Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Magni, R.; Espina, Benjamin H.; Liotta, Lance A.; Luchini, Alessandra; Espina, Virginia

doi:10.3791/51789

Cited by 8 publications

(6 citation statements)

References 34 publications

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“…PRM is a targeted mass spectrometry analysis that is able to directly detect peptides derived from specific proteins of interest [ 39 , 40 ]. Prior LC-MS/MS experiments identified fragment ions b7, b9, b11, y9 as the best ones for detection of GRA1 peptide VERPTGNPDLLK during PRM analysis based on the abundance of these ions.…”

Section: Discussionmentioning

confidence: 99%

Detection of toxoplasmic encephalitis in HIV positive patients in urine with hydrogel nanoparticles

et al. 2021

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Background Diagnosis of toxoplasmic encephalitis (TE) is challenging under the best clinical circumstances. The poor clinical sensitivity of quantitative polymerase chain reaction (qPCR) for Toxoplasma in blood and CSF and the limited availability of molecular diagnostics and imaging technology leaves clinicians in resource-limited settings with few options other than empiric treatment. Methology/principle findings Here we describe proof of concept for a novel urine diagnostics for TE using Poly-N-isoproplyacrylamide nanoparticles dyed with Reactive Blue-221 to concentrate antigens, substantially increasing the limit of detection. After nanoparticle-concentration, a standard western blotting technique with a monoclonal antibody was used for antigen detection. Limit of detection was 7.8pg/ml and 31.3pg/ml of T. gondii antigens GRA1 and SAG1, respectively. To characterize this diagnostic approach, 164 hospitalized HIV-infected patients with neurological symptoms compatible with TE were tested for 1) T. gondii serology (121/147, positive samples/total samples tested), 2) qPCR in cerebrospinal fluid (11/41), 3) qPCR in blood (10/112), and 4) urinary GRA1 (30/164) and SAG1 (12/164). GRA1 appears to be superior to SAG1 for detection of TE antigens in urine. Fifty-one HIV-infected, T. gondii seropositive but asymptomatic persons all tested negative by nanoparticle western blot and blood qPCR, suggesting the test has good specificity for TE for both GRA1 and SAG1. In a subgroup of 44 patients, urine samples were assayed with mass spectrometry parallel-reaction-monitoring (PRM) for the presence of T. gondii antigens. PRM identified antigens in 8 samples, 6 of which were concordant with the urine diagnostic. Conclusion/significances Our results demonstrate nanoparticle technology’s potential for a noninvasive diagnostic test for TE. Moving forward, GRA1 is a promising target for antigen based diagnostics for TE.

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Section: Discussionmentioning

confidence: 99%

Detection of toxoplasmic encephalitis in HIV positive patients in urine with hydrogel nanoparticles

et al. 2021

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“…In Chagas and Lyme disease urine nanoparticle diagnostics, larger volumes of urine increased the sensitivity and specificity at least 10-fold. [38][39][40] Finally, the lack of a true gold standard limits our ability to calculate sensitivity and specificity. CSF qPCR is <70% sensitive, so a definitive diagnosis of TE was impossible in most study participants.…”

Section: Discussionmentioning

confidence: 99%

Detection of Toxoplasmic Encephalitis in HIV Positive Patients in Urine with Hydrogel Nanoparticles

Steinberg

Bowman²,

Diestra

et al. 2020

Preprint

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Diagnosis of toxoplasmic encephalitis (TE) is challenging under the best clinical circumstances. The poor sensitivity of quantitative polymerase chain reaction (qPCR) for Toxoplasma in blood and CSF and the limited availability of molecular diagnostics and imaging technology leaves clinicians in resource-limited settings with few options other than empiric treatment. Here we describe proof of concept for a novel urine diagnostics for TE using Poly-N-isoproplyacrylamide nanoparticles dyed with Reactive Blue-221 to concentrate antigens, substantially increasing the limit of detection. After nanoparticle-concentration, a standard western blotting technique with a monoclonal antibody was used for antigen detection. Limit of detection was 7.8pg/ml and 31.3pg/ml of T. gondii antigens GRA1 and SAG1, respectively. To characterize this diagnostic approach, 164 hospitalized HIV-infected patients with neurological symptoms compatible with TE were tested for 1) T. gondii serology (121/147, positive samples/total samples tested), 2) qPCR in cerebrospinal fluid (11/41), 3) qPCR in blood (10/112), and 4) urinary GRA1 (30/164) and SAG1 (12/164). GRA1 appears to be superior to SAG1 for detection of TE antigens in urine. Fifty-one HIV-infected, T. gondii seropositive but asymptomatic persons all tested negative by nanoparticle western blot and blood qPCR, suggesting the test has good specificity for TE for both GRA1 and SAG1. In a subgroup of 44 patients, urine samples were assayed with mass spectrometry parallel-reaction-monitoring (PRM) for the presence of T. gondii antigens. PRM identified antigens in 8 samples, 6 of which were concordant with the urine diagnostic. Our results demonstrate nanoparticle technology potential for a noninvasive diagnostic test for TE. Moving forward, GRA1 is a promising target for antigen based diagnostics for TE.

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“…In one step, in solution, hydrogel nanoparticles perform molecular size sieving, exclude high abundance proteins, capture classes of proteins based on the dye's functional group properties, and protect the captured proteins from enzymatic degradation [142]. Hydrogel nanoparticles are now commercially available in large quantities with uniform size distribution (NanoTrap ®) for harvesting and concentrating proteins in body fluids [143]. …”

Section: Hydrogel Nanoparticlesmentioning

confidence: 99%

Current state of the art for enhancing urine biomarker discovery

Harpole

Davis

Espina

2016

Expert Review of Proteomics

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Urine is a highly desirable biospecimen for biomarker analysis because it can be collected recurrently by non-invasive techniques, in relatively large volumes. Urine contains cellular elements, biochemicals, and proteins derived from glomerular filtration of plasma, renal tubule excretion, and urogenital tract secretions that reflect, at a given time point, an individual's metabolic and pathophysiologic state. High-resolution mass spectrometry, coupled with state of the art fractionation systems are revealing the plethora of diagnostic/prognostic proteomic information existing within urinary exosomes, glycoproteins, and proteins. Affinity capture pre-processing techniques such as combinatorial peptide ligand libraries and biomarker harvesting hydrogel nanoparticles are enabling measurement/identification of previously undetectable urinary proteins. Future challenges in the urinary proteomics field include a) defining either single or multiple, universally applicable data normalization methods for comparing results within and between individual patients/data sets, and b) defining expected urinary protein levels in healthy individuals.

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Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Cited by 8 publications

References 34 publications

Detection of toxoplasmic encephalitis in HIV positive patients in urine with hydrogel nanoparticles

Detection of toxoplasmic encephalitis in HIV positive patients in urine with hydrogel nanoparticles

Detection of Toxoplasmic Encephalitis in HIV Positive Patients in Urine with Hydrogel Nanoparticles

Current state of the art for enhancing urine biomarker discovery

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