Western Blotting Analysis of Antibodies to Prostate-Specific Antigen: Specificities for Prostate-Specific Antigen and Prostate-Specific Antigen Fragments

Wang, T.J.; Linton, Harry J.; Sokoloff, Roger L.; Grauer, Lana S.; Rittenhouse, Harry G.; Wolfert, Robert L.

doi:10.1159/000056534

Cited by 6 publications

(4 citation statements)

References 12 publications

(13 reference statements)

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“…In a parallel report for the TD-3 Workshop, we have shown that many PSA epitopes are dependent on conformation [9]. The present study shows that those anti-PSA antibodies recognizing conformation-dependent epitopes are unlikely to exhibit cross-reactivity with hK2.…”

Section: Resultssupporting

confidence: 50%

Western Blotting Analysis of Antibodies to Prostate-Specific Antigen: Cross-Reactivity with Human Kallikrein-2

Wang¹,

Linton²,

Rittenhouse³

et al. 1999

Tumor Biol

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Immunoassays for prostate-specific antigen (PSA) using monoclonal or polyclonal antibodies have clinical applications, such as monitoring and early detection of prostate cancer. However, PSA shares 80% sequence homology with human glandular kallikrein (hK2). In the present study, we have used SDS-PAGE and Western blotting of a recombinant form of hK2 to determine the degree of cross-reactivity in a panel of 83 antibodies submitted to the ISOBM TD-3 PSA Workshop. From this panel, 24 of the 83 antibodies showed cross-reactivity with hK2. The majority of these antibodies showed binding to conformationally independent or linear epitopes. Fourteen of these antibodies appear to map to the same epitope group, indicating the existence of a dominant and linear immunogenic domain shared by PSA and hK2.

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Section: Resultssupporting

confidence: 50%

Western Blotting Analysis of Antibodies to Prostate-Specific Antigen: Cross-Reactivity with Human Kallikrein-2

Wang¹,

Linton²,

Rittenhouse³

et al. 1999

Tumor Biol

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“…We also observe additional minor peaks at a lower and higher MW. We attribute the smaller MW peak to known biological cleavage PSA isoforms 28−31 and/or sample degradation and the larger MW peak to proPSA 28−30 and/or PSA aggregates (Figure 3A). Validation of the μWestern was completed via conventional Western blot.…”

Section: Resultsmentioning

confidence: 99%

Microfluidic Western Blotting of Low-Molecular-Mass Proteins

Gerver

Herr

2014

Anal. Chem.

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We describe a microfluidic Western blot assay (μWestern) using a Tris tricine discontinuous buffer system suitable for analyses of a wide molecular mass range (6.5–116 kDa). The Tris tricine μWestern is completed in an enclosed, straight glass microfluidic channel housing a photopatterned polyacrylamide gel that incorporates a photoactive benzophenone methacrylamide monomer. Upon brief ultraviolet (UV) light exposure, the hydrogel toggles from molecular sieving for size-based separation to a covalent immobilization scaffold for in situ antibody probing. Electrophoresis controls all assay stages, affording purely electronic operation with no pumps or valves needed for fluid control. Electrophoretic introduction of antibody into and along the molecular sieving gel requires that the probe must traverse through (i) a discontinuous gel interface central to the transient isotachophoresis used to achieve high-performance separations and (ii) the full axial length of the separation gel. In-channel antibody probing of small molecular mass species is especially challenging, since the gel must effectively sieve small proteins while permitting effective probing with large-molecular-mass antibodies. To create a well-controlled gel interface, we introduce a fabrication method that relies on a hydrostatic pressure mismatch between the buffer and polymer precursor solution to eliminate the interfacial pore-size control issues that arise when a polymerizing polymer abuts a nonpolymerizing polymer solution. Combined with a new swept antibody probe plug delivery scheme, the Tris tricine μWestern blot enables 40% higher separation resolution as compared to a Tris glycine system, destacking of proteins down to 6.5 kDa, and a 100-fold better signal-to-noise ratio (SNR) for small pore gels, expanding the range of applicable biological targets.

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“…The 9 antibodies in this group show different patterns of reactivity with the 6 isoenzymes [14]. Most of the monoclonal antibodies recognize linear epitopes [26,28,29], although antibody 2H11 (#41) appears to bind a conformational epitope distant from the active site. The antibodies do not react with hK2 [33], but react equally with free and complexed PSA and form good immunoradiometric assays with antibodies from groups 1-4 [25].…”

Section: Footnote To Tablementioning

confidence: 98%

“…In Western blotting studies [27][28][29]31], there was weak reactivity with reduced PSA for two antibodies (92-28464, #68; 5E4, #80), suggesting they may react with linear epitopes. Antibodies 5A10 (#25) and 9B10 (#26), recently shown to recognize linear peptides of PSA [23], showed only weak reactivity with reduced PSA by one workshop group [29].…”

Section: -D Mapping Of Antibody Groupsmentioning

confidence: 98%

Summary Report of the TD-3 Workshop: Characterization of 83 Antibodies against Prostate-Specific Antigen

et al. 1999

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Twelve research groups participated in the ISOBM TD-3 Workshop in which the reactivity and specificity of 83 antibodies against prostate-specific antigen (PSA) were investigated. Using a variety of techniques including cross-inhibition assays, Western blotting, BIAcore, immunoradiometric assays and immunohistochemistry, the antibodies were categorized into six major groups which formed the basis for mapping onto two- and three-dimensional (2-D and 3-D) models of PSA. The overall findings of the TD-3 Workshop are summarized in this report. In agreement with all participating groups, three main antigenic domains were identified: free PSA-specific epitopes located in or close to amino acids 86–91; discontinuous epitopes specific for PSA without human kallikrein (hK2) cross-reactivity located at or close to amino acids 158–163; and continuous or linear epitopes shared between PSA and hK2 located close to amino acids 3–11. In addition, several minor and partly overlapping domains were also identified. Clearly, the characterization of antibodies from this workshop and the location of their epitopes on the 3-D model of PSA illustrate the importance of selecting appropriate antibody pairs for use in immunoassays. It is hoped that these findings and the epitope nomenclature described in this TD-3 Workshop are used as a standard for future evaluation of anti-PSA antibodies.

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Western Blotting Analysis of Antibodies to Prostate-Specific Antigen: Specificities for Prostate-Specific Antigen and Prostate-Specific Antigen Fragments

Cited by 6 publications

References 12 publications

Western Blotting Analysis of Antibodies to Prostate-Specific Antigen: Cross-Reactivity with Human Kallikrein-2

Western Blotting Analysis of Antibodies to Prostate-Specific Antigen: Cross-Reactivity with Human Kallikrein-2

Microfluidic Western Blotting of Low-Molecular-Mass Proteins

Summary Report of the TD-3 Workshop: Characterization of 83 Antibodies against Prostate-Specific Antigen

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