Surface plasmon resonance-based inhibition assay for real-time detection of Cryptosporidium parvum oocyst

Abstract

A surface plasmon resonance (SPR)-based inhibition assay method using a polyclonal anti-mouse IgM arrayed Cryptosporidium sensor chip was developed for the real-time detection of Cryptosporidium parvum oocysts. The Cryptosporidium sensor chip was fabricated by subsequent immobilization of streptavidin and polyclonal anti-mouse IgM (secondary antibody) onto heterogeneous self-assembled monolayers (SAMs). The assay consisted of the immunoreaction step between monoclonal anti-C. parvum oocyst (primary antibody) and oocysts, followed by the binding step of the unbound primary antibody onto the secondary antibody surface. It enhanced not only the immunoreaction yield of the oocysts by batch reaction but also the accessibility of analytes to the chip surface by antibody–antibody interaction. Furthermore, the use of optimum concentration of the primary antibody maximized its binding response on the chip. An inversely linear calibration curve for the oocyst concentration versus SPR signal was obtained in the range of 1×10⁶–1×10² oocysts ml⁻¹. The oocyst detection was also successfully achieved in natural water systems. These results indicate that the SPR-based inhibition assay using the Cryptosporidium sensor chip has high application potential for the real-time analysis of C. parvum oocyst in laboratory and field water monitoring.

Introduction

Cryptosporidium parvum, a protozoan parasite of the mammalian intestinal epithelium, has been responsible for numerous waterborne and foodborne outbreaks of diarrheal disease. Most of these outbreaks were connected with contamination of water supplies or recreational water (Fayer et al., 2000; Carey et al., 2004). C. parvum oocysts, resistant to standard chlorination disinfection (Korich et al., 1990), can survive in aquatic environments for several months (Robertson et al., 1992; Carey et al., 2004). C. parvum infection is initiated by ingestion of oocysts in the intestine, which then undergo excystation to release sporozoites. Attachment of the sporozoites to intestinal mucosal epithelial cells and their subsequent invasion are crucial primary steps in the pathogenesis of cryptosporidiosis (Carey et al., 2004; Deng et al., 2004). According to the research of Okhuysen et al. (1999), the median infective dose (ID₅₀) in healthy human volunteers was calculated to be 87 oocysts for the Iowa calf isolates. Therefore, to minimize the impact of this parasite on human health due to its presence in environmental source water, an accurate detection procedure with a high sensitivity is required to detect and enumerate oocysts (Fayer et al., 2000; Quintero-Betancourt et al., 2002; Carey et al., 2004).

Surface plasmon resonance (SPR) biosensor systems have been widely used for bio-affinity monitoring using protein chip technology. The principle of the SPR biosensor is based on the change in the refractive index on a thin metal film surface modified with various materials. The change of SPR signals is proportional to the refractive index close to the chip surface and is therefore related to the amount of bound analyte. Two major advantages of SPR biosensors over conventional immunosensors such as ELISA methods are that they do not require any labeling or staining of the analytes and that binding can be monitored in real time (Salmon et al., 1997; Homola et al., 1999; Mullett et al., 2000; Choi et al., 2005; Subramanian et al., 2006).

The Biacore biosensor is a widely used powerful sensor using SPR, allowing the real-time detection of biomolecular interaction analysis due to its flow type of sample injection. However, when the flow-type Biacore system was used to detect pathogens, it has disadvantages over protein detection due to their inherently large sizes as follows: (i) SPR signals are weakly generated by poor penetration of cells within the evanescent field and (ii) sufficient binding avidity is required to overcome hydrodynamic resistive forces between cells and the chip surface (Leonard et al., 2004; Kang et al., 2006; Skottrup et al., 2007). Sensitive SPR biosensors for pathogen detection are batch-type sensors which require a certain incubation time for the immobilization of antibody against each pathogen and the binding of pathogens on the antibody layer (Oh et al., 2003, Oh et al., 2004, Oh et al., 2005). Therefore, the development of a novel assay method based on flow-type SPR biosensors is essential for the direct and real-time identification of pathogens. Leonard et al. (2004) proposed a real-time subtractive inhibition assay using a Biacore SPR biosensor for the rapid detection of Listeria monocytogenes, which consisted of a stepwise centrifugation process and free antibody detection, allowed the detection of 1.0×10⁵ cells ml⁻¹.

In this study, we also propose an alternative immunoreaction-mediated inhibition assay method for the rapid detection of Cryptosporidium oocysts in solution based on the Biacore SPR biosensor system. In comparison to Leonard et al. (2004), our method allows for (i) the construction of heterogeneous self-assembled monolayers (SAMs) on the bare gold chip, (ii) the fabrication of the secondary antibody-arrayed Cryptosporidium sensor chip using biotin–streptavidin binding with high affinity on the SAM surface (Fig. 1), and (iii) the maximization of the sensitivity of Cryptosporidium oocyst detection in the flow-type SPR biosensor by optimizing the primary antibody concentration.

Heterogeneous SAM surface reduces the steric hindrance of chip surface due to homogeneous chain length of SAMs, and increases the accessibility of biomolecules to the chip surface. The long-chain carboxylic-terminated thiols are used as a linker to immobilize the target biomaterials and the short-chain hydroxyl-terminated thiols are used as a spacer to construct the accessible chip surface (Mittler-Neher et al., 1995; Patel et al., 1998; Lee et al., 2005; Choi et al., 2005). Moreover, hydroxyl-terminated SAMs are more resistant to the non-specific binding of undesired biological materials than carboxylic-terminated SAMs (Silin et al., 1997; Choi et al., 2005).

Biotin–streptavidin chemistry has been used to improve the orientation of the captured molecules and to reduce the level of non-specific binding (Frey et al., 1995). For this reason, in this study, biotin–streptavidin chemistry was utilized for the immobilization of streptavidin onto the mixed SAMs and the secondary antibody onto the streptavidin layer. During the SPR-based inhibition assay, the immune reaction between the oocysts and the primary antibody was achieved in a short time and the unbound residual antibodies were sequentially separated. The analyte or the residual primary antibody was detected on the Cryptosporidium sensor chip in the flowing buffer system of the Biacore SPR biosensor. We also tested the performance of the Cryptosporidium sensor chip under harsh environments, such as coexistence of the oocyst with other microorganisms and existence of the oocyst in real water systems.