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.

Download full-text


Available from: Sakari Rannikko, Mar 18, 2014
104 Reads
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Prostate cancer (PCa) is the most commonly diagnosed non-skin cancer and second leading cause of cancer-related death of men in developed countries. Measurement of prostate specific antigen (PSA) is a very sensitive method for diagnosing and monitoring of prostate cancer (PCa), but the specificity needs improvement. Measurements of different molecular forms of PSA have been shown to improve differentiation between PCa and benign prostatic diseases. However, accurate measurement of some isoforms has not been achieved in previous assays. The aim of the present study was to develop new assays that reliably measure enzymatically active PSA, PSA-α1-chymotryposin (PSA-ACT) and PSA-α1-protease inhibitor (PSA-API), and to evaluate their diagnostic value. Double-label immunofluorometric assays using a novel monoclonal antibody (MAb) and another antibody to either free PSA (fPSA) or total PSA (tPSA) were developed and used to measure PSA-ACT and fPSA or tPSA at the same time. These assays provide enough sensitivity for measurement of PSA-ACT in sera with low PSA levels. The results obtained confirmed that proportion of PSA-ACT to tPSA (%PSA-ACT) was as useful as proportion of fPSA to tPSA (%fPSA) for discrimination between PCa and benign prostatic hyperplasia (BPH). We developed an immunoassay for detection of PSA-API based on proximity ligation, which improved assay sensitivity 10-fold compared with conventional assays. Our results confirmed previous findings that the PSA-API level is somewhat lower in men with than without PCa, and the combination of %fPSA and proportion of PSA-API to tPSA (%PSA-API) provides diagnostic improvement compared with either method alone. Assays based on this principle should be applicable to other immunoassays in which the nonspecific background is a problem. An immunopeptidometric sandwich assay (IPMA) was developed to measure the enzymatically active PSA. This assay showed high specificity, but sensitivity was not good enough for measurement of PSA concentrations in the gray zone, 2-10 µg/L, in which tPSA does not efficiently differentiate between PCa and BPH. We further developed a solid-phase proximity ligation immunoassay, which provided a 10-fold improvement in sensitivity. This proof of concept study shows that peptides reacting with proteins are potentially useful for sensitive and specific measurement of protein variants for which specific MAbs cannot be obtained. Prostataspesifisen antigeenin (PSA) määrittäminen seerumista on herkkä menetelmä eturauhassyövän diagnostiikkaan, mutta se ei sovellu hyvin eturauhassyövän ja eturauhasen hyvänlaatuisen liikakasvun erottamiseen. Spesifisyyttä on pystytty lisäämään erilaisten PSA-muotojen mittaamisella. Kuitenkaan useiden PSA-muotojen luotettava mittaaminen ei ole ollut mahdollista aikaisemmilla menetelmillä. Tutkimukseni tarkoituksena on ollut kehittää uusia määritysmenetelmiä näiden PSA-muotojen määrittämiseksi [entsymaattisesti aktiivinen PSA, sekä PSA-α1-kymotrypsiini (PSA-ACT) ja PSA-α1-proteaasi inhibiittori (PSA-API) kompleksit]. Lisäksi tutkin näiden menetelmien diagnostista merkitystä. Tuotimme uusia vasta-aineita ja pystytin herkkiä määritysmenetelmiä PSA-ACT ja PSA-API kompleksien mittaamiseksi. Kehittämilläni menetelmillä voidaan PSA-ACT:n määrityksen kanssa samanaikaisesti mitata vapaata PSA:ta tai kokonais PSA:ta. Tästä on etua mm. määritettäessä PSA-ACT:n suhdetta kokonais PSA:han, minkä havaittiin myös tässä tutkimuksessa olevan eturauhassyövän diagnostiikassa yhtä hyödyllinen kuin vapaan PSA:n suhde kokonais PSA:han ja huomattavasti parempi kuin pelkkä kokonais PSA:n mittaus. PSA-API kompleksin mittaamiseksi käytettiin ns. läheisyys-ligaatioon perustuvaa immunomääritystä, jossa vasta-aineiden tulee sitoutua toistensa lähelle ja signaali saadaan aikaan DNA:n monistamiseen perustuvalla menetelmällä. Tällä tavoin saavutettiin 10 kertaa parempi herkkyys kuin perinteisillä menetelmillä ja päästiin eroon aiempia menetelmiä vaivanneesta korkeasta taustasta. Kliiniset tuloksemme vahvistavat aikaisemmat löydökset, joiden mukaan PSA-API tasot ovat jonkin verran korkeammat miehillä, joilla on eturauhassyöpä. Menetelmä soveltunee myös muiden immunomääritysten parantamiseksi. Kehitin myös kaksi immunopeptidometristä määritysmenetelmää, joissa PSA:n tunnistamiseen käytettiin aktiiviseen PSA:han sitoutuvaa peptidiä. Ensimmäinen menetelmä oli spesifinen, muttei tarpeeksi herkkä. Parantaakseni herkkyyttä kehitin läheisyys-ligaatio menetelmän, jossa peptidi ja kaksi vasta-ainetta tunnistavat PSA:n. Tämä menetelmä paransi herkkyyttä 10-kertaisesti. Tämä uuteen periaatteeseen perustuva menetelmä osoittaa että peptidejä voidaan käyttää herkkiin ja spesifeihin määritysmenetelmiin, myös mitattaessa sellaisia proteiini-variantteja, joita vastaan on vaikea kehittää vasta-aineita.
  • [Show abstract] [Hide abstract]
    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
Show more