Development of a highly sensitive chemiluminescence enzyme immunoassay using enhanced luminol as substrate

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<ul><li><p>Research article</p><p>Received: 3 February 2013, Revised: 10 April 2013, Accepted: 7 May 2013 Published online in Wiley Online Library: 20 June 2013</p><p>(wileyonlinelibrary.com) DOI 10.1002/bio.2544</p><p>Development of a highly sensitivechemiluminescence enzyme immunoassayusing enhanced luminol as substrateXiaoqi Tao,a Wenjun Wang,b Zhanhui Wang,a Xingyuan Cao,a Jinghui Zhu,a</p><p>Lanlan Niu,b Xiaoping Wu,b Haiyang Jianga* and Jianzhong Shena*</p><p>ABSTRACT: In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancerswas developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed byhorseradish peroxidase (HRP-C) in the presence of 3-(10-phenothiazinyl) propane-1-sulfonate (SPTZ) and 4-morpholinopyridine(MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33pg/well, whichis lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail cansignificantly increase the light output of HRP-catalyzed ECR, which can be translated into a corresponding improvement insensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR ofN-azoles as secondary enhancer was also presented. Copyright 2013 John Wiley &amp; Sons, Ltd.</p><p>Keywords: 3-(10-phenothiazinyl) propane-1-sulfonate; 4-morpholinopyridine; horseradish peroxidase; chemiluminescence enzymeimmunoassay; chloramphenicol</p><p>* Correspondence to: Jianzhong Shen, Department of Pharmacology andToxicology, College of Veterinary Medicine, China Agricultural University,Yuan Ming Yuan West Road NO.2, Beijing 100193, China. Tel: +86 01062732803; Fax: +86 010 62731032. E-mail: sjz@cau.edu.cn</p><p>Haiyang Jiang, Department of Pharmacology and Toxicology, College ofVeterinary Medicine, China Agricultural University, Yuan Ming Yuan WestRoad NO.2, Beijing 100193, China. Tel: +86 010 62732802; Fax: +86 01062731032. E-mail: haiyang@cau.edu.cn</p><p>Xiaoqi Tao and Wenjun Wang contributed equally to this work.</p><p>a Department of Pharmacology and Toxicology, College of VeterinaryMedicine, China Agricultural University, Beijing100193, China</p><p>b Beijing WDWK Biotech Co., Ltd., Beijing100085, China</p><p>30</p><p>IntroductionThe horseradish peroxidase (HRP)-catalyzed chemiluminescentoxidation of luminol is used widely in bioanalytical methods,such as western blots, immunohistochemistry and chemilumi-nescent enzyme immunoassays (CLEIA). A chemiluminescence(CL) assay is often more sensitive than other methods (1,2) andmuch effort has been made to further improve its efficiencyand analytical performance. In particular, the addition ofenhancers to the reaction substrate greatly increases lightoutput and duration kinetics (3). Luminol oxidation leads tothe formation of a 3-aminophthalate ion in an excited state,which emits light on returning to the ground state (Fig. 1,reaction 10). The emission spectrum shows a maximumwavelength at 425 nm (4). The mechanism of the enhancedchemiluminescence reaction (ECR) has been described inprevious reports (5,6), in which luminol and an enhancer wereoxidized simultaneously. During the first step of ECR, theenhancer molecule, which is a better substrate for HRP thanluminol, is oxidized by hydrogen peroxide in the presence ofHRP according to the ping-pong mechanism (Fig. 1).</p><p>Several analytes have been studied as enhancers for theluminolperoxidase system, including phenolic and aminederivatives (79), indophenols (10), 4-phenylylboronic acid (11),4-methoxyphenol, 4-hydroxy-biphenyl, 4-(1H-pyrrol-1-yl)-phenol(12) and different polymers (13). Use of 3-(10-phenothiazinyl)propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP)(Fig. 2) as primary and secondary enhancers, respectively,allowed the development of a sensitive CL method for thedetermination of different plant peroxidases (1417). Subsequently,ECR with the above-mentioned enhancers was successfully usedin the development of ultrasensitive immunochemical methodsfor the determination of human thyroglobulin, ochratoxin A and</p><p>Luminescence 2014; 29: 301306 Copyright 2013 John</p><p>bacterium Yersinia enterocolitica (16,18,19). Although previousstudies optimized the ECR conditions and obtained good sensitivity(1417), follow-up stability, significant for a mature product, wasnot determined.Chloramphenicol (CAP) is an effective broad-spectrum</p><p>antibiotic which was widely used in both human and veterinarypractice for the prevention and treatment of many bacterialinfections. However, CAP is a hemotoxic substance for humansand can cause bone marrow depression, aplastic anemia andacute leukemia (20). These potential hazards led to a prohibitionon its use in food-producing animals in many countries,including China, the USA and the EU (21,22). However, CAP is stillillegally used as an antibiotic in animal husbandry because of itslow cost and excellent antibacterial effect. Therefore, there is anurgent need to develop a rapid and sensitive method for thedetermination of CAP at trace levels in animal-derived food. In aprevious study, we developed a competitive direct chemiluminescent</p><p>Wiley &amp; Sons, Ltd.</p><p>1</p></li><li><p>Figure 1. HRP catalyzed the oxidation of luminol by the ping-pong mechanismwith the enhancer.</p><p>Figure 2. Chemical structures of 3-(10-phenothiazinyl)ropane-1-sulfonate (SPTZ)(a) and 4-morpholinopyridine (MORP) (b).</p><p>X. Tao et al.</p><p>302</p><p>enzyme-liked immunosorbent assay (CL-ELISA) for detecting CAPresidues in milk, milk powder, honey, eggs and chicken muscle usingthe SuperSignal CL substrate (23).</p><p>Under optimized conditions, a highly sensitive CLEIA for thedetermination of CAP in milk was developed. This was basedon the enzymatic oxidation of luminol by sodium peroxide inthe presence of SPTZ and MORP as enhancers and HRP-C asthe biocatalyst. The obtained results (low detection limit ofCAP, good stability, etc.) demonstrated that this establishedmethod gave a significant improvement in the sensitivity of CLdetermination of HRP-C activity.</p><p>Materials and methods</p><p>Apparatus</p><p>The CL reader, Veritas Microplate Luminometer was from TurnerBioSystems (Sunnyvale, CA, USA). White opaque microplates (highbindinggrade; Costar,Washington, DC, USA)were used for the CLEIAassay together with a Milli-Q system (Millipore, Billerica, MA, USA).</p><p>Reagents</p><p>Standards were as follows: CAP (99% purity, Sigma Aldrich, St.Louis, MO, USA); luminol sodium (98.3% purity), SPTZ (98.0%purity) and MORP (97% purity) were purchased from SeebioBiotech, Inc (Shanghai, China). HRP (RZ 3.0) was purchasedfrom Sigma. Imidazole (99% purity), 1,2,3-triazole (99% purity),1,2,4-triazole (99% purity), 1-methylimidazole (99% purity),</p><p>Copyright 2013 Johnwileyonlinelibrary.com/journal/luminescence</p><p>imidazole (99% purity), sodium perborate (NaBO3, 98% purity)and 30% H2O2 were purchased from Aladdin (Shanghai, China).All other compounds were obtained from commercial sourcesand used without further purification. Other chemicals andorganic solvents were of reagent grade and were from BeijingChemical Co. (Beijing, China).</p><p>The SuperSignal CL substrate solution was purchased fromPierce (Rockford, IL, USA).</p><p>The polyclonal anti-CAP serum (PAb) antibody was obtainedfrom WDWK Biotech Co. (Beijing, China).</p><p>Buffers</p><p>The following buffers and solutions were used in CLEIA assay:(a) coating buffer (CB, pH 9.6) 0.05M carbonate buffer, madewith 1.59 g Na2CO3 and 2.93 g NaHCO3 in 1 L of purified water;(b) blocking buffer 0.01M PBS containing 0.5% casein;(c) washing solution (PBST) 0.01M PBS containing 0.05%Tween-20; (d) 0.2M sodium phosphate solution (pH 7.2)containing 11.0 g NaH2PO4 2H2O, 51.6 g Na2HPO4 12H2O in1 L of purified water; (e) PBS (pH 7.4) 0.01M PBS was preparedby dissolving 8.0 g NaCl, 0.2 g KCl, 0.24 g KH2PO4 and 3.63 gNa2HPO4 12H2O in 1 L of purified water; (f) solution A 0.36MK4Fe(CN)6 3H2O, solution B 1.04M ZnSO4 7H2O. All bufferswere prepared using MilliQ H2O (18 M/cm).</p><p>Catalytic luminol oxidation condition</p><p>Catalytic luminol oxidationwas assayed as follows: 0.050.3mmol/Lluminol, 0.93.1mmol/L SPTZ, 0.525.0mmol/L MORP and1.03.0mmol/L sodium peroxide (both reagents were prelim-inarily dissolved in Tris buffer at the relevant pH and concen-tration) were prepared with 10100mmol/L Tris, pH 8.09.0for the enzyme immunoassay. The enzymatic reaction wasinitiated by adding 10 mL of peroxidase solution (2 mg/mL)dissolved in Tris at relevant pH and concentration. CL kineticswas measured at 425 nm with a CL reader, 3min after theaddition of the substrate at room temperature; the resultswere expressed in relative light units (RLU). The CL signalformed in the absence of enzyme was used as a control.</p><p>Effect of nucleophilic acylation and N-azoles catalysts onSPTZ-enhanced substrates</p><p>The working solution was freshly prepared with the followingconcentrations: 0.17mM luminol sodium salt, 2.1mM SPTZ and2mM sodium perborate in 0.10M Tris (pH 8.5). This solutionwas split into portions, and the following compounds wereadded to each portion to a series concentration: (a) MORP(0.525.0mM), (b) 1,2,3-triazole (385mM), (c) 1,2,4-triazole(1.585mM), (d) imidazole (1.575mM), (e) 1-methylimidazole(1.575mM), and (f) reference (without enhancer). The enzymeaddition protocol described above was used.</p><p>Light emission from working solutions containing MORP:effect of pH</p><p>Working solutions of MORP were freshly prepared. The MORPworking solution contained 0.17mM luminol sodium salt,2.1mM SPTZ, 1.5mM MORP and 2mM sodium perborate in0.10M Tris (pH 8.5). The pH for each working solution wasadjusted with negligible amounts of HCl (1M) or NaOH (1M) in</p><p>Luminescence 2014; 29: 301306Wiley &amp; Sons, Ltd.</p></li><li><p>A high sensitivity CLEIA based on novelty enhancers</p><p>the interval pH 89. The enzyme addition protocol describedabove was used.</p><p>Standard curve for HRP</p><p>Ten microliters of HRP diluted in Tris buffer (0.1M, pH 8.5) in therange 0100 pg/well were analyzed in a microplate usingalternatively NoMORP, MORP and SuperSignal working solutions(100 mL) prepared as described above.</p><p>Application of the MORPSPTZluminol CL reaction in adirect competitive CLEIA</p><p>A direct competitive CLEIA for CAP previously developed inour laboratory (23) was used to evaluate the analyticalperformance of the new CL cocktail. Briefly, high-bindingwhite plates were coated overnight at 4 C with 100 mL ofthe polyclonal anti-CAP serum (PAb) dissolved in buffer a(1.5 mg/mL). The plates were washed with 260 mL/well of bufferc manually three times, blocked with 150 mL/well of buffer band incubated at 37 C for 1 h. After the plates were washedas described above (conditioned ELISA plates can be storedat 4 C for one week), then 80 mL/well of standard in buffer dor sample solution, followed by 20 mL/well of HRP-conjugatedCAP at a dilution of 1/160 000 in buffer d were added, respec-tively. The competitive reaction took place for 15min at roomtemperature. After washing five times, the HRP tracer activitywas revealed by adding 100 mL/well of a freshly preparedsubstrate mixture of SuperSignal substrate solution or MORPSPTZluminol CL cocktail reaction. The intensity of lightemission was measured at 425 nm with a CL reader, 3min afteraddition of the substrate and the results were expressed in RLU.Calibration curves were obtained by plotting B/B0 againstthe logarithm of analyte concentration and fitted to a four-parameter logistic equation using Origin (v. 8.0; Microcal,Northampton, MA, USA) software packages.</p><p>30</p><p>Milk sample preparation</p><p>For extraction of CAP from milk, 500mL of solution A and and500mL of solution B were added to 10mL of milk, mixed thor-oughly and then centrifuged for 10min at 4000g and 4 C. Analiquot (4.4mL) of aqueous supernatant (amount to 4mL milk)was thoroughly mixed with 8.0mL of ethyl acetate for 10minin a new tube. Following centrifugation at 4000 g for 10min,4mL of organic supernatant (amount to 2mL milk) wastransferred to a new tube and dried by nitrogen at 60 C. Theresidue was dissolved in 2mL of buffer d. The sample solutionwas used for determination.</p><p>Results and discussion</p><p>Ping-pong mechanism</p><p>Use of SPTZ and MORP as primary and secondary enhancers,respectively, allowed the development of the sensitivechemiluminescent method for the determination of horseradishperoxidase; which catalyzed the oxidation of luminol by theping-pong mechanism (Fig. 1). According to the mechanism,the native Fe(III) enzyme (HRP) is oxidized by peroxide to HRP-Iin a two-electron oxidation (Fig. 1, reaction 1). HRP-I then returnsto its native state, HRP, by reaction with the primary enhancer (E)</p><p>Luminescence 2014; 29: 301306 Copyright 2013 John</p><p>or luminol anion (LH) in two one-electron transfer steps,with HRP-II as an intermediate (Fig. 1, reactions 2 and 4).Subsequently, HRP-II oxidized another primary enhancer (E) orluminol anion (LH), returning to its native state in which it willparticipate in another oxidization cycle (Fig. 1, reactions 3 and5). In each of these steps, the primary enhancer (E) or luminolanion (LH) is oxidized to its radical form (E). The reaction ofluminol with HRP-II is ~ 100-fold less rapid than the reaction ofluminol with HRP-I. Luminescence enhancers react more rapidlywith HRP-II than luminol does, thereby accelerating enzymeturnover. In the next step, the enhancer radical E oxidizesluminol anion LH to the key intermediate L (Fig. 1, reaction 6).Dismutation of L regenerates LH while producing atwo-electron oxidized luminol species, a diazaquinone (L) (Fig. 1,reaction 7). The diazaquinone is then attacked by hydrogenperoxide anion HO2</p><p> (Fig. 1, reaction 8). An intermediateperoxide species is formed and then collapses, with loss ofnitrogen, to 3-aminophthalate in its excited state, AP* (Fig. 1,reaction 9). The decay of AP* to aminophthalate, AP, isresponsible for the chemiluminescent light emission at 425nm(Fig. 1, reaction 9).</p><p>Optimization of catalytic luminol oxidation</p><p>In order to get the lowest value of the lower detection limit (LDL)(i.e. the HRP concentration required for CL to be twice that of thesame solution without HRP), concentrations of luminol, sodiumperoxide, SPTZ and Tris as well as the pH of the reaction mixturewere optimized. In each case, the ratios of peroxidase-catalyzedCL to background were determined (Figs 3,4). The concentrationof MORP was also optimized (Fig. 5). The most favorableconditions for HRP were 100mM Tris buffer (pH 8.5) containing0.17mM luminol, 2mM NaBO3, 2.1mM SPTZ and 1.5mM MORP.In the presence of SPTZ combined with MORP, HRP-induced CLreached a maximum value at 3min after the initiation of luminoloxidation and then decreased slowly (Fig. 6).Actually, signal enhancement for MORP is quite strong even at</p><p>very low concentrations. When the concentration is 1.5mM, thevalue of CL reaches a maximum. There is a sudden decrease inCL signal intensity with a further increase in the concentrationof MORP. By contrast, N-azoles either decrease their enhancedeffect slowly (imidazole) or reach a stable level at a very highconcentration (1-methy...</p></li></ul>

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