-8128941-B2
Publication Date
2012-03-06
Expiration Date
Abstract
Systems, methods, and compositions for a diagnostic method for detecting TB in non-human primates that is easy to use, sensitive, and specific. The method utilizes recombinant mycobacterial antigens, such as polyfusion proteins. The method utilizes an antigen-antibody-antigen arrangement to detect TB infection in nonhuman primates. The method can detect IgM antibodies to TB, in addition to IgG antibodies, providing ability to detect TB earlier in nonhuman primate TB infection as compared to conventional TB tests.
Core Innovation
The invention relates to a tuberculosis diagnostic in non-human primates for detecting antibodies to one or more mycobacterial antigens. It uses recombinant mycobacterial antigen mixtures, including polyfusion proteins and polyepitope fusion proteins, immobilized on a solid support, and is directed to detecting IgM and IgG responses by forming antibody-antigen complexes that are subsequently detected.
A central aspect is contacting a non-human primate sample with a first composition comprising an antigen-particle conjugate carrying multiple mycobacterial antigens, and then contacting the sample with a second composition comprising multiple mycobacterial antigens immobilized at one or more locations on a solid support. The binding permits formation of an antibody-antigen-particle complex and then an antibody-antigen-particle complex bound to immobilized antigens, and detecting the formation of the bound complex is used to detect antibodies to the mycobacterial antigens.
A further aspect addresses non-specific antibody interactions by incorporating non-specific binding components and additional detection of trapped non-specific complexes. One method uses a first composition that forms a non-specific antibody-conjugate complex, a second composition with immobilized mycobacterial antigens, and a third composition immobilized on the solid support capable of trapping non-specific antibody-conjugate complex that does not bind the second composition, followed by detecting both immobilized antigen-antibody complexes and trapped non-specific antibody-conjugate complexes.
Claims Coverage
The independent claims cover diagnostic methods and diagnostic compositions for detecting antibodies to one or more mycobacterial antigens in non-human primates. The inventive features focus on multi-antigen formats using an antigen-particle conjugate combined with a solid-support immobilized antigen composition, and on polyfusion protein or non-specific binding and trapping detection schemes.
Antigen-particle conjugate followed by immobilized matching antigens on a solid support
A method for detecting antibodies to one or more mycobacterial antigens in a sample from a non-human primate by obtaining the sample, contacting it with a first composition comprising an antigen-particle conjugate comprising multiple mycobacterial antigens conjugated to a particle, contacting the sample with a second composition comprising multiple mycobacterial antigens immobilized at one or more locations on a solid support, and detecting the formation of the bound antibody-antigen-particle complex, wherein each antigen present in the first composition is present in the second composition.
Sequential non-specific antibody-conjugate formation, immobilized antigen binding, and trapping of non-specific complexes
A method for detecting antibodies to one or more mycobacterial antigens in a sample from a non-human primate by obtaining the sample, contacting the sample with a first composition comprising particles conjugated to one or more molecules capable of non-specifically binding non-human primate antibodies to permit formation of a non-specific antibody-conjugate complex, contacting the sample with a second composition comprising a mixture of mycobacterial antigens immobilized on a solid support to permit formation of an immobilized mycobacterial antigen-antibody-conjugate complex, contacting the sample with a third composition comprising one or more molecules capable of non-specifically binding non-human primate antibodies immobilized on the solid support to trap the non-specific antibody-conjugate complex that did not bind to the second composition, and detecting both complexes on the solid support.
Polyfusion protein combining multiple immobilized antigens on a solid support
A composition for detecting antibodies to one or more mycobacterial antigens in nonhuman primates comprising a mixture of mycobacterial antigens immobilized on a solid support wherein at least two of the mycobacterial antigens are combined in a polyfusion protein which binds to non-human primate antibodies, the polyfusion protein selected from CFP10/ESAT-6, TBF10, and Arc1/MPB83.
Particle-conjugated individual antigens within a selected multi-antigen mixture
A composition for detecting antibodies to one or more mycobacterial antigens in nonhuman primates comprising a plurality of particles and a mixture of mycobacterial antigens selected to bind to nonhuman primate antibodies comprising at least two of amino acid sequences selected from ESAT-6, CFP-10, Mo-29, MPB70, MPT70, MPB83, MPT83, MPT63, MPB63, MPB64 and MPT64, each individual antigen of the mixture conjugated to at least one of the plurality of particles, and each particle conjugated to at least one individual antigen.
The independent claims collectively cover a match between antigens carried on an antigen-particle conjugate and antigens immobilized on the solid support for bound complex detection, an additional non-specific binding and trapping scheme with detection of both immobilized antigen-antibody complexes and trapped non-specific antibody-conjugate complexes, and diagnostic compositions that implement multi-antigen binding through polyfusion protein immobilized antigen mixtures or individually antigen-conjugated particles.
Stated Advantages
Earlier and more sensitive/specific detection by detecting IgM and IgG responses.
Reduction of non-specific antibody effects by incorporating trapping and non-specific binding components with detection of trapped non-specific antibody-conjugate complex.
Documented Applications
Detecting antibodies to mycobacterial antigens in a sample from non-human primates for TB diagnostics, including an M. bovis outbreak context involving rhesus macaques.
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