Antibody detection by agglutination–PCR (ADAP) enables early diagnosis of HIV infection by oral fluid analysis

Tsai, Cheng‐ting; Robinson, Peter V.; Cortez, Felipe de Jesus; Elma, Maria; Seftel, David; Pourmandi, Narges; Pandori, Mark; Bertozzi, Carolyn R.

doi:10.1073/pnas.1711004115

Cited by 42 publications

(45 citation statements)

References 30 publications

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“…Finally, the barcoding capacity of the oligonucleotide signals allows for multiplexed labeling, oligonucleotide amplification, and quantitative measurements in the same sample. These attributes are specifically attractive for complex, limited-abundance samples like clinical tissues and fluids, which are an active area of focus for ultrasensitive protein detection technologies (32)(33)(34)(35)(36). The high-throughput nature of this proof-of-principle application to clinical samples highlights the ability to perform hundreds to thousands of activity measurements with minimal sample preparation, high sensitivity, and good reproducibility.…”

Section: Discussionmentioning

confidence: 99%

Ultrasensitive, multiplexed chemoproteomic profiling with soluble activity-dependent proximity ligation

Eckert

Chang

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Chemoproteomic methods can report directly on endogenous, active enzyme populations, which can differ greatly from measures of transcripts or protein abundance alone. Detection and quantification of family-wide probe engagement generally requires LC-MS/MS or gel-based detection methods, which suffer from low resolution, significant input proteome requirements, laborious sample preparation, and expensive equipment. Therefore, methods that can capitalize on the broad target profiling capacity of family-wide chemical probes but that enable specific, rapid, and ultrasensitive quantitation of protein activity in native samples would be useful for basic, translational, and clinical proteomic applications. Here we develop and apply a method that we call soluble activity-dependent proximity ligation (sADPL), which harnesses family-wide chemical probes to convert active enzyme levels into amplifiable barcoded oligonucleotide signals. We demonstrate that sADPL coupled to quantitative PCR signal detection enables multiplexed “writing” and “reading” of active enzyme levels across multiple protein families directly at picogram levels of whole, unfractionated proteome. sADPL profiling in a competitive format allows for highly sensitive detection of drug–protein interaction profiling, which allows for direct quantitative measurements of in vitro and in vivo on- and off-target drug engagement. Finally, we demonstrate that comparative sADPL profiling can be applied for high-throughput molecular phenotyping of primary human tumor samples, leading to the discovery of new connections between metabolic and proteolytic enzyme activity in specific tumor compartments and patient outcomes. We expect that this modular and multiplexed chemoproteomic platform will be a general approach for drug target engagement, as well as comparative enzyme activity profiling for basic and clinical applications.

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Section: Discussionmentioning

confidence: 99%

Ultrasensitive, multiplexed chemoproteomic profiling with soluble activity-dependent proximity ligation

Eckert

Chang

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…1c). [18][19][20] Innovative recent examples of PLA assays applied to chemical biology include ultrasensitive detection of posttranslational modifications 19,21 (e.g., Glycoseek 22 ) and antibodies, 23,24 as well as assessing hydrolytic 20 and kinase 25 enzyme activity (e.g., activity-dependent proximity ligation, ADPL).…”

Section: Resultsmentioning

confidence: 99%

An Activity-Based Methionine Bioconjugation Approach to Developing Proximity-Activated Imaging Reporters

Ohata¹,

Krishnamoorthy²,

González³

et al. 2019

Preprint

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Chemical probes that report on protein activity, rather than protein abundance, with spatial and temporal resolution can enable studies of their native function in biological contexts as well as provide opportunities for developing new types of biochemical reporters. Here we present a sensing platform, termed proximity-activated imaging reporter (PAIR), which combines activity-based methionine bioconjugation and antibody labeling with proximity-dependent oligonucleotide-based amplification to monitor dynamic changes of a given analyte in cells and animals through context-dependent methionine labeling of specific protein targets. We establish this PAIR method to develop sensors for imaging reactive oxygen species (ROS) and calcium ions through oxaziridine-directed labeling of reactive methionine residues on β-actin and calmodulin (CaM), respectively, where the extent of methionine bioconjugation on these protein targets can serve as an indicator of oxidative stress or calcium status. In particular, application of PAIR to activity-based CaM detection provides a method for imaging integrated calcium activity in both in vitro cell and in vivo zebrafish models. By relying on native protein biochemistry, PAIR enables redox and metal imaging without introduction of external small-molecules or genetically encoded indicators that can potentially buffer the natural/existing pools. This approach can be potentially generalized to target a broader range of analytes by pairing appropriate activity-based protein probes with protein detection reagents in a proximity-driven manner, providing a starting point not only for designing new sensors but also for monitoring endogenous activity of specific protein targets in biological specimens with spatial and temporal fidelity.

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“…Overall, thermostable preparations of TSHR260 described in this study are promising tools for developing new methods of TRAb detection including agglutination PCR (Tsai et al 2016(Tsai et al , 2018. Also they should be useful in therapeutic interventions such as specific immunoadsorption of TSHR autoantibodies.…”

Section: M22mentioning

confidence: 91%

Crystal structure of a ligand-free stable TSH receptor leucine-rich repeat domain

Miller-Gallacher

Sanders

Young

et al. 2019

Journal of Molecular Endocrinology

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The crystal structures of the thyroid-stimulating hormone receptor (TSHR) leucine-rich repeat domain (amino acids 22-260; TSHR260) in complex with a stimulating human monoclonal autoantibody (M22 TM ) and in complex with a blocking human autoantibody (K1-70™) have been solved. However, attempts to purify and crystallise free TSHR260, that is not bound to an autoantibody, have been unsuccessful due to the poor stability of free TSHR260. We now describe a TSHR260 mutant that has been stabilised by the introduction of six mutations (H63C, R112P, D143P, D151E, V169R and I253R) to form TSHR260-JMG55 TM , which is approximately 900 times more thermostable than wild-type TSHR260. These six mutations did not affect the binding of human TSHR monoclonal autoantibodies or patient serum TSHR autoantibodies to the TSHR260. Furthermore, the response of full-length TSHR to stimulation by TSH or human TSHR monoclonal autoantibodies was not affected by the six mutations. Thermostable TSHR260-JMG55 TM has been purified and crystallised without ligand and the structure solved at 2.83 Å resolution. This is the first reported structure of a glycoprotein hormone receptor crystallised without ligand. The unbound TSHR260-JMG55 TM structure and the M22 and K1-70 bound TSHR260 structures are remarkably similar except for small changes in side chain conformations. This suggests that neither the mutations nor the binding of M22 TM or K1-70 TM change the rigid leucine-rich repeat domain structure of TSHR260. The solved TSHR260-JMG55 TM structure provides a rationale as to why the six mutations have a thermostabilising effect and provides helpful guidelines for thermostabilisation strategies of other soluble protein domains.

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Antibody detection by agglutination–PCR (ADAP) enables early diagnosis of HIV infection by oral fluid analysis

Cited by 42 publications

References 30 publications

Ultrasensitive, multiplexed chemoproteomic profiling with soluble activity-dependent proximity ligation

Ultrasensitive, multiplexed chemoproteomic profiling with soluble activity-dependent proximity ligation

An Activity-Based Methionine Bioconjugation Approach to Developing Proximity-Activated Imaging Reporters

Crystal structure of a ligand-free stable TSH receptor leucine-rich repeat domain

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