Analytica Chimica Acta 883 (2015) 74–80

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A rapid sandwich immunoassay for human fetuin A using agarose-3-aminopropyltriethoxysilane modified microtiter plate Sandeep Kumar Vashist a,b, * , E. Marion Schneider c , John H.T. Luong d a Laboratory for MEMS Applications, Department of Microsystems Engineering—IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany b HSG-IMIT—Institut für Mikro- und Informationstechnik, Georges-Koehler-Allee 103, 79110 Freiburg, Germany c Sektion Experimentelle Anaesthesiologie, University Hospital Ulm, Albert Einstein Allee 23, 89081 Ulm, Germany d Innovative Chromatography Group, Irish Separation Science Cluster (ISSC), Department of Chemistry and Analytical, Biological Chemistry Research Facility (ABCRF), University College Cork, Cork, Ireland

H I G H L I G H T S

G R A P H I C A L A B S T R A C T

 Agarose-based signal enhanced immunoassay for human fetuin A (HFA).  Wide linear range of 1–243 ng mL1 with high sensitivity and specificity.  Limit of detection of 0.02 ng mL1 and limit of quantification of 0.3 ng mL1.  Detection of clinically-relevant HFA levels in human blood and serum in 200 mg mL1 were diluted 1:1000 and 1:3000, respectively, to fit the linear range of DIA (1–243 ng mL1). Both dilutions of 1:1000 and 1:3000 were employed for each of the unknown clinical samples, as per the standard sample preparation guidelines for the clinical ELISA. The assay was performed at 37  C (Thermostat, Labnet International, USA), while the absorbance was measured by the Tecan Infinite M200 Pro microplate reader from Tecan (Austria). DIW and PBS washings were performed five times with 300 mL of each solution, while the blocking was done with 300 mL of 5% BSA. Similarly, 100 mL was taken for the dispensing of 1% KOH, EDCactivated capture anti-HFA Ab mixed with agarose in 1% (v/v) APTES, HFA, biotinylated anti-HFA Ab conjugated to SA-HRP, and TMB. EDC was reconstituted in 0.1 M MES. The biotinylated antiHFA detection Ab (0.2 mg mL1) was admixed and incubated with SA-HRP (diluted 1:200), 1:1 (v/v), resulting in the conjugation of biotinylated anti-HFA detection Ab to SA-HRP. 2.2. Preparation of anti-HFA Ab-bound agarose-functionalized MTP The treatment of the MTP wells with 1% (w/v) KOH at RT for 10 min followed by washing with UPW generates the desired hydroxyl groups for subsequent binding of APTES (Fig. 1). Unless otherwise indicated, the following steps were performed at 37  C. EDC-activated anti-HFA Ab was prepared by incubating 990 mL of anti-HFA Ab (8 mg mL1) with 10 mL of EDC (4 mg mL1) for 15 min. The EDC-activated anti-HFA Ab was then mixed with agarose (1 mg mL1) in 1% (v/v) APTES in the ratio of 1:1 (v/v). Thereafter, 100 mL of this anti-HFA Ab solution (4 mg mL1, 0.5 mg mL1 agarose and 0.5% APTES) was dispensed into each well, incubated for 30 min and washed with PBS. The anti-HFA Ab-bound agarosefunctionalized wells were blocked with 5% (v/v) BSA for 30 min and washed with PBS. The stability of anti-HFA Ab-bound and BSApreblocked MTPs, stored in 0.1 M PBS at 4  C, was assessed for 6 weeks.

2.1. Materials 2.3. Developed sandwich IA procedure Phosphate buffered saline (PBS, 0.1 M, pH 7.4), 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC), bovine serum albumin (BSA), 2-(N-morpholino)ethanesulfonic acid (MES, pH 4.7), and 3,30 ,5,50 -tetramethylbenzidine (TMB) substrate kit were purchased from Thermo Scientific. Agarose (85 mm in diameter), 3-aminopropyltriethoxysilane (APTES, purity 98%, w/v), H2SO4 (97.5%, v/v), and Nunc microwell 96-well polystyrene plates (flat bottom, nontreated and sterile) were procured from Sigma–Aldrich. The HFA

The anti-HFA Ab-bound and BSA-preblocked MTP was provided with biotinylated anti-HFA Ab preconjugated to SA-HRP, and HFA (0.1–243 ng mL1) in buffer, diluted human serum or diluted human whole blood. It was then incubated for 15 min and subsequently washed with PBS. This was followed by the addition of TMB (as per manufacturer’s guidelines) and stopping of the enzyme–substrate reaction after 14 min by adding 50 mL of 2N

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Fig. 1. Schematic of the bioanalytical procedure used in the developed HFA IA.

H2SO4. The absorbance was measured at 450 nm with reference at 540 nm. All experiments were performed in triplicate and the blank reading (no HFA) was subtracted from all assay values. The cELISA was performed as per the manufacturer’s guidelines. Various controls were also performed to determine the BSA blocking efficiency; nonspecific interaction of BSA with HFA, biotinylated anti-HFA Ab and SA-HRP; and nonspecific interactions of DIA with other biomarkers. Datasets were plotted by SigmaPlot software version 11.2 using a four-parameter logistic-based standard curve analysis function. The maximal half-effective concentration (EC50), the correlation coefficient (R2) and the Hill slope values were determined by the reports generated by

SigmaPlot. The LOD and limit of quantification (LOQ) values were determined by the standard formulae i.e., 3  (standard deviation of the blank/slope) and 10  (standard deviation of the blank/ slope), respectively. 3. Results and discussion 3.1. Developed HFA sandwich IA The precise and rapid determination of HFA is important in the diagnosis and management of diabetes, obesity, metabolic syndrome, cardiovascular diseases, atherosclerosis, hepatocellular

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carcinoma, arthritis and other diseases/disorders. The DIA format based on agarose microparticles provides a simple and costeffective procedure to detect HFA in 30 min (Fig. 1). In brief, the hydroxyl groups were generated on the polystyrene MTP’s surface by treatment with KOH [50]. Agarose serves as an excellent matrix for enzyme/protein immobilization due to its hydrophilic character and high porosity, i.e., a high capacity for enzymes/proteins. Besides its ease of derivatization, agarose has no charged groups, which would circumvent nonspecific binding as the major driving forces behind such nonspecific adsorption are hydrophobicity, and ionic or electrostatic interaction. There are the two prerequisites for its use in immunoassays. Agarose has been advocated in this study as its abundant hydroxyl groups will react with the alkoxy groups of APTES to form a stable complex. In brief, silanization will occur on any substrate that has chemically active hydroxyl groups for silane grafting [51]. Simultaneously, the remaining alkoxy groups of APTES react with the hydroxyl groups present on the KOH-treated MTP surface by a hydrolysis-dependent reaction [52,53]. Agarose alone also exhibits strong adhesion with a solid substrate with  OH groups, e.g., glass slide [54]. In the DIA, the EDC-activated anti-HFA capture Ab are covalently attached to the amino groups on APTES by the formation of an amide bond [53].

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This heterobifunctional crosslinking chemistry enables the highly specific binding of Ab to APTES with minimal nonspecific reaction products [55]. The modified MTP is blocked by incubating with 5% BSA for 30 min. Thereafter, HFA and biotinylated Ab preconjugated to SA-HRP are sequentially dispensed into the MTP wells and incubated for 15 min to form a sandwich immune complex. After washing with PBS, the resulting MTP well is incubated with TMB enzyme substrate for 14 min. This is followed by the termination of enzyme–substrate reaction and the measurement of absorbance of resulting colorimetric solution. DIA detected HFA in the range of 0.01–243 ng mL1 with linearity from 1 to 243 ng mL1. The LOD, LOQ, and EC50 of DIA are 0.02 ng mL1, 0.3 ng mL1, and 18.6 ng mL1, respectively (Table S1) (Fig. 2A). The precise determination of HFA spiked in diluted human whole blood and serum attests its capability to detect the entire pathophysiological range of HFA (0.15–600 mg mL1) with appropriate sample dilution. An apparent matrix effect was noted as the LOD and LOQ for the detection of HFA spiked in diluted human whole blood and plasma were 0.03 vs 0.40 and 0.03 vs 0.34, respectively (Fig. 2A). The intraday and interday variability for five assay repeats (in triplicate) in a single day and five consecutive days, respectively, were 1.5–8.2 and 2.7–11.8, respectively. The

Fig. 2. Analytical performance of the developed HFA IA. (A) Determination of HFA in PBS (10 mM, pH 7.4), diluted human serum and diluted human whole blood by the DIA (with agarose). (B) Experimental process controls used in the DIA (with agarose) to analyze the efficiency of blocking, and non-specific interactions between IA components and with other biomarkers. Anti-HFA1 and anti-HFA2 are capture and detection antibodies, respectively, while HSA, LCN2 and CRP are human serum albumin, human lipocalin 2 and C-reactive protein, respectively. (C) A comparison of DIA (with agarose) with DIA (without agarose). (D) Correlation of DIA with cELISA used in commercial HFA kit for the detection of HFA-spiked in diluted human serum. All experiments were performed in triplicate with the error bars indicating the standard deviation.

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procedure was highly specific for HFA without any interaction with tested immunological reagents and other related biomarkers (Fig. 2B). The tested nonspecific biomarkers, i.e., HSA, CRP, LCN2, IL-1b, IL-6, IL-8 and TNF-a, are usually found along with HFA in patients suffering from inflammation, infection, CVD, diabetes, obesity and other metabolic disorders. Therefore, the nonspecificity of DIA was tested using the pathophysiologically high concentration of these biomarkers taking into account the dilution factor being used for human whole blood/serum. The overall analysis time of DIA was only 2 h (including the 45 min Ab binding and 30 min BSA blocking steps), which is 14fold faster than the cELISA (28 h) using commercial HFA Duoset ELISA kit. This could be attributed to the rapid Ab immobilization and the one-step kinetics-based IA format requiring only minimal process steps [56]. The Ab immobilization required only 45 min, i.e., 18-fold faster than conventional Ab immobilization by overnight passive adsorption (used in the commercial kit). The subsequent IA duration was just about 30 min, which is 10-fold faster than that of cELISA (5 h) being used in the commercial kit (Tables S1–S2). The DIA is a rapid and highly-sensitive IA based on our previously developed one-step kinetics-based IA format [56]. However, it is different from the cELISA, which requires prolonged time and multiple consecutive steps, as it is based on the formation of a sandwich immune complex by one-step kinetics in just a single incubation step. The main characteristic features of one-step kinetics-based IA format are discussed elsewhere [56]. The optimized concentration of agarose used for the DIA was 1 mg mL1 (Fig. S2A), while the optimized duration of the single incubation step was 15 min (Fig. S2B). The Ab-bound and BSApreblocked MTP was prepared within 75 min, which is significantly shorter than that of our recently demonstrated IA format based on the use of magnetic beads [56], which employs a two day protocol as suggested by the commercial manufacturer. This limitation was obviated by employing the capture anti-HFA Abbound MTP [57], thereby leading to an improved IA format. However, the use of agarose in the DIA has critically improved the bioanalytical performance, as demonstrated by a 10-fold lower LOD (0.03 ng mL1) and a wider linear range (1–243 ng mL1) in comparison to the HFA IA without agarose (LOD 0.3 ng mL1, linear range (3–243 ng mL1) (Fig. 2C). The improved performance could be attributed to the high surface area of agarose, which leads to a higher density of the capture Ab (Fig. S1) that in turn enhances detection sensitivity. The higher response signal, and the superior bioanalytical performance than other materials,

Table 1 Determination of HFA in the EDTA plasma samples of patients using DIA and cELISA. Samples

Conventional (ng mL1)

Developed (ng mL1)

Percentage recovery

1 2 3 4 5 6 7 8 9 10

4.1  0.7 8.4  1.9 26.1  2.9 39.6  3.5 72.3  4.9 89.7  4.5 124.4  6.9 158.6  6.7 169.6  7.2 210.0  7.4

3.8  0.6 7.9  2.4 24.5  2.3 41.4  3.1 67.0  5.7 86.2  4.9 119.0  6.3 156.3  6.1 174.4  7.8 224.5  8.1

92.7 94.0 93.9 104.5 92.7 96.1 95.7 98.5 102.8 106.9

such as polystyrene beads and magnetic Dynabeads1 (unpublished results), will further contribute to its preferential application in the determination of HFA in inflammatory diseases [37] and other pathophysiological conditions. The DIA is analyticallysuperior to the previously developed SPR-based IAs [47,48] in terms of higher analytical sensitivity and wider linear range. Considering the use of the commercially-available agarose with fixed dimensions in this study, it is possible to further improve the analytical performance of DIA by employing agarose microparticles of smaller diameters that have increased total surface area for greater binding of capture anti-HFA Ab. Apparently, the DIA procedure using agarose modified with APTES has clearly demonstrated all the important bioanalytical features as required for potential applications in clinical diagnostics. 3.2. Bioanalytical parameters The results obtained by DIA correlated well with those of cELISA for the detection of HFA in anonymized EDTA plasma samples of patients (Table 1). The percentage recoveries of HFA in the samples are in the range of 96.7–110.0 for DIA, when the HFA values determined by cELISA are used as standard. Similarly, the results obtained for HFA spiked in diluted human serum agree well with those of cELISA (Fig. 2D). The agarose-based signal enhancement results in remarkable detection sensitivity and a wide linear range. The anti-HFA Ab-bound and BSA-blocked MTPs retained their functional activity when stored at 4  C in 0.1 M PBS for up to 6 weeks (Fig. 3A), confirming the leach-proof covalent binding of anti-HFA Ab to the agarose-functionalized MTP. Therefore, DIA is best suited for clinical diagnostics, where the Ab-prebound MTPs

Fig. 3. (A) Storage stability of anti-HFA1-bound agarose-functionalized MTP stored at 4  C in PBS (10 mM, pH 7.4) for 6 weeks. (B) Batch-to-batch variability for various preparations of agarose-anti-HFA Ab, as demonstrated for the detection of 27 ng mL1 HFA by DIA. All experiments were performed in triplicate with the error bars representing the standard deviation.

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are being employed on a large scale and usually stored up to 4 weeks to facilitate rapid analyte detection. Moreover, there was no appreciable batch-to-batch variability for various preparations of agarose-anti-HFA Ab when tested for the detection of 27 ng mL1 HFA (Fig. 3B), which demonstrated high production reproducibility. Being generic, the rapid DIA procedure can be extended for the detection of other disease biomarkers and analytes. Based on its high analytical precision and stability, it would be of immense utility for the development of fully-integrated in vitro diagnostic formats using advanced biosensors, microfluidics and lab-on-achip technologies. Moreover, it would be a potential IA format for the smartphone-based colorimetric reader [58] that provides similar detection sensitivity as the microtiter plate reader. 4. Conclusions The developed agarose-based signal enhanced IA enables the detection of HFA in just about 30 min, which is 16-fold faster than a cELISA procedure employed in the commercial HFA kit. The DIA procedure is highly simplified, employs minimal process steps, and can detect the entire clinically-relevant pathophysiological range of HFA. Agarose forms a stable complex with APTES to serve as an excellent matrix for bioconjugation and offers high precision, high production reproducibility, and ability to detect HFA in diluted whole blood, serum or plasma. Being generic, DIA procedure can be employed for the detection of other disease biomarkers and analytes. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2015.04.060. References [1] J.D. Young, J. Martel, D. Young, A. Young, C.M. Hung, L. Young, Y.J. Chao, J. Young, C.Y. Wu, Characterization of granulations of calcium and apatite in serum as pleomorphic mineralo-protein complexes and as precursors of putative nanobacteria, PLoS One 4 (2009) e5421. [2] L. Kalabay, L. Jakab, Z. Prohászka, G. Füst, Z. Benkö, L. Telegdy, Z. Lörincz, P. Závodszky, P. Arnaud, B. Fekete, Human fetuin/a2HS-glycoprotein level as a novel indicator of liver cell function and short-term mortality in patients with liver cirrhosis and liver cancer, Eur. J. Gastroenterol. Hepatol. 14 (2002) 389–394. k, J. Masopust, K. Kithier, J. Rádl, Hepatocellular carcinoma in [3] J. Houšte association with a specific fetal a-1-globulin, fetoprotein, J. Pediatr. 72 (1968) 186–193. [4] M. Singh, P.K. Sharma, V.K. Garg, S.C. Mondal, A.K. Singh, N. Kumar, Role of fetuin-A in atherosclerosis associated with diabetic patients, J. Pharm. Pharmacol. 64 (2012) 1703–1708. [5] R. Westenfeld, W. Jahnen-Dechent, M. Ketteler, Vascular calcification and fetuin-A deficiency in chronic kidney disease, Trends Cardiovasc. Med. 17 (2007) 124–128. [6] K. Mori, Y. Ikari, S. Jono, M. Emoto, A. Shioi, H. Koyama, T. Shoji, E. Ishimura, M. Inaba, K. Hara, Fetuin-A is associated with calcified coronary artery disease, Coron. Artery Dis. 21 (2010) 281–285. [7] C.K. Glass, J.M. Olefsky, Inflammation and lipid signaling in the etiology of insulin resistance, Cell Metab. 15 (2012) 635–645. [8] A.-S. Goustin, A.B. Abou-Samra, The thrifty gene encoding Ahsg/Fetuin-A meets the insulin receptor: insights into the mechanism of insulin resistance, Cell Signal. 23 (2011) 980–990. [9] S. Grandy, R.H. Chapman, K.M. Fox, S.S. Group, Quality of life and depression of people living with type 2 diabetes mellitus and those at low and high risk for type 2 diabetes: findings from the study to help improve early evaluation and management of risk factors leading to diabetes (SHIELD), Int. J. Clin. Pract. 62 (2008) 562–568. [10] D.P. Lorant, M. Grujicic, C. Hoebaus, J.M. Brix, F. Hoellerl, G. Schernthaner, R. Koppensteiner, G.H. Schernthaner, Fetuin-A levels are increased in patients with type 2 diabetes and peripheral arterial disease, Diabetes Care 34 (2011) 156–161. [11] J.I. Odegaard, A. Chawla, Pleiotropic actions of insulin resistance and inflammation in metabolic homeostasis, Science 339 (2013) 172–177. [12] D. Pal, S. Dasgupta, R. Kundu, S. Maitra, G. Das, S. Mukhopadhyay, S. Ray, S.S. Majumdar, S. Bhattacharya, Fetuin-A acts as an endogenous ligand of TLR4 to promote lipid-induced insulin resistance, Nat. Med. 18 (2012) 1279–1285.

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A rapid sandwich immunoassay for human fetuin A using agarose-3-aminopropyltriethoxysilane modified microtiter plate.

A rapid sandwich immunoassay (IA) with enhanced signal response for human fetuin A (HFA) was developed by modifying the surface of a KOH-treated polys...
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