Research paper
One-step homogeneous C-reactive protein assay for saliva

https://doi.org/10.1016/j.jim.2011.07.013Get rights and content

Abstract

Background

Cardiovascular disease is the leading cause of death in the world. Human C-reactive protein (CRP) has been used in the risk assessment of coronary events. Human saliva mirrors the body's health and well-being and is non-invasive, easy to collect and ideal for third world countries as well as for large patient screening. The aim was to establish a saliva CRP reference range and to demonstrate the clinical utility of salivary CRP levels in assessing the coronary events in a primary health care setting.

Methods

We have used a homogeneous bead based assay to detect CRP levels in human saliva. We have developed a rapid 15 min (vs 90 min), sequential, one-step assay to detect CRP in saliva. Saliva was collected from healthy volunteers (n = 55, ages 20–70 years) as well as from cardiac patients (n = 28, ages 43–86 years).

Results

The assay incubation time was optimised from 90 min to 15 min and generated a positive correlation (n = 29, range 10–2189 pg/mL, r2 = 0.94; Passing Bablok slope 0.885, Intercept 0, p > 0.10), meaning we could decrease the incubation time and produce equivalent results with confidence. The mean CRP level in the saliva of healthy human volunteers was 285 pg/mL and in cardiac patients was 1680 pg/mL (p < 0.01). Analysis of CRP concentrations in paired serum and saliva samples from cardiac patients gave a positive correlation (r2 = 0.84, p < 0.001) and the salivary CRP concentration capable of distinguishing healthy from diseased patients.

Conclusions

The results suggest that this minimally invasive, rapid and sensitive assay will be useful in large patient screening studies for risk assessment of coronary events.

Highlights

► We have developed 15-min salivary CRP assay compatible for near patient testing. ► Salivary CRP levels are significantly higher in cardiac patients compared to healthy individuals. ► Salivary CRP levels are useful in risk assessment of coronary events.

Introduction

C-reactive protein (CRP) is a marker of inflammation. CRP is a member of the class of acute-phase reactants that mediates innate and adaptive immunity (Chomdej et al., 2004). It is produced by the hepatocytes in response to a variety of inflammatory cytokines (Du Clos, 2000) and may rise rapidly as much as 1000-fold or more after an acute inflammatory stimulus (Black et al., 2004). It rises above normal levels within 6 h, and peaks at 48 h during inflammatory processes. C-reactive protein is the best characterized biomarker of inflammation (Devaraj et al., 2010). Its half-life is stable, and there is no diurnal variation in the blood (Meier-Ewert et al., 2001). Therefore, the levels are mainly determined by the rate of production and the severity of the underlying cause (Mallat et al., 2002, Pepys and Hirschfield, 2003).

CRP has been shown to be an independent predictor of cardiovascular events, and the marker has also been proven to add prognostic value to cardiovascular risk (Ridker et al., 2008) (Wilson et al., 2006). This has been greatly aided by the development of highly sensitive CRP (hsCRP) assays that have enabled reliable detection at low levels. Measurement of serum CRP using a high sensitivity assay can demonstrate subclinical inflammatory states, which may reflect vascular inflammation (Wilson et al., 2006). Current American Heart Association/CDC guidelines identified CRP as the best of all existing inflammatory markers for use in clinical practice (American-Heart-Association, 2010). A recent randomized double blinded trial (Milani et al., 1996, Aydin et al., 2009) “JUPITER” which included over 17,000 “apparently healthy women and men” from 26 countries, revealed that in “apparently healthy” persons without hyperlipidemia but with elevated CRP levels, rosuvastatin [a lipid lowering drug] significantly reduced the incidence of major cardiovascular events.

The mainstream clinical laboratory practice today is to analyse blood (serum or plasma) samples to identify underlying causes for diseases. Although blood samples are collected by persons skilled in the field to minimise adverse outcomes, the practice is still associated with discomfort and risk, and in certain circumstances it is not even possible (i.e. neonates, elderly, obese etc.). The ability to monitor health status, disease onset, progression, and treatment outcomes through a non-invasive method is a most desirable goal in health care promotion and delivery. Human saliva as a diagnostic fluid has gained interest in the last decade (Malamud, 1992, Mandel, 1993a, Mandel, 1993b, Mandel, 1993c, Hofman, 2001, Pink et al., 2009, Schipper et al., 2007, Wong, 2008). Saliva can be considered as a mirror to a person's health and well-being. It is composed of water (99%), electrolytes, immunoglobulins, proteins, enzymes, lipids, and nitrogenous products such as urea and ammonia (Burtis, 1999). The concentrations of the analytes of interest tend to be significantly lower (usually one tenth to one thousandth) in saliva than in serum (Malamud, 1992). Also, the salivary environment is complicated by the presence of contaminants such as mucins, degraded proteins, bacteria, and undigested food particles. However saliva collection is relatively non-invasive and allows for collection of multiple samples a day, and can easily yield sufficient volumes for analysis. This makes it more suitable for collection by personnel with limited training particularly in the third world countries and geographically remote sites.

We have found three studies that have documented the CRP levels in human saliva (Floriano et al., 2009, Dillon et al., 2010, Rao et al., 2010). However, a search of the literature failed to show establishment of reference ranges for salivary concentration or correlations studies between human salivary CRP levels and serum CRP levels if salivary CRP is to be used in a diagnostic setting.

The main goals of this study were to develop a high sensitivity, one-step immunoassay to rapidly quantify CRP levels in human saliva, and to demonstrate that salivary CRP levels can be used for screening patients with elevated blood CRP levels such as in cardiac patients. The results demonstrate the feasibility of salivary CRP levels measurements in large patient screening studies at a primary health care setting.

Section snippets

Participants

This study was approved by the University of Queensland Medical Ethical Institutional Board. All participants gave informed consent before sample collection. All study participants were > 18 years of age. In total 55 healthy volunteers were recruited (age range 18–70 years) for human saliva reference interval establishment. The exclusion criteria for the participants were based on a simple questionnaire asking prospective participants to indicate the existence of any comorbid diseases and oral

Salivary total protein concentrations in the healthy and the cardiac populations

Total protein concentration in the saliva of healthy volunteers 1440 ± 158 (μg/mL) was significantly (p < 0.05) lower for cardiac patients', 2371 ± 209 (μg/mL).

Development of a CRP assay

The assay incubation time was reduced from 90 to 15 min and produced a positive correlation (n = 29, range 10–2189 pg/mL, r2 = 0.94; Passing Bablok slope 0.885, Intercept 0, p > 0.10), meaning we could decrease the incubation time and produced equivalent results with confidence.

In-house developed CRP assay vs Beckman CRP assay

The comparison using serum samples vs the Beckman serum/plasma CRP assay

Discussion

Our study confirmed that the bead based homogeneous assay is a suitable method for measuring salivary CRP concentrations, and our in-house developed method with the reduced incubation time, from 90 to 15 min, provided comparable results. The assay imprecision data provided confidence and our results were reproducible from run to run and the assay limit of detection was 10 pg/mL. Furthermore, the CVs for the human salivary CRP measurements are in agreement with the current commercially available

Acknowledgements

The authors would like to acknowledge the financial support of the Queensland Government Smart Futures Fellowship Programme (QGSFF), and the University of Queensland New Staff Research Funds (UQNSRSF 601252). The authors wish to acknowledge Professor Maree Smith and her team at TetraQ for the access to the Envison plate reader.

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