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Intrinsic Bioprobes Inc.

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Trenchevska O.,Intrinsic Bioprobes Inc. | Trenchevska O.,University of Macedonia | Kamcheva E.,Intrinsic Bioprobes Inc. | Nedelkov D.,Intrinsic Bioprobes Inc.
Journal of Proteome Research | Year: 2010

Protein biomarkers are essential in assessing pathogenic processes. The impetus for finding new biomarkers has been accelerated by the arrival of the "omics" technologies. However, equally important is the rediscovery of existing biomarkers with these new approaches as novel variants can be discovered that can improve their utility. Presented here is a mass spectrometric immunoassay method for quantitative determination of β-2-microglobulin, an established biomarker used in the diagnosis of active rheumatoid arthritis and kidney disease, and its structural variant, cleaved at and deficient in lysine-58 (ΔK58-b2m). β-Lactoglobulin was incorporated into the assay as an internal reference standard, serving as normalization point for β-2-microglobulin quantification. The precision, linearity, and recovery characteristics of the assay were established. The new assay was also benchmarked against existing β-2-microglobulin ELISA. The assay was utilized to determine the individual concentration of β-2-microglobulin and its variant across a larger cohort of samples, demonstrating the ability to simultaneously quantify both proteins. © 2010 American Chemical Society.


Nedelkov D.,Intrinsic Bioprobes Inc. | Nedelkov D.,Institute for Population Proteomics
Expert Review of Molecular Diagnostics | Year: 2012

Mass spectrometry-based protein assays hold great promise for in vitro diagnostic testing. Technological advances in mass spectrometry have given rise to instruments and methods that are fully capable of automated and high-throughput protein assaying. Yet, the numerous steps involved in such assays can lead to difficulties in assay characterization and validation, and can also make them unnecessarily complex and prohibitively expensive for everyday use. Simplification of both approaches and instrumentation seems to be the solution to the fast introduction of the mass spectrometry-based assays into the clinical laboratories. One such simplified approach is the mass spectrometric immunoassay, which couples targeted immunoaffinity protein separation with the power of mass spectrometry detection. Several mass spectrometric immunoassays have been extensively characterized and have found their way into clinical laboratory improvement amendments-certified laboratories in the form of laboratory developed tests. Reviewed in this special report is the development and validation of one of those assays - a Cystatin mass spectrometric immunoassay. With the added advantage of protein variant detection and quantification, these assays can redefine our view of protein diversity, with clear implications in biomarker discovery, validation, and ultimately, in vitro diagnostic testing. © 2012 Expert Reviews Ltd.


Kiernan U.A.,Intrinsic Bioprobes Inc. | Phillips D.A.,Intrinsic Bioprobes Inc. | Trenchevska O.,Intrinsic Bioprobes Inc. | Nedelkov D.,Intrinsic Bioprobes Inc.
PLoS ONE | Year: 2011

Background: Retinol Binding Protein 4 (RBP4) is an exciting new biomarker for the determination of insulin resistance and type 2 diabetes. It is known that circulating RBP4 resides in multiple variants which may provide enhanced clinical utility, but conventional immunoassay methods are blind to such differences. A Mass Spectrometric immunoassay (MSIA) technology that can quantitate total RBP4 as well as individual isoforms may provide an enhanced analysis for this biomarker. Methods: RBP4 was isolated and detected from 0.5 uL of human plasma using MSIA technology, for the simultaneous quantification and differentiation of endogenous human RBP4 and its variants. Results: The linear range of the assay was 7.81-500 ug/mL, and the limit of detection and limit of quantification were 3.36 ug/mL and 6.52 ug/mL, respectively. The intra-assay CVs were determined to be 5.1% and the inter-assay CVs were 9.6%. The percent recovery of the RBP4-MSIA ranged from 95 - 105%. Method comparison of the RBP4 MSIA vs the Immun Diagnostik ELISA yielded a Passing & Bablok fit of MSIA = 1.05× ELISA - 3.09, while the Cusum linearity p-value was >0.1 and the mean bias determined by the Altman Bland test was 1.2%. Conclusion: The novel RBP4 MSIA provided a fast, accurate and precise quantitative protein measurement as compared to the standard commercially available ELISA. Moreover, this method also allowed for the detection of RBP4 variants that are present in each sample, which may in the future provide a new dimension in the clinical utility of this biomarker. © 2011 Kiernan et al.


Nedelkov D.,Intrinsic Bioprobes Inc.
Methods in molecular biology (Clifton, N.J.) | Year: 2010

The combination of surface plasmon resonance (SPR) and mass spectrometry (MS) creates a comprehensive protein investigation approach wherein SPR is employed for protein quantification and MS is utilized to structurally characterize the proteins. In such, MS utterly complements the SPR detection and reveals intrinsic protein structural modifications that go unregistered via the SPR detection. Protein complexes and non-specific binding can also be delineated via the SPR-MS approach. Described here are the protocols and know-how for successful and reproducible integration of SPR and MS. The individual steps of the entire SPR-MS process are illustrated via an example showing analysis of myoglobin from human plasma.


Trenchevska O.,Intrinsic Bioprobes Inc. | Nedelkov D.,Intrinsic Bioprobes Inc.
Proteome Science | Year: 2011

Background: Post-translational modifications and genetic variations give rise to protein variants that significantly increase the complexity of the human proteome. Modified proteins also play an important role in biological processes. While sandwich immunoassays are routinely used to determine protein concentrations, they are oblivious to protein variants that may serve as biomarkers with better sensitivity and specificity than their wild-type proteins. Mass spectrometry, coupled to immunoaffinity separations, can provide an efficient mean for simultaneous detection and quantification of protein variants.Results: Presented here is a mass spectrometric immunoassay method for targeted quantitative proteomics analysis of protein modifications. Cystatin C, a cysteine proteinase inhibitor and a potential marker for several pathological processes, was used as a target analyte. An internal reference standard was incorporated into the assay, serving as a normalization point for cystatin C quantification. The precision, linearity, and recovery characteristics of the assay were established. The new assay was also benchmarked against existing cystatin C ELISA. In application, the assay was utilized to determine the individual concentration of several cystatin C variants across a cohort of samples, demonstrating the ability to fully quantify individual forms of post-translationally modified proteins.Conclusions: The mass spectrometric immunoassays can find use in quantifying specific protein modifications, either as a part of a specific protein biomarker discovery/rediscovery effort to delineate the role of these variants in the onset of the disease, progression, and response to therapy, or in a more systematic study to delineate and understand human protein diversity. © 2011 Trenchevska and Nedelkov; licensee BioMed Central Ltd.


Patent
Intrinsic Bioprobes Inc. | Date: 2015-01-30

The present invention is directed to diagnosing, determining, and/or monitoring type 2 diabetes, pre-diabetes, insulin resistance, and their misted conditions by detecting levels and modulations of ApoCIII and its variants. The present invention is also directed to methods for identifying and evaluating therapeutic treatments for type 2 diabetes, pre-diabetes, insulin resistance, and their related conditions by monitoring ApoCIII and its variants.


Patent
Intrinsic Bioprobes Inc. | Date: 2013-11-04

Described is an affinity microcolumn comprising a high surface area material, which has high flow properties and a low dead volume, contained within a housing and having affinity reagents bound to the surface of the high surface area material that are either activated or activatable. The affinity reagents bound to the surface of the affinity microcolumn further comprise affinity receptors for the integration into high throughput analysis of biomolecules.


Patent
Intrinsic Bioprobes Inc. | Date: 2013-02-11

Described is an affinity microcolumn comprising a high surface area material, which has high flow properties and a low dead volume, contained within a housing and having affinity reagents bound to the surface of the high surface area material that are either activated or activatable. The affinity reagents bound to the surface of the affinity microcolumn further comprise affinity receptors for the integration into high throughput analysis of biomolecules.


Patent
Intrinsic Bioprobes Inc. | Date: 2015-09-14

Described is an affinity microcolumn comprising a high surface area material, which has high flow properties and a low dead volume, contained within a housing and having affinity reagents bound to the surface of the high surface area material that are either activated or activatable. The affinity reagents bound to the surface of the affinity microcolumn further comprise affinity receptors for the integration into high throughput analysis of biomolecules.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 373.30K | Year: 2011

This proposal sets forth a precise and rapid methodology for identifying the interaction sites between monoclonal antibodies and proteins. The basis of the system is a high-throughput mass spectrometry-based analysis of epitope determinants to identify theinteracting protein sequences using immunoaffinity capture combined with enzymatic digestion of the target protein; either pre- or post-capture. This combination will be used to rapidly determine the approximate location of each binding epitope. The precise location of the binding epitope will then be determined by immunoaffinity capture and mass spectrometric readout of sets of synthetic ladder peptides that span the approximate epitope established from the initial analysis. The project will develop reproducible methods to demonstrate epitope mapping for monoclonal antibodies of interest to NCI tested by multiple sampling and quantifiable results. Mapped epitopes will be functionally characterized by amino acid substitutions in the epitope sequence using high-throughput spotted array surface plasmon resonance imaging and SPR-Biomolecular Interaction Analysis to identify the key amino acids that are critically involved in binding the antibody to the antigenic epitope. Thus, this approach will provide an efficient and cost effective platform from which to accomplish high-throughput epitope mapping of monoclonal antibodies for use in diagnosis of cancer.

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