Research paper
Immunoassay of total prostate-specific antigen using europium(III) nanoparticle labels and streptavidin–biotin technology

https://doi.org/10.1016/j.jim.2004.09.002Get rights and content

Abstract

Nanoparticle labels conjugated with biomolecules are used in a variety of different assay applications. We investigated the possibility of using europium(III)-labeled 68-nm nanoparticles coated with monoclonal antibodies or streptavidin (SA) to detect prostate-specific antigen (PSA) in serum. The selection of a suitable antibody pair and interference caused by the combination of nanoparticle label and structurally complex analyte were of special interest.

A set of antibodies recognizing different epitope areas of PSA was mapped to find the optimal antibody pair for the immunometric nanoparticle-based assay. Different assay configurations were tested to obtain a good correlation with a conventional method based on biotinylated detection antibodies and europium(III) chelate-labeled streptavidin. Monoclonal capture antibody 5E4 was covalently coated on a microtitration well surface; biotinylated 5H6 monoclonal antibody (Mab) was used for detection, and europium(III)-labeled streptavidin-coated nanoparticles were utilized for signal generation. Total PSA concentrations were determined from a panel of male serum samples to test the developed assay. The correlation of the nanoparticle-based and reference assays was good; y=0.9844x−0.1252, R2=0.98, n=27; and the lowest limit of detection of the assay (LLD=0.83 ng/l) was 35-fold lower than for the reference method. The assay application presented here, where a structurally complex analyte is detected, combines the exceptionally high affinity of streptavidin–biotin technology and the high specific activity of long lifetime fluorescence nanoparticle labels. The general characteristics of this combination should permit the development of various immunoassay applications featuring high sensitivity, rapidity, and low consumption of reagents.

Introduction

Conventional immunometric solid-phase assays are being developed to achieve higher sensitivity, shorter total assay times, and lower reagent/sample consumption. A widely used approach to improve the sensitivity of immunoassay is the utilization of indirect detection reagents with high specific activities. Probably, the most popular example of indirect detection reagent-application is the avidin–biotin technology, which is based on the extremely high affinity of avidin and streptavidin (SA) towards biotin (KA 1.7×1015 and 2.5×1013 1/M, respectively; Wilchek and Bayer, 1999). In addition, avidin and SA permit radical chemical modifications without losing the biotin-binding characteristics (Green, 1990, Hermanson, 1996). Applications of SA–biotin technology are diverse. Enzyme-linked assays, where SA is typically conjugated to horseradish peroxidase or alkaline phosphatase, are widely used (Wilchek and Bayer, 1990). The application of direct labeling of SA with europium(III) chelate in a two-site immunoassay was first presented by Chan et al. (1987). In two-site immunoassays, europium(III) chelate-labeled SA is typically utilized for the binding of biotinylated antibodies and signal generation. The indirect labeling of SA with europium(III) chelate-labeled BSA–polypeptide (Glu:Lys) conjugate has enabled further improvements in assay sensitivity. The designed fluorescent conjugate has been suggested as a valuable tool in the development of ultrasensitive immunoassays. (Qin et al., 2001) the spatial proximity of SA–biotin has been applied in fluorescence resonance energy transfer based homogeneous assay models (Alpha-Bazin et al., 2000, Boisclair et al., 2000). Typically, in these models, a donor of fluorescence energy and a respective acceptor are brought close to each other via SA–biotin binding. The SA–biotin system has also been applied in protein- and single-nucleotide polymorphism arrays. The protein-array model utilizes the universal nature of labeled SA in the detection of biotinylated analytes captured on a surface by a set of analyte-specific monoclonal capture antibodies (Luo and Diamandis, 2000). The single-nucleotide polymorphism array utilizes SA as a solid phase capture agent, in which biotin is incorporated in one primer of each PCR primer pair to generate biotinylated PCR products. The biotinylated strands of PCR products are bound to the solid phase and detected with specific reporters (Santacroce et al., 2002). Thus, the possible applications of SA–biotin technology are diverse.

The characteristics and use of SA or monoclonal antibody (Mab)-coated, highly fluorescent europium(III) nanoparticles in immunoassays with the free form of prostate-specific antigen (F-PSA) as a model analyte have been thoroughly described (Härmä et al., 2000, Härmä et al., 2001, Soukka et al., 2001a, Soukka et al., 2001b). The use of SA-coated nanoparticles has improved the sensitivity of the assay due to the high specific activity of label and low nonspecific binding. It has also permitted rapid assays based on the high affinity of SA–biotin complex and on the high number of biotin binding sites per particle (Härmä et al., 2000). An increase in the density of antigen binding sites on the Mab-coated nanoparticles enhanced the reaction kinetics (Soukka et al., 2001b). The association rate constants of nanoparticles increased as a function of the binding site density and exceeded the respective constant of europium(III) chelate-labeled Mab. The dissociation rate constants of nanoparticles were not dependent on the number of binding sites but were still lower than that of the labeled antibody. These changes in the kinetic properties brought an eightfold increase in the affinity constant when the nanoparticle–antibody conjugate and the labeled antibody were compared (Soukka et al., 2001b).

Although nanospheres are used increasingly in different diagnostic applications, e.g., in many qualitative rapid tests, the possible effects on assays caused by the spatial requirements of spherical labels have not been reported. Therefore, we have studied the possibility of using highly fluorescent nanoparticle labels in a sandwich assay, where the analyte is structurally complex. Due to the spatial constraints between the nanoparticle and the analyte, the importance of epitope location on the analyte is also emphasized. Structurally complex α1-antichymotrypsin (ACT)-bound PSA (PSA-ACT) was selected as an analyte because it has been studied thoroughly, it has several well-mapped epitopes, and there are a considerable number of antibodies available (Stenman et al., 1999). The selection of PSA-ACT also ensured the comparability of results between the present study and our earlier work with nanoparticle-label-based F-PSA immunoassays. Furthermore, PSA-ACT has a high clinical importance in prostate cancer diagnostics (Papsidero et al., 1980, Wang et al., 1981), where it is used in combination with F-PSA to measure the total PSA (T-PSA) level of serum and to calculate the percentage of F-PSA (Catalona et al., 1994, Björk et al., 1996).

The aim of the study was to evaluate the possible structural limitations of the T-PSA sandwich assay caused by the particulate labels. We hypothesized that Mab-coated nanoparticles might be unable to reach some epitopes of PSA that are either directly covered by ACT or located in the vicinity of it. Instead, applying the nanoparticle labels in combination with the SA–biotin technology was assumed to overcome the constraints. A set of antibodies recognizing different epitopes of the PSA molecule and diverse nanoparticle-based assay configurations were used to determine the spatial constraints. The results showed that the 68-nm Mab-coated nanoparticles might induce limitations in immunometric sandwich assay design when a structurally complex analyte is detected. However, the limitations were resolved by employing SA-coated nanoparticle labels, together with biotinylated detection antibodies and ultrasensitivity, comparable with the previously presented results of Mab-coated nanoparticle-based assays (Soukka et al., 2003) was obtained. Our results are generally applicable to other particle-based technologies when a structurally complex analyte needs to be determined.

Section snippets

Reagents

Polystyrene nanoparticles, 68 nm in diameter, containing europium(III) chelates, were purchased from Seradyn (Indianapolis, IN). Tween-20 and -85 were from Merck (Darmstadt, Germany), SA was from SPA-Società Prodotti Antibiotici (Milano, Italy), and bovine serum albumin fraction V (BSA) from Bioreba (Nyon, Switzerland). Assay buffer and washing solution were from InnoTrac Diagnostics (Turku, Finland). Europium(III) chelate-labeled SA, DELFIA® Enhancement Solution, DELFIA Enhancer, SA-coated

Antibody selection for the sandwich assay

The epitope nomenclature proposal presented by the ISOBM TD-3 workshop on monoclonal antibodies against prostate-specific antigen (Stenman et al., 1999) was used as a basis for antibody selection. The proposal classified the anti-PSA antibodies into six major groups and placed the respective epitopes on a three-dimensional PSA model presented byVilloutreix et al. (1996; Fig. 2). Here, antibodies from four out of six groups were tested (Table 1): 2E9 and 9E8 (group 2); 5F7, 5H6, and 7G1 (group

Discussion

The use of particles in different assay applications is increasing, as is the number of particle-based assays performed. With particle-based immunoassays becoming more common, the range of analytes also broadens, raising new questions concerning the assay conditions. Here, we have studied the applicability of nanoparticles as labels in an immunometric sandwich assay where the analyte is structurally complex. We describe a model where the use of SA–biotin technology and the selection of a

Acknowledgements

This work was supported by the National Technology Agency of Finland (TEKES).

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