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Analytica Chimica Acta 798 (2013) 89– 96

Contents lists available at ScienceDirect

Analytica Chimica Acta

j ourna l ho mepage: www.elsev ier .com/ locate /aca

agneto immunofluorescence assay for diagnosis of celiac disease

ilvina V. Kergaravata, Luis Beltraminob, Nidia Garnerob, Liliana Trottac,arta Wagenerc, Silvia N. Fabianoa, Maria Isabel Pividorid, Silvia R. Hernandeza,∗

Laboratorio de Sensores y Biosensores, Cátedra de Química Analítica I, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral,000 Santa Fe, ArgentinaSección Serología e Inmunología del Laboratorio Central, Hospital Iturraspe, 3000 Santa Fe, ArgentinaServicio Gastroenterología y Nutrición, Hospital Dr. O. Alassia, 3000 Santa Fe, ArgentinaGrup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Catalonia, Spain

i g h l i g h t s

A magneto immunoassay for thedetection of anti-transglutaminaseantibodies (ATG2).The ATG2 were captured by trans-glutaminase enzyme immobilized onmagnetic beads.The fluorescent response of the reve-lation system was related to ATG2.The novel assay can discriminate cor-rectly between celiac and non-celiacpatients.The novel assay is four times moresensitive than the commercial ELISAassay.

g r a p h i c a l a b s t r a c t

r t i c l e i n f o

rticle history:eceived 18 January 2013eceived in revised form 2 September 2013ccepted 5 September 2013vailable online 10 September 2013

a b s t r a c t

A magneto immunofluorescence assay for the detection of anti-transglutaminase antibodies (ATG2) inceliac disease was developed. The ATG2 were recognized by transglutaminase enzyme immobilized on themagnetic beads and then the immunological reaction was revealed by antibodies labeled with peroxidase.The fluorescent response of the enzymatic reaction with o-phenylenediamine and H2O2 as substrates wascorrelated with anti-transglutaminase titer, showing EC50 and LOD values of 1:11,600 and 1:74,500 ofantibody titers, respectively. A total number of 29 sera samples from clinically confirmed cases of celiac

eywords:nti-transglutaminase antibodiesagnetic beads

luorescence responseLISAeliac disease

disease and 19 negative control samples were tested by the novel magneto immunofluorescence assay.The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicatedthat 8.1 U was the most effective cut-off value to discriminate correctly between celiac and non-celiacpatients. The immunofluorescence assay exhibited a sensitivity of 96.6%, a specificity of 89.5% and anefficiency 93.8% compared with the commercial optical ELISA kit.

eceiver-operating characteristic

∗ Corresponding author. Tel.: +54 0342 4575205; fax: +54 0342 4575205.E-mail addresses: shernand@fbcb.unl.edu.ar, shernand22@yahoo.com.ar

S.R. Hernandez).

003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.aca.2013.09.009

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Celiac disease (CD) is one of the most prevalent geneticallydetermined clinical conditions. It is characterized by an exces-

sive immunological reaction against dietary gluten, which startsin the small intestine [1]. Even though the disease was consideredrelatively rare in most European countries, the availability of sensi-tive noninvasive serological tests has allowed detecting the celiac

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isease in the general population nowadays. Most tests indicate arevalence of 1 celiac disease patient for each 200 healthy indi-iduals [2]. On the order hand, the prevalence is high in Americanountries whose population has European ancestors [3]. The anti-ndomysium antibodies (AEA) of IgA isotype are usually used aserological CD markers due to their highly specificity. In 1997, theransglutaminase (TG2) was identified by Dieterich [4] as the majorarget antigen recognized by AEA in patients with untreated CD5]. Several studies demonstrate good correlation between anti-G2 antibodies of IgA isotype (IgA-ATG2) and IgA-AEA [6,7]. SinceTG2 recognize the same antigen as anti-endomysium antibod-

es, they only can be differentiated in terms of detection method.gA-AEA antibodies are tested by the indirect immunofluorescenceIIF) method on cryostatic sections of monkey esophagus and this

ethod has historically been considered as the gold standard sero-ogic assay for CD. These autoantibodies recognize an intermyofibrilubstance found in primate smooth muscle connective tissue. Theissue of choice for detection of endomysial antibodies is monkeysophagus. On the other hand, ATG2 measurement by (an enzyme-inked immunosorbent assay) ELISA is easier and it is objectivelynterpreted therefore most laboratories have opted to perform thisssay rather than the endomysial antibodies by IIF. Therefore, theommon methodology for the diagnostic of CD is the IgA-ATG2etection by ELISA with optical detection in the VIS region [8,9].ince the IgA-AEA test is highly specific, but less sensitive than anti-gA-TG2 ELISA, IIF method should therefore preferably be used as aonfirmation test and not be used as a routine method [10].

Lately electrochemical immunosensors based on the capture ofTG2 by immobilization of TG2 onto electrode surface and mag-etic beads (MB) were reported [11–13].

Although it is well known that fluorescence spectroscopy isore sensitive than UV–vis spectroscopy, an ELISA with fluorescent

etection for quantification of ATG2 has not been reported yet [14].he most common system for revealing of immunological event inLISA is based on the enzymatic reaction of horseradish peroxi-ase (HRP) in presence of H2O2 as substrate and a cosubstrate. Inhe family of fluorescent cosubstrates we can name HPPA (3-(p-ydroxyphenyl)propionic acid) [15], OPD (o-phenylenediamine)14,16] or TMB (3,3′,5,5′-tetrametylbenzidine) [17].

Recently numerous reports related to magnetic bead-basedmmunoassays for different analytes, were reported; but not all

ethods comprise the use of a disposable microplate and have theame characteristics as format, kind of magnetic beads, detectionechnique, label and immobilization procedure. Therefore, each

ethod is a different one with different analytical performance (ashown in Supplementary Material, Table i). The use of magneticeads coupled to the disposable 96-well plate to improve the sen-itivity of the fluorescent detection ELISA systems and reduces theime of the pre-enrichment phase are also used [17,18]. In addition,he matrix effect is minimized in complex samples due to the facthat the washing and separation steps are improved [19,20].

According to our knowledge, no study has focused on the appli-ation of magnetic bead-based immunoassay for the ATG2 withuorescence detection. Therefore, the present work proposed theevelopment of an immunoassay of this kind. The immunologicaleaction is performed on MB as solid support in which the TG2 isovalently immobilized. The biorecognition strategy is based on anndirect assay in two steps. In the first step, the ATG2 are added toG2-MB and in the second step, the antiIg-HRP conjugate is incor-orated for the revelation. The modified magnetic beads are thenaptured by a magnet located at the bottom of microplates. Theubsequent detection is achieved through the response caused by

he HRP-catalyzed reaction with H2O2 as substrate and OPD orMB as cosubstrate. The performance of the ELISA with fluores-ent detection is reported. Blood serum samples were evaluatedy this methodology and the optimum cut-off value of ATG2 was

mica Acta 798 (2013) 89– 96

calculated by ROC analysis. Then, the results were correlated withthe ones obtained by commercial optical ELISA kit.

2. Materials and methods

2.1. Chemicals and immunochemicals

Magnetic beads (diameter = 2.8 �m) modified with tosyl groups(Dynabeads MyOneTM Tosylactivated, Cat. no. 655.01) werepurchased from Invitrogen Dynal AS (Oslo, Norway). Transgluta-minase type II (TG2) from Guinea Pig Liver was purchased fromSigma–Aldrich (Cat. n◦ T5398-2UN and T5398-1UN) and stock solu-tions were prepared in phosphate buffer 0.01 mol L−1 and NaCl0.15 mol L−1 at pH 7.4 and stored at −15 ◦C until used. Bovine serumalbumin (BSA), anti-transglutaminase type II antibody (whole anti-serum, developed in goat) (T7066), anti-goat IgG (whole molecule)Peroxidase (antibody developed in rabbit) (A5420) and anti-humanIgA (alpha-chain specific) – Peroxidase (antibody produced ingoat) (A0295) were purchased from Sigma-Aldrich. Commercialanti-TG2 isotype IgA ELISA detection kit (QUANTA Lite® 708760INOVA Diagnostics, Inc.) was supplied by Ianus s.a. Bradfordsolution (Coomasie Bradford Assay Kit, Cat.n◦ 23200) and the per-oxide, Triton X-100, TMB (3,3′,5,5′-tetrametylbenzidine) and OPD(o-phenylenediamine) solutions utilized for the optical and fluo-rescent measurements were purchased from Sigma.

All aqueous solutions were prepared with milliQ water. Thecomposition of the solutions used for the immobilization stepswere: coating buffer (0.1 M sodium borate, pH 9.5); PBST (0.01 Mphosphate buffer, 0.15 M NaCl, 0.05% v/v Tween 20, pH 7.4); block-ing and washing buffer (PBST with 0.5% and 0.1% w/v of BSA added,respectively); storage buffer (PBST with 0.1% w/v of BSA and 0.02%w/v of sodium azide added). The OPD–H2O2 and Triton X-100 solu-tions were prepared in citric-phosphate buffer 0.1 mol L−1 pH 5.0while the TMB-H2O2 solution was dissolved in citric-phosphatebuffer 0.1 mol L−1 with dimetilsulfoxide 1% (v/v) at pH 4.2.

The antigenic proteins were covalently coupled to tosyl-activated magnetic beads by the reaction between the amine orsulfhydryl amino acids residues of the protein and the tosyl groupson the beads surface [21]. The binding of TG2 to tosyl-activatedmagnetic beads was performed using 10 mg mL−1 TG2 stock solu-tion, as detailed explained in Supplementary Material. The beadswere finally resuspended in storage buffer to reach a 25 mg mL−1

stock solution, and stored at 4 ◦C. Before each assay, TG2-MB werewashed (3×) with PBST and resuspended to a final volume in orderto obtain the desired concentration of magnetic beads.

After the immobilization the efficiency of the coupling strate-gies was evaluated by the Bradford test [22], analyzing the proteinconcentration in the supernatant before and after the immobiliza-tion. The assay was performed by comparing the samples with astandard curve obtained with bovine serum albumin (BSA) and byanalyzing two standard solutions of TG2 in the same experiment, asdetailed described in Supplementary Material. Finally, the stabilityof the TG2 modified MB was also studied following a weekly basisover a 1-month period, as described in Supplementary Material.

2.2. Instrumentation

Both the TG2 immobilization on the magnetic beads and theincubations and washing steps were carried out using an IKA MS3basic rotator (Model SA0741724) with controlled rotation. Blackand white F96 maxisorp microwell plates were purchased from

Nunc (Denmark). The magnetic separation of the beads duringthe washing steps were carried out using magnetic separator formicroplate designed in our lab (Fig. 1B). In order to do that, anacrylic piece (227 mm of long and 85 mm width) was cut by LEC

S.V. Kergaravat et al. / Analytica Chimica Acta 798 (2013) 89– 96 91

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ig. 1. Picture of the ELISA array: (A) schematic representation of the immunologiesigned magnetic support including 8 neodymium magnets placed underneath th

achine (Laser engraving and cutting machine – Consys Laser Mod030 40 W – China that was purchased from Control System SRL,rgentina). Then eight perforations (4 mm of diameter and 8 mmf height) were performed by LEC machine in the center of thecrylic platform. In these perforations, eight neodymium magnets4 mm of diameter and 8 mm of height) were incorporated. All cutsere performed to 16 W of laser power and 20 mm s−1 of engrav-

ng speed. In order to improve the aspiration of the supernatant, theagnets were localized between two microwells in the magnetic

eparation steps. This allowed the introducing of pipette tip andhe aspirating of the supernatant without suck modified magneticarticles (see Fig. 1A and B).

Electronic absorption measurements were carried out on aerkin-Elmer Lambda 20 UV-VIS spectrophotometer. Fluorescenceeasurements were carried out on two Perkin-Elmer (Llantrisant,nited Kingdom) LS 55 luminescence spectrometers with surface

eaders (named A and B). The standard curves were analyzed with four-parameter logistic equation using the Graph Prism softwareGraphPad Software, San Diego, CA).

.3. Fluorescent measurement

Then, the enzymatic response of the HRP with OPD as cosub-trate and H2O2 as substrate was measured in the ELISA plate by

uorescent methods. In order to do that, solutions of OPD (up to

× 10−3 mol L−1) and H2O2 (up to 1 × 10−3 mol L−1) were addedo HRP-containing solutions (or immunocomplex-MB) and incu-ated for 20 min in the dark. Then, Triton X-100 was added to

ctions onto the surface of the magnetic beads localized on the plate; (B) speciallyline of microplate with 96 well microplates.

reach concentration of 2% (w/v) and afterward was incubated for2 min. The final volume was 300 �L and the reaction medium wascitric–phosphate buffer 0.1 mol L−1 pH 5.0.

Afterward, the fluorescence of the OPD product was recorded atabsorption and fluorescence wavelengths of 440 nm and 540 nm,respectively.

2.4. Magneto immunofluorescence assay

The magneto immunofluorescence assay is schematically out-lined in Fig. 1A. The steps were: (i) incubation step: 100 �L ofthe TG2-MB solution (0.1 mg mL−1) were mixed with 100 �L ofthe ATG2 (titers ranged from 1:68,000 to 1:300) in shaking condi-tions for 30 min at room temperature, and then washed (3×) with100 �L of PBST, by applying a magnetic field between each washing(ii) Immunological reaction: 100 �L of the anti IgG-HRP antibody1:20,000 was added and incubated for 30 min at room temperatureunder mixing conditions. Posteriori, the washed step was appliedagain. (iii) Fluorescence measurement (see Section 2.3).

The standard curve was fitted to a four-parameter logistic equation according to the formulay = {(A − B)/[1 + 10 exp((log C − log X) × D)]} + B, where A is themaximal fluorescence intensity, B is the minimum fluorescenceintensity, C is the concentration producing 50% of the maximal

fluorescence intensity, X is the ATG2 titer and D is the slopeat the inflection point of the sigmoid curve. The limit of detec-tion (LOD) and quantification (LOQ) and half maximal effectiveconcentration (EC50) were calculated as the 10, 20 and 50% of

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uorescence intensity from the standard curve, respectively. Inddition, the dynamic range was established between 20 and 80%f fluorescence intensity.

.5. Assay validation

The precision tests were performed by analyzing ATG2 titers of:40,000, 1:15,000 and 1:4000. The intra-assay test was evaluatedy multiple analyses (n = 3) in one assay while the inter-assay wasssessed by analysis in five separate analytical runs. The exper-mental conditions for this assay were 0.05 mg mL−1 of TG2-MBnd anti IgG-HRP secondary antibody diluted 1:20,000. Moreoverntermediate precision was performed by two lots of TG2 (6.28nd 3.14 mU mg MB−1 per assay) on two separate days and twoifferent instruments. The experimental conditions for this assayere 0.05 mg mL−1 of TG2-MB, 1:15,000 of ATG2 and anti IgG-HRP

econdary antibody diluted 1:20,000.For evaluating matrix effect, the calibration curves for the ATG2

iters ranged from 1:70,000 to 1:300 were built in PBS, negativelasma diluted 1:25 and negative serum diluted 1:25, respectively.

Recovery experiments were performed by spiking at three ATG2evels in negative serum diluted 1:25 and in negative plasmailuted 1:25, respectively.

Data for each group (29 positive and 19 negative clinically con-rmed cases for celiac disease) were analyzed for normality byolmogorov-Smirnov test [23]. Because deviations from normalityere statistically significant for both groups (P < 0.05), the compar-

son between groups was performed by U of Mann–Whitney test.he best ATG2 cut-off value that discriminated between patientsith or without celiac disease was determinated by the ROC plot

y SPSS and MedCalc softwares. Then forty-eight blood serum sam-les were evaluated and the results were correlated with the onesbtained by commercial optical ELISA kit.

. Results and discussion

.1. TG2 binding on the magnetic beads

The efficiency of the coupling of TG2 to the magnetic beads wasvaluated by Bradford test. A calibration curve was prepared withSA standard solution (as shown in Supplementary Material Fig.

). Although BSA is habitually used as standard protein in the Brad-ord method, in a particular assay the absorbance readings of targetnd standard protein can be slightly different (as shown in Supple-entary Material Fig. ii). In order to achieve a greater accuracy in

he efficiency estimation among batches of immobilized transglu-aminase, in each assay two solutions (0.15 and 0.55 mg mL−1) ofG2 also were used as standard (or control) solutions.

In this way, we avoid performing a complete calibration curvef TG2 for each batch. Therefore, this procedure allows us to dimin-sh the consumption of an expensive protein as transglutaminasend it allows guaranteeing the accuracy in the determination. Then,G2 before and after binding on the magnetic beads was evaluatednd the protein concentration was calculated from the calibrationurve. The obtained efficiency was around 80%, as described in Sup-lementary Material. The batches of TG2-MB were stable over aeriod of at least one month, when stored as recommended by theanufacturer at 4 ◦C (as shown in Fig. iii, Supplementary Material).

.2. Fluorescent measurement optimization

Since the proposed system for revealing of immunological event

s based on the enzymatic reaction of HRP, two cosubstrates suchs OPD and TMB were studied. Hence, several factors with relationo the fluorescent response and the enzymatic kinetics were evalu-ted for both systems (HRP–OPD–H2O2 and HRP–TMB–H2O2). On

mica Acta 798 (2013) 89– 96

the one hand, OPD is one of the most effective chromogenic sub-strate for HRP-mediated ELISA, being the 2,3-diaminophenazine(DAP) the fluorescent product of the HRP-catalysed oxidation [16].Therefore, the fluorescent response is directly related to the enzy-matic activity when OPD is used as cosubstrate. On the other hand,the TMB is a methylated derivative of benzidine. The extendedunsaturated ring system of TMB can emit strong fluorescence inthe ultraviolet region, which can be related to resonance interac-tion at different positions of the molecule and the formation of aquinine structure with a planar configuration in the excited stage.The product of the HRP-catalysed oxidation of TMB is a nonflu-orescence compound [17]. Therefore, the fluorescent response isindirectly related to the enzymatic activity when TMB is used ascosubstrate.

The Table ii in Supplementary Material shows factors relatedto the optimization of fluorescent response. The absorption andfluorescence spectra for DAP and TMB allow selecting the wave-lengths of fluorescence (emission) and absorption (excitation) foreach system (as shown in Fig. 2A and B, respectively).

In order to reach a compromise between sensitivity and reactiontime, the reaction kinetics and the effect of addition of Triton-100were assessed. The addition of Triton-100 was studied at four con-centrations levels (from 1 to 4% w/v) together to correspondingblank tests (as described in Supplementary Material, Fig. ivA andB). The TMB-H2O2 system was not significantly affected by Tri-ton. On the other hand, when non-ionic Triton X-100 is added tothe OPD-H2O2 system, the DAP fluorescence intensity increasesconsiderably. This is due to Triton X-100 can provide a good envi-ronment to encapsulate and restrict the intramolecular rotationof DAP to fluorescence. In addition, the polyethylene glycol chainof Triton X-100 shares similar functional groups as diethyl ethermay provide a microenvironment and polarity favorable for DAPfluorescence. Therefore for the HRP–OPD–H2O2 system, 20 and2 min were times selected before and after Triton X-100 addi-tion, respectively. The reaction time was defined as the time ofsampling the fluorescence signal for each system. After this study,22 min was selected for the OPD system and 5 min was fixed forthe TMB system. Both revelation systems need a reaction timelower than its corresponding (32 min) required by commercial opti-cal ELISA (HRP–TMB–H2O2–H2SO4) that was used as a comparisonmethod.

Finally, the optimal combinations of the enzymatic concentra-tion (A), cosubstrate concentration (B) and H2O2 concentration (C)were then researched in each system to optimize the enzymaticresponse coupled to fluorescence detection. Therefore a centralcomposite design of 34 experiments with six center points wasperformed. In this design, an ANOVA test was applied to eachHRP-cosubstrate-H2O2 system, to obtain a significant fitted modeland not significant lack of fit (p-values must be minor and majorto 0.05, respectively). After obtaining the fitted model, the factorcombination that provides the best “values of desirable response”should be investigated [24]. In the optimization stage the flu-orescent response directly related to the enzymatic activity isdesirable therefore substrate concentrations must be present in anexcess amount, i.e. the reaction must be independent of substrateconcentrations (zero-order kinetics). The Table iii in Supplemen-tary material shows the optimal results obtained by the responsesurface methodology for the systems using OPD and TMB as cosub-strate. The predicted and experimental responses in all cases werenot significantly different when compared by a mean comparisontest with alpha level of 0.05 [25,26]. In this context, the global desir-ability function (D) could be considered as a combined efficiency

of two events, the enzymatic activity and the fluorescence signal,respectively. The value of D was higher for the system with OPD ascosubstrate than for TMB as cosubstrate. The minor performanceof HRP–TMB–H2O2 system is probably due to the fact that the

S.V. Kergaravat et al. / Analytica Chimica Acta 798 (2013) 89– 96 93

Fig. 2. (A) The absorption spectrum of DAP by UV–vis spectrophotometer (blackline). Conditions in absorption: OPD 4 × 10−4 mol L−1, H2O2 5 × 10−4 mol L−1 yand HRP 2.5 × 10−11 mol L−1 in citric phosphate buffer 0.1 mol L−1 at pH 5.0. Theabsorption and fluorescence spectra of DAP by LS 55 luminescence spectrome-ter (blue and cyan lines for absorption and fluorescence spectra). Conditions influorescence using a microplate: OPD 5 × 10−4 mol L−1, H2O2 5 × 10−4 mol L−1 andHRP 9.5 × 10−10 mol L−1 in citric-phosphate buffer 0.1 mol L−1 at pH 5.0. (B) Theabsorption spectrum of TMB by UV–vis spectrophotometer (black line). Condi-tions in absorption: TMB 2.5 × 10−4 mol L−1, H2O2 2.5 × 10−4 mol L−1 y and HRP1.9 × 10−12 mol L−1 in citricphosphate buffer 0.1 mol L−1 with dimetilsulfoxide 1%(v/v) at pH 4.2. The absorption and fluorescence spectra of TMB by LS 55 lumi-nescence spectrometer (blue and cyan lines for absorption and fluorescencespectra). Conditions in fluorescence using a microplate: TMB 1 × 10−6 mol L−1, H2O2

1 × 10−6 mol L−1 and HRP 3.8 × 10−8 mol L−1 in citric–phosphate buffer 0.1 mol L−1

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Fig. 3. Calibration curves obtained by two fluorescent detection systems: (A)HRP–OPD–H2O2 and (B) HRP–TMB–H2O2. Reaction conditions: 0.5 mg mL−1 ofTG2-MB, ATG2 ranged from 1:70,000 to 1:300 and 1:20,000 of antiIgG-HRP. Detec-tion system conditions: (A) OPD 1 × 10−3 mol L−1 and H2O2 1 × 10−3 mol L−1 in

ith dimetilsulfoxide 1% (v/v) at pH 4.2. (For interpretation of the references to colorn this figure legend, the reader is referred to the web version of the article.)

agnitude of interest, the enzymatic activity for this system, isndirectly related to fluorescent readings.

.3. Immunoassay optimizations

For the detection stage of immunoassay, an optic fiber system (as surface reader) was used. This setup allows using of disposableicro plate and the fluorescent detection in the presence of MBith no separation step. In contrast to other methods that use MBe did not observe any interference under experimental conditions

sed. Therefore, this effectively simplifies the analysis procedurend thus avoids unnecessary steps and potential losses of materials,n comparison to some methods previously reported [27,28].

citric–phosphate buffer 0.1 mol L−1 at pH 5.0 and (B) TMB 5 × 10−5 mol L−1 and H2O2

5 × 10−5 mol L−1 in citric–phosphate buffer 0.1 mol L−1 at pH 4.2.

In order to optimize the indirect immunoassay for ATG2, thedetection system, the TG2-MB concentration and the anti IgG-HRP titer were studied. First, in order to select the more sensitivedetection system, OPD and TMB were used as cosubstrates ofHRP in the optimal conditions according to section 3.2. Therefore,calibration curves were built by using 0.5 mg mL−1 of TG2-MB,ATG2 ranged from 1:70,000 to 1:300 and a dilution of 1:20,000of anti IgG-HRP and finally their performances were compared.The OPD–H2O2–Triton system was chosen for further studies, giv-ing an EC50 1:18,300 and higher fluorescence readings than theTMB–H2O2 system, which presents an EC50 1:8500, as shown inFig. 3A and B.

Second, increasing concentrations of TG2-MB ranged from 0.025to 0.75 mg mL−1 were tested against different dilutions of ATG2and the anti IgG-HRP secondary antibody diluted 1:20,000. Thestandard curves of ATG2 for four TG2-MB concentrations are shownin Supplementary material (Fig. iii). The curve for TG2-MB concen-tration of 0.025 mg mL−1 shows the lower sensitivity (expressed asEC50). The fluorescence readings corresponding to 0.05 mg mL−1

and 0.75 mg mL−1 of TG2-MB concentrations, especially for highATG2 titer were very imprecise. In addition, the curve for TG2-MBconcentration of 0.75 mg mL−1 has a steep shape and shows lowerfluorescence readings than the curve for TG2-MB concentration of0.5 mg mL−1, it can be explained by fluorescence quenching causedby an excess of immunoreagents. On the other hand, the curve with0.05 mg mL−1 of TG2-MB presented wider dynamic range (1:11,000to 1:1700 of ATG2) with lower LOD value and EC50 (1:18,900 and1:4150 of ATG2, respectively). Therefore, 0.05 mg mL−1 of TG2-MBwas chosen for further studies.

Third, the anti IgG-HRP titer was also studied, since it should

be in excess to ensure the complete reaction with the maximumamount of ATG2 that can be captured by TG2-MB. In order to opti-mize this parameter, calibration curves were built by incubation of

94 S.V. Kergaravat et al. / Analytica Chimica Acta 798 (2013) 89– 96

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Table 1Assay validation (A) intra- and inter-assay precision for ATG2 title. (B) Intermediateprecision results. (C) Recovery test of ATG2 titer in serum and plasma matrixes.

Statistics ATG2 title

1:40,000 1:15,000 1:4000

Intra-assayb Mean 1:41,600 1:16,500 1:3800R.S.D. (%) 9.8 17.4 19.4Recovery (%) 104 110 95

Inter-assaya Mean 1:40,800 1:15,900 1:3920R.S.D. (%) 16.8 20.0 17.3Recovery (%) 105 108 98

Test day Instrumentc Lot of TG2d Recovery (%) (n = 3)

1 A C (106 ± 4)%1 A D (94 ± 10)%1 B D (107 ± 8)%2 A C (115 ± 6)%2 B C (84 ± 7)%2 A D (94 ± 5)%

Means, R.S.D. (%) 100% (±11%)

Statistics ATG2 titer

1:4000 1:2000 1:1000

Serum matrix Mean 1:3280 1:1804 1:1143R.S.D. (%) 9 4 20Recovery (%) 82.2 90.2 114.3

Statistics ATG2 titer

1:8000 1:4000 1:2000

Plasma matrix Mean 1:9568 1:3120 1:2082R.S.D. (%) 8 12 10Recovery (%) 119.6 78.1 104.1

a The intra-assay test was evaluated by multiple analyses (n = 3) in one assay.b

curves (see Fig. 5). For the estimation of this effect, the compari-son of EC50s was performed by test ANOVA, which indicated nosignificant difference (P < 0.05) between the different matrices. The

ig. 4. Standard curve of ATG2 by magneto immunofluorescence assay. Experi-ental conditions: 0.05 mg mL−1 of TG2-MB and anti IgG-HRP secondary antibody

iluted 1:20,000.

ifferent dilutions of anti IgG-HRP antibody with different dilutionsf ATG2 and TG2-MB in 0.05 mg mL−1. In addition, when ANOVAas applied, no significant difference between blank tests (cor-

esponding to different secondary antibody levels in absence ofTG2) was obtained (P < 0.05). Fig. vi in Supplementary Materialhows the calibration curves constructed under these experimentalonditions. The calibration curve with the better analytical perfor-ance was obtained using a titer of 1:20,000 of anti IgG-HRP. This

urve shows the lower LOD (1:56,300 of ATG2), the wider dynamicange (1:50,000 to 1:2500 of ATG2) and the best-fit value for theill slope was 0.95, close to the expected value of 1.0 [26]. Thether curves presented minor analytical performance. The curvebtained with anti IgG-HRP titer of 1:10,000 has a flattened shapeith the narrowest dynamic range. The readings of fluorescencender this condition were lower than those that we expected but aolor development was observed. Various phenomena can generatehis evidence. For instance, at higher secondary antibody titer, annefficient washing step might cause an excessive (non-specific)etention of conjugate therefore high enzymatic catalysis mightenerate an excess fluorescent product and then this might causeuorescence quenching [29]. However, in a future research a deepertudy should be carried out in order to elucidate this evidence.

.4. Assay validation

.4.1. Calibration standards and figures of meritThe linearity of the method was evaluated by analyzing ten

alibration standards using the four-parameter logistic regressionlgorithm in order to fit the response [fluorescence intensity nor-alized (FI)] versus ATG2 titer. “Goodness of fit” was indicated by

n average correlation coefficient of 0.97 from ten standard curvesnd a Hill slope of 0.84. A typical standard curve of ATG2 titer ishown in Fig. 4. The LOD and LOQ were 1:74,500 and 1:42,700 ofntibody, respectively. The dynamic range from 1:42,700 to 1:2700with EC50 = 1:11,600) was very wide for the application of this

ethod in the detection of this antibody in celiac patients.

.4.2. Inter-assay and intra-assay precisionInter-assay and intra-assay data are shown in Table 1A. The

ecoveries (%) were ranged from 98% to 108% and from 95% to 110%or inter- and intra-assay, respectively. Inter-assay and intra-assayrecision as R.S.D. (%) were ranged from 16.8% to 20.0% and from.8% to 19.4%, respectively. These values are considered acceptableccording to recommendations for bioanalytical method validationbout precision requirements in the phase of method development30].

.4.3. Intermediate precisionIntermediate precision was performed by two separate days and

sing two different instruments and two lots of TG2-MB. Fresh

The inter-assay was assessed by analysis in five separate analytical runs (n = 15).c Instrument A y Bd Lot of TG2: 6.28 mU mg MB−1 per assay (C); 3.14 mU mg MB−1 per assay (D).

sample and standard solutions were independently prepared oneach day of analysis. Afterward, the Kruskal–Wallis test wasapplied and no significant difference between groups was obtained(P < 0.05). The intermediate precision results are shown in Table 1B.Therefore, these results were considered acceptable with a meanrecovery of 100% and a R.S.D. (%) of 11% [30].

3.4.4. Matrix interference testsFor the evaluation of the matrix effect, the ATG2 calibration

curves obtained in negative serum diluted 1:25 and negativeplasma diluted 1:25 were compared with the calibration curve per-formed in PBS. Similar dynamic ranges were obtained for three

Fig. 5. Calibration curves for different matrixes obtained by magneto immunofluo-rescence assay at optimized conditions. The error bars show the standard deviationfor n = 3.

S.V. Kergaravat et al. / Analytica Chimica Acta 798 (2013) 89– 96 95

Table 22 × 2 contingency table relating the detection of celiac disease by optical ELISA kit and the one predicted from magneto immunofluorescence assay (cut-off at ATG2 = 8.1 U).

Optical ELISA kit PPV (%) NPV (%) Efficiency (%) Youden’s index

Positive Negative Total

Magneto immunofluorescence assay

Positive (ATG2 > 8.1 U) 28TP 2FP 30 93.3 94.4 93.8 0.88Negative (ATG2 < 8.1 U) 1FN 17TN 18

Total 29 19 48

T ive preS PV = TC

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P: true positive, FP: false positive, FN: false negative, TN: true negative, PPV: posite = TP × 100/(TP + FN) = 96.6%; Sp = TN × 100/(TN + FP) = 89.5%; PPV = TP/(TP + FP); Nhi-square = 36.2; P < 0.0001 Cohen’s Kappa = 0.868 [38].

bsence of matrix effect can be related to the use of magnetic bead.n this case, the magnetic beads could play a role in the preconcen-ration and separation of the antibody in the complex samples oferum and plasma.

For the recovery experiment in negative serum diluted 1:25,hree ATG2 titers (1:1000, 1:2000 and 1:4000) were spiked andecovery values were ranged from 82.2% to 114.3%. On the otherand, for the same experiment in negative plasma diluted 1:25,nother three ATG2 titers (1:2000, 1:4000 and 1:8000) were spikednd recovery values were ranged from 78.1% to 119.6%. The R.S.D.%) was lower than 20% in all cases (Table 1C). These results arecceptable and confirm the absence of matrix effect between theifferent matrices tested.

.5. Blood serum samples

The diagnostic of celiac disease can be determinated by acreening test as the method proposed where the samples areichotomised into positive or negative according to a cut-off valuen the continuous scale derived from the ROC plot. As reportedn literature, this plot is a good tool for the selection of cut-offalue based in the sensitivity (Se) and specificity (Sp) therefore its a method of describing the intrinsic accuracy of a diagnostic testpart from the decision threshold [31,32].

As it was previously mentioned, the magneto immunofluores-ence assay was developed and validated by using commerciallyvailable ATG2 (whole antiserum, developed in goat). Conse-uently, in order to demonstrate the applicability of assay proposedor the detection of human anti-TG2 in the real patient samples,e must change the anti-goat IgG-HRP secondary antibody by

n anti-human IgA (alpha-chain specific)-HRP antibody. Then, theatient samples were analyzed both by the magneto immunofluo-escence assay and by the INOVA optical ELISA kit. To comparehe results obtained by both methods, two calibration curves inhe range of ATG2 concentration from 7.3 to 30 U were built. Oneas built versus fluorescence intensity (FI) and the other was built

ersus absorbance unit (Abs). To estimate the non-specific bindingnd to study the repeatability, two control samples (prepared bypiking a negative serum of healthy person) of 10 and 20 U weressessed, as detailed described in Supplemetary Material, see Fig.ii. In order to do that, the positive control of the INOVA opticalLISA kit that has a labeled activity level of ATG2 of 150 UnitsU) was used as calibrator solution. For the magneto immuno-uorescence assay the linear regression equation was adjusted toI = 1.292 × CATG2(U) − 6.033 with a LOD of 7 U using the 3.3SB cri-erion (n = 12, R2 = 0.995) and a R.S.D. (%) of 6.8 (n = 6) for controlample (10 U). On the other hand, for ELISA assay the regression lin-ar was Abs = 0.00674 × CATG2(U) + 0.1206 with a LOD of 15 U usinghe 3.3SB criterion (n = 12, R2 = 0.997) and a R.S.D. (%) of 5.9 (n = 6)or control sample (20 U). The analytical sensitivity values were

.57 U−1 and 0.36 U−1 for the immunofluorescence assay and theNOVA optical ELISA kit, respectively. This demonstrates that theroposed method is approximately four times more sensitive thanhe standard ELISA assay. The recovery values for control samples

dictive value, NPV: negative predictive value.N/(FN + TN); Youden’s index = 1 − –[(1 − PPV) + (1 − NPV)] [37].

(10 and 20 U) were 97.7% and 105.4% (n = 6), respectively, givingvalues of R.S.D. (%) lower than 7% in two cases; these results wereconsidered acceptable [30].

Afterward, 29 and 19 sera of celiac and non-celiac patientsrespectively were diluted 1:50 (as described in SupplementaryMaterial, see Fig. viii) and analyzed. The data obtained weresubsequently submitted to the ROC plot (as shown in Fig. ix,Supplementary Material), but previously it was necessary toascertain the actual differences between groups. Therefore, theU of Mann–Whitney test was applied and significant differencebetween groups was obtained [33].

From ROC plot obtained, the upper-left tract yields a Se equalto 96.6% and a Sp equal to 94.7%. These two values correspond toa cut-off of 8.1 U ATG2. This value is lower than its correspond-ing obtained by the mean plus two-fold standard deviation of thenegative sample (9.8 U, n = 19) [34] and another the cut-off valueobtained by one-tailed t test at a 99% confidence level (11 U) [35].In addition the cut-off value from ROC analysis is lower than its cor-responding (16.95 U) obtained by using electrochemical magnetoimmunosensor recently reported for ATG2 detection [13]. Takinginto consideration these results, a cut-off value of 8.1 U ATG2 fromROC analysis was chosen for further studies. On the other hand,the overall efficiency of the method is indicated by the area underthe ROC plot that assumes values between 0.5 (inaccurate) and 1.0(perfect). The value of 0.97 has been characterized as “highly accu-rate” by Greiner [36]. Likelihood ratios calculated at the fixed cut-offpoint were +LR = 18.34 and −LR = 0.036. These values are classifiedas “very useful” by Chien [31].

Correspondence of predicted results by optical ELISA kit andthe obtained results by the magneto immunofluorescence assay,according to the cut-off value from ROC analysis is summarized inTable 2. The table of contingency evidences a good correlation withcommercial optical ELISA kit. The values calculated of Se and Spwere 96.6% and 89.5%, respectively. The positive predictive (PPV)and negative predictive (NPV) values obtained were greater than90%. This indicates that very few (<10%) of the positive and negativeresults are false positive and false negative, respectively. Youdenindex (J) was 0.88 indicating that the effectiveness is relativelylarge [37]. Moreover, the test efficiency [(true positive + true nega-tive)/total] was 93.7%. Cohen’s Kappa agreement value was 0.868,classified as “excellent” by published guidelines [38].

4. Conclusion

In this work a magneto immunofluorescence assay based ontransglutaminase-coated magnetic beads was developed for theATG2 detection, using a total assay time of 2 h for each 96-wellplate.

Regarding performance advantages, the approach presentedhere contains several benefits. First, the revelation system used

in the fluorescent immunoassay allowed decreasing 10 min thetotal assay time in respect to commercial ELISA assay. Second,the method presented is approximately four times more sen-sitive and faster than the commercial ELISA assay. Third, the

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ransglutaminase-modified magnetic beads show a good stabil-ty and a low non-specific adsorption level of labeled secondaryntibody. Fourth, the cut-off value (8.1 U ATG2) obtained by ROClot (with a sensitivity of 96.6%, a specificity of 94.7% and an effi-iency of 97.0% expressed as area under ROC curve) and used toiscriminate between celiac and non-celiac patients is lower thanhat of the commercial kit. Fifth, the clinical usefulness of fluo-escent immunoassay was demonstrated by analyzing celiac andon-celiac patients. The immunoassay exhibited a sensitivity of6.6%, a specificity of 89.5% and an efficiency of 93.8% comparedo the commercial optical ELISA kit.

According to all these features, the immunoassay proposed cane considered an analytical tool with high-throughput potentialor clinical diagnosis of celiac disease. However, in the near future,n order to improve the diagnostic power (sensitivity and speci-city) of the assay, the TG2 (from Guinea Pig Liver) used as antigenhould be changed by human TG2. Moreover, smaller magneticeads could be used in conjunction with an automated manipu-

ation of these particles to avoid manual intervention.

cknowledgments

This work is supported by the Universidad Nacional del LitoralUNL), Santa Fe, Argentina for financial support from the (ProjectsAID 2009 N◦ 8/41 and CATT 2010 código 1545), the Ministry ofcience and Innovation (MEC), Madrid, Espana (Project BIO2010-7566) and the from Generalitat de Catalunya, Espana (Projects SGR23). S.V. K. acknowledges UNL for providing the doctoral fellow-hip. We thank to Dr. A. Beccaria (Fermentations Laboratory -UNL)y allowing the use of incubation room and to Gómez G.A. studenty participating in the magnetic separator building. We also thanko “Iturraspe” and “Dr. O. Alassia” Hospitals of Santa Fe (Argentina)or the blood serum samples. The authors also wish to thank Prof.. Fernández (Math Departament-UNL) for your assistance in sta-istical pretreatment of data.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.aca.2013.09.009.

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