The present invention relates to a method for detecting a pathogen in cellular lysate by measuring pathogen-specific enzyme activity. The method comprises contacting the cellular lysate with a substrate the pathogen of interest recognizes and modifies, and obtaining a measurable, recordable, signal. The method may comprise detection of SARS-CoV viruses using the activity of SARS PLpro enzyme in tongue scrape lysate as a readout.

The present application provides compositions and methods for determining a disease or condition in a subject. The method comprises contacting a body fluid with a molecule comprising a reporter thereof and the reported is cleaved by an agent in the body fluid. Diseases and conditions that can be determined by the method are also described.

Aspects of the disclosure relate to methods and compositions useful for in vivo and/or in vitro enzyme profiling. In some embodiments, the disclosure provides methods of in vivo enzymatic processing of exogenous molecules followed by detection of signature molecules as representative of the presence of active enzymes associated with diseases or conditions. In some embodiments, the disclosure provides compositions and in vitro methods for localization of enzymatic activity in a tissue sample.

Aspects of the disclosure relate to methods and compositions useful for in vivo and/or in vitro enzyme profiling. In some embodiments, the disclosure provides methods of in vivo enzymatic processing of exogenous molecules followed by detection of signature molecules as representative of the presence of active enzymes associated with diseases or conditions. In some embodiments, the disclosure provides compositions and in vitro methods for localization of enzymatic activity in a tissue sample.

In this vein, we present CTLAP, a fluorogenic probe that is rapidly activated by CTL and displays good selectivity over CTB and CTV, the closest competing analytes for CTL activity probes. CTLAP exhibits intrinsically low background fluorescence, which we attribute to the notably low quantum yield measured for the probe. CTLAP demonstrates markedly higher turn-on ratios (24-fold) and moderately improved enzyme selectivity (6- to 10-fold) when compared to Z-FR-AMC (10-fold turn-on ratio, 6- to 7-fold selectivity), a commercially available CTL-selective probe commonly used to detect CTL activity in mixed samples. Optimum selectivity for CTL is achieved within 10 min of incubation with the enzyme, suggesting that CTLAP is amenable for rapid detection of CTL, even in the presence of competing cathepsins.

The present invention provides a method in which a porous body having a monoclonal antibody to be measured immobilized in pores thereof is brought into contact with nanoparticles having a protease immobilized thereonto in a liquid to perform selective protease digestion of the monoclonal antibody and a peptide fragment obtained by the digestion is detected by liquid chromatography mass spectrometry (LC-MS), wherein the monoclonal antibody is digested with the protease in the presence of an antibody specifically binding to the monoclonal antibody or a target molecule of the monoclonal antibody.

The present invention provides a method in which a porous body having a monoclonal antibody to be measured immobilized in pores thereof is brought into contact with nanoparticles having a protease immobilized thereonto in a liquid to perform selective protease digestion of the monoclonal antibody and a peptide fragment obtained by the digestion is detected by liquid chromatography mass spectrometry (LC-MS), wherein the monoclonal antibody is digested with the protease in the presence of an antibody specifically binding to the monoclonal antibody or a target molecule of the monoclonal antibody.

A probe for measuring the activity of caspase-1 according to the present disclosure can specifically image cells or tissues where inflammatory response is induced because it is cleaved by reacting specifically with the active caspase-1 enzyme in vivo and in vitro and re-emits fluorescence. The probe for measuring the activity of caspase-1 can be used for various purposes, such as for imaging of cells or tissues where inflammatory response is induced, as a drug carrier, for screening of a drug inhibiting inflammatory response, etc. The probe for measuring the activity of caspase-1 is applicable both in vivo and in vitro, and can be used for various applications such as high-throughput screening for new drug development, early diagnosis of diseases, etc.

A probe for measuring the activity of caspase-1 according to the present disclosure can specifically image cells or tissues where inflammatory response is induced because it is cleaved by reacting specifically with the active caspase-1 enzyme in vivo and in vitro and re-emits fluorescence. The probe for measuring the activity of caspase-1 can be used for various purposes, such as for imaging of cells or tissues where inflammatory response is induced, as a drug carrier, for screening of a drug inhibiting inflammatory response, etc. The probe for measuring the activity of caspase-1 is applicable both in vivo and in vitro, and can be used for various applications such as high-throughput screening for new drug development, early diagnosis of diseases, etc.

The present invention relates to compounds of formula I, wherein D is a detectable moiety, or salts thereof, which can be used as activity-based probes for neutrophil elastase, as well as to methods for detecting neutrophil elastase (NE) activity in a tissue sample lysate, and related diagnostic methods using compounds of formula I.

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