Characterization and immunological determination of the complex between prostate-specific antigen and α2-macroglobulin

Department of Clinical Chemistry, Helsinki University Central Hospital, Helsinki, Finland.
Clinical Chemistry (Impact Factor: 7.91). 01/1999; 44(12):2471-9.
Source: PubMed


Prostate-specific antigen (PSA) rapidly forms a complex with alpha2-macroglobulin (A2M) in vitro; however, PSA complexed with A2M (PSA-A2M) is not detected by conventional immunoassays for PSA because it is encapsulated by the A2M. In this study, we show that denaturation of PSA-A2M at high pH renders PSA immunoreactive. Part of the complexed PSA is released in free form and part remains bound to denatured A2M. These forms can be measured by a conventional immunoassay for PSA. This finding enabled us to design a dissociation assay for the detection of PSA-A2M, which was based on the removal of immunoreactive PSA in serum by immunoadsorption, denaturation of PSA-A2M at high pH, and measurement of the released PSA immunoreactivity by a conventional PSA immunoassay. This PSA-A2M assay was calibrated with PSA-A2M formed in vitro. The detection limit of the assay was 0.14 microg/L. Inter- and intraassay coefficients variation were 4-9% and 8-14%, respectively. When purified PSA was incubated with A2M, the loss of PSA immunoreactivity was highly correlated with the PSA-A2M formed, as measured by the dissociation assay for PSA-A2M (r = 0.99; P <0.0001). The concentration of PSA-A2M in serum correlated with that of total PSA both in prostate cancer (PCa) and benign prostatic hyperplasia (BPH); however, the ratio of PSA-A2M in relation to total PSA was significantly higher in BPH than in PCa (P <0.0003). ROC curve analysis suggested that measurement of the ratio of PSA-A2M to total PSA in serum improves the diagnostic accuracy for PCa compared with assays for total PSA only.

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Available from: Sakari Rannikko, Mar 18, 2014
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    • "Complexes of PSA with API [54] have been shown to occur at a 1:1 ratio and, therefore, should not induce a systemic reaction. Tetrameric A2M can bind two PSA molecules; however, the A2M-PSA complex cannot be detected in the blood by immunoassays since PSA is encapsulated by A2M [55,56]. Therefore, it is expected that the anti-PSA IgE would not be able to bind the encapsulated PSA in this complex and thus, it would not be expected to trigger a systemic anaphylactic reaction. "
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    ABSTRACT: Background Prostate cancer (PCa) is the second leading cause of cancer deaths in men in the United States. The prostate-specific antigen (PSA), often found at high levels in the serum of PCa patients, has been used as a marker for PCa detection and as a target of immunotherapy. The murine IgG1 monoclonal antibody AR47.47, specific for human PSA, has been shown to enhance antigen presentation by human dendritic cells and induce both CD4 and CD8 T-cell activation when complexed with PSA. In this study, we explored the properties of a novel mouse/human chimeric anti-PSA IgE containing the variable regions of AR47.47 as a potential therapy for PCa. Our goal was to take advantage of the unique properties of IgE in order to trigger immune activation against PCa. Methods Binding characteristics of the antibody were determined by ELISA and flow cytometry. In vitro degranulation was determined by the release of β-hexosaminidase from effector cells. In vivo degranulation was monitored in human FcεRIα transgenic mice using the passive cutaneous anaphylaxis assay. These mice were also used for a vaccination study to determine the in vivo anti-cancer effects of this antibody. Significant differences in survival were determined using the Log Rank test. In vitro T-cell activation was studied using human dendritic cells and autologous T cells. Results The anti-PSA IgE, expressed in murine myeloma cells, is properly assembled and secreted, and binds the antigen and FcεRI. In addition, this antibody is capable of triggering effector cell degranulation in vitro and in vivo when artificially cross-linked, but not in the presence of the natural soluble antigen, suggesting that such an interaction will not trigger systemic anaphylaxis. Importantly, the anti-PSA IgE combined with PSA also triggers immune activation in vitro and in vivo and significantly prolongs the survival of human FcεRIα transgenic mice challenged with PSA-expressing tumors in a prophylactic vaccination setting. Conclusions The anti-PSA IgE exhibits the expected biological properties and is capable of triggering immune activation and anti-tumor protection. Further studies on this antibody as a potential PCa therapy are warranted.
    BMC Cancer 04/2013; 13(1):195. DOI:10.1186/1471-2407-13-195 · 3.36 Impact Factor
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    ABSTRACT: Prostate specific antigen is one-chained glicoprotein, consisted of 240 aminoacids, with molecular weight of 33-34 kDa and exhibiting a serin protease activity. In blood circulation a complex PSA-PHT predomi- nates, with 86% relative ratio of the total PSA, whereas only a small fraction is presented by the com- plex PSA-a2-macroglobulin and by free PSA. The complexed form of PSA is in general more stable than the free form. In blood from patients without neo- plastic changes, there is larger amount of free PSA, opposite from patients with malignant disease, where increase of complexed PSA and decrease of the free IZVADOK
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