Elsevier

Analytica Chimica Acta

Volume 722, 13 April 2012, Pages 114-118
Analytica Chimica Acta

Chemiluminescence enzyme immunoassay using magnetic nanoparticles for detection of neuron specific enolase in human serum

https://doi.org/10.1016/j.aca.2012.02.007Get rights and content

Abstract

To detect a biomarker for small cell lung carcinoma, neuron specific enolase (NSE), a sensitive and specific chemiluminescence enzyme immunoassay was developed. Fluorescein isothiocyanate (FITC) labeled NSE capture antibody connected with NSE and alkaline phosphatase (ALP) labeled NSE detection antibody in a sandwich-type detection manner. This immune complex was further reacted with anti-FITC coated magnetic beads. In a magnetic field, the complex was enriched, and the sensitivity was thus enhanced. The limit of detection (LOD) of this method was <0.2 ng mL−1. The proposed immunoassay was highly selective, and not interfered by hook effect. The recovery was >83.0% and the coefficient of variation was <10.0%. Human sera from 120 patients were tested with the presented and traditional chemiluminescence enzyme immunoassay. An excellent linear relationship was obtained between two techniques. Overall, this immunoassay offers a promising alternative for NSE detection than traditional clinical examinations.

Highlights

► The FITC labeled NSE capture antibody and ALP labeled NSE detection antibody were prepared to develop a sandwich detection format. ► The immune complex formed in aqueous solution and then bound with anti-FITC immobilized magnetic beads for detection of NSE by chemiluminescence intensity. ► The presented method showed high sensitivity and satisfactory recovery and coefficient of variation. ► A linear relationship was obtained for detection results of 120 patients’ sera by the proposed method and traditional chemiluminescence immunoassay.

Introduction

In recent years, protein biomarkers have shown useful information for early diagnosis and effective treatment of cancer [1], [2]. Neuron specific enolase (NSE) is a glycolytic neurospecific isoenzyme of enolase. It consists of two polypeptide chains, each with molecular weight of 39 kDa. NSE could provide information to cancer course and treatment outcome of small cell lung carcinoma (SCLC) [3] and neuroblastoma [4]. Clinically, NSE level in healthy person is below 15 ng mL−1. High levels (>100 ng mL−1) of NSE indicate the presence of SCLC with high probability [5]. NSE levels in patients with these two diseases could give information about the extent of the disease much earlier than chest X-ray inspection (4–12 weeks earlier). Furthermore, NSE detection is much easier and cost-effective than the current clinical examinations. So, the measurement of NSE in serum has been applied in SCLC and neuroblastoma diagnosis and the examination of the patient's recovery to treatments [6], [7].

Immunoassays hold enormous potentials in diagnostic applications since they are more convenient and cost-effective than traditional analytical techniques [8]. The common immunochemical methods for NSE were radioimmunoassay (RIA) [9] and enzyme-linked immunosorbent assay (ELISA) [10]. The RIA method is sensitive (sensitivity = 3.0 ng mL−1) and reliable, but it requires specialized equipments, sophisticated separation steps and long performance period. The operators may suffer from the harmful effect of radioactive labels. ELISA method is less sensitive (sensitivity = 12.5 ng mL−1) and probably interfered by false negative results, especially for patients with light infection. Therefore, a more sensitive and convenient screening method is required.

The aim of this study was to develop a magnetic bead-based chemiluminescence immunoassay (MPs-CLEIA). This method is advantageous in the increased surface area for connecting antigen or antibody. It possesses the high sensitivity and specificity without using radioactive reagents [11]. MPs-CLEIA technique has been used to rapidly screen several tumor biomarkers before routine clinical examination because of its higher sensitivity and wider dynamic range [12], [13]. Recently, Zhang et al. performed a magnetic beads-based chemiluminescence immunoassay for sequential dual determination of NSE and S100β for diagnosis of ischemic stroke [14]. Capture antibodies of S100β and NSE were coupled to magnetic beads. After connecting with NSE or S100β, the magnetic beads were divided into two parts. The concentration of NSE and S100β was separately determined by further reacting with detection antibodies labeled by ALP and HRP, respectively. In the “sandwich-type” detection strategy, magnetic beads (mean diameter of 1.5 μm) were used, which means the immunoreaction happened in an aqueous suspension. When the magnetic beads were divided for CL signal measurement, the beads in two different parts might be not same, especially in automatic analysis. In addition, the method was not actually tested in the biological samples of patients. In our research, NSE was designed connect with capture antibody and detection antibody in a liquid solution before coupling with magnetic beads, so the stability and reproducibility could be satisfactory for automated separation. The proposed MPs-CLEIA method was further applied to detect NSE in patient serum and the results obtained are in excellent linear relationship with those from traditional CLEIA method.

Section snippets

Materials

Fluorescein isothiocyanate (FITC), luminal, cysteine, bovine serum albumin (BSA), N-Hydroxysuccinimide (NHS), Sephadex G-25 and 2-(N-morpholino)ethanesulfonic acid (MES) were from Sigma–Aldrich (St. Louis, MO, USA). The carboxylated immunomagnetic particles (diameter of 1.0 μm) were purchased from Merck (Beijing, China). Monoclonal capture and detection antibodies for NSE were obtained from HyTest Ltd. (Turku, Finland). Alkaline phosphatase (ALP) was from BBI Enzymes (Wisconsin, USA). Lumigen

Sensitivity, accuracy and repeatability

A sandwich immunoassay was designed. Before binding with FITC antibody, the carboxylated magnetic beads were treated with EDC. As a result, FITC tagged capture antibody bound with the MPs surface after reaction with NSE and detection antibodies. The method was optimized for the concentration of magnetic beads, capture antibody and detection antibody. To optimize amount of magnetic beads, the CL response ratio was calculated according to the CL signal of positive group (300 ng mL−1 NSE) to that of

Conclusions

In this study, a magnetic particle-based chemiluminescence enzyme immunoassay (MPs-CLEIA) was developed and applied to detect NSE in patient sera. The method showed excellent sensitivity (LOD < 0.2 ng mL−1) and high accuracy, stable (CVs < 10%), and specific. When comparing the consistency with the traditional CLEIA method, the MPs-CLEIA method was also accurate and reliable. As a result, the MPs-CLEIA method is advantageous in low cost and easy automation. It is expected to be a promising method for

Acknowledgment

We would like to thank the financial support of National Natural Science Foundation (no. 81173017, no. 31101277), the National High-Tech Research and Development Program of China (863 Program, no. 2010AA10Z402, no. 2007AA06A407), Tianjin Science and Technology Program (no. 09ZCKFSH07500), the Scientists-Company Cooperation Project of the Ministry of Science and Technology of China (SQ2009GJA0002591) and the Fundamental Research Funds for the Central Universities (no. 65011121, no. 65011751).

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