New Approaches for Detection of PSA

5.1. Identification of Novel PSA-Binding Ligands

Immunoassays are very sensitive and specific facilitating accurate measurement of individual proteins in the presence of more than 1 million-fold excess of other proteins [159]. The use of monoclonal antibodies has made it possible to develop assays that differentiate between variants of the same protein, but apparently because antibodies recognize epitopes on the surface of proteins, recognition of differences in internal structures that are not well exposed is hard to achieve with antibodies. In contrast, small pep-tides are able to recognize differences in such structures and peptides with specific binding properties can be identified with phage display libraries [160, 161].

Phage display is a molecular diversity technology and a powerful approach for selection of novel ligands for target molecules, such as enzymes [162-164], antibodies [165] and receptors [166, 167]. Foreign peptides or proteins are

Fig. 2. Schematic illustration of selection of PSA-binding peptides by phage display.

displayed on the surface of the phage by inserting the displayed genes (DNA fragments or synthetic oligonucleotides) between the DNA encoding the signal peptide and the structural gene encoding major (pVIII) or minor (pIII) coat proteins [168]. This facilitates linking the phenotype of the phage to its genotype [169]. Phage libraries can display over 109 different peptides expressed as a fusion with phage surface proteins and offer a means of searching for ligands possessing unique binding specificities. By the phage display approach, we have identified various peptides binding to PSA (Fig. 2) [160]. A monoclonal antibody to PSA, which does not block the active site of PSA, was used to capture PSA on the solid phase. By this technique, peptides binding specifically to enzymatically active free PSA, but not to internally cleaved or pro forms of free PSA were identified [160, 161]. The peptides do not bind to chymotrypsin or cathepsin G that have an enzymatic specificity similar to that of PSA. They did not bind to hK2, which is structurally closely related to PSA, showing 79% identity at the amino acid level [36]. In addition, the peptides did not bind to PSA-serine protease inhibitor (serpin) complexes occurring in serum, that is, PSA-ACT and PSA-API, in which the serpins

Fig. 3. Principle of immunopeptidometric assay (IPMA) for active free PSA.

cover the active site of PSA. The binding of peptides to PSA was also blocked by antibodies that react with free PSA through epitopes covered in PSA-serpin complexes; these antibodies inhibit the enzymatic activity of PSA [107]. Thus, the peptides are specific for enzymatically active free PSA.

5.2. Immunopeptidometric Assay for Active Free PSA

Immunopeptidometric assay (IPMA) is a new assay principle based on the use of PSA-binding peptides as a tracer in combination with a monoclonal capture antibody. In the assay all forms of PSA are captured by the immobilized monoclonal antibody, but only enzymatically active free PSA is recognized by the PSA-binding peptide (Fig. 3). The IPMA detects enzymat-ically active PSA, but not internally cleaved PSA or proPSA, which are enzymatically inactive. Between 1% and 10% of free PSA in serum from prostate cancer patients is detected by this assay [170]. The finding of active

PSA in patient samples also indicates that the reaction between PSA and inhibitors leaves part of the active PSA free. The IPMA offers a potential approach for detection of cancer-associated forms of PSA, but the sensitivity of the assay needs to be improved in order to detect active PSA at clinically important concentrations, that is, 2-10 yg/liter of total PSA.

5.3. Detection of Protein Analytes by a Nanoparticle-Based Bio-Bar-Code Approach

Nam et al. [171] have reported a nanoparticle-based bio-bar-code method to detect PSA at very low concentrations [171]. This method uses two types of probes, one is a magnetic microparticle coupled to a monoclonal PSA antibody, and the other one is a gold nanoparticle that carries multiple copies of a unique DNA and a polyclonal antibody to PSA. In the assay, PSA is first captured by magnetic microparticle probes, then gold nanoparticle probes are added. The probes and PSA form a complex that can be separated with a magnet. The PSA concentration is measured by detecting the oligonucleotide in the nanoparticle probe. The nanoparticle probe carries a large number of oligonucleotides per PSA molecule and this amplifies the signal. The signal can be further amplified by the polymerase chain reaction. By this approach, PSA could be detected at very low concentrations, that is, 3 attomol/liter, which is about six orders of magnitude more sensitive than that of conventional immunoassays. The method is also suitable for simultaneous detection of multiple analytes. If applicable to analysis of serum samples, this assay might be useful for simultaneous detection of different cancer-associated forms of PSA that are present at very low levels in sera and are not readily detectable by conventional immunoassays.

5.4. Detection of Subfractions of Free PSA by Two-Dimensional Gel Electrophoresis

Charrier et al. [20, 146] have studied free PSA in serum from men with prostate cancer and BPH by two-dimensional gel electrophoresis and showed that BPH sera contain more low-molecular-weight forms of free PSA than prostate cancer sera. Jung et al. [172] reported a method, in which PSA was captured by immunoadsorption on streptavidin-coated magnetic beads with a biotinylated monoclonal antibody to PSA and separated by two-dimensional electrophoresis followed by detection of PSA by Western blotting. They separated and quantified free PSA isoforms in men with total PSA concentrations of 2-20 yg/liter and found 15 forms of free PSA differing with respect to molecular weight and isoelectric point.

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