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The development and use of an ELISA-based method to follow the distribution of cellulase monocomponents… Pribowo, Amadeus Y; Hu, Jinguang; Arantes, Valdeir; Saddler, Jack N May 20, 2013

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METHODOLOGY Open AccessThe development and use of an ELISA-basedmethod to follow the distribution of cellulasemonocomponents during the hydrolysis ofpretreated corn stoverAmadeus Y Pribowo, Jinguang Hu, Valdeir Arantes and Jack N Saddler*AbstractBackground: It is widely recognised that fast, effective hydrolysis of pretreated lignocellulosic substrates requiresthe synergistic action of multiple types of hydrolytic and some non-hydrolytic proteins. However, due to thecomplexity of the enzyme mixture, the enzymes interaction with and interference from the substrate and a lack ofspecific methods to follow the distribution of individual enzymes during hydrolysis, most of enzyme-substrateinteraction studies have used purified enzymes and pure cellulose model substrates. As the enzymes present in atypical “cellulase mixture” need to work cooperatively to achieve effective hydrolysis, the action of one enzyme islikely to influence the behaviour of others. The action of the enzymes will be further influenced by the nature ofthe lignocellulosic substrate. Therefore, it would be beneficial if a method could be developed that allowed us tofollow some of the individual enzymes present in a cellulase mixture during hydrolysis of more commerciallyrealistic biomass substrates.Results: A high throughput immunoassay that could quantitatively and specifically follow individual cellulaseenzymes during hydrolysis was developed. Using monoclonal and polyclonal antibodies (MAb and PAb,respectively), a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed tospecifically quantify cellulase enzymes from Trichoderma reesei: cellobiohydrolase I (Cel7A), cellobiohydrolase II(Cel6A), and endoglucanase I (Cel7B). The interference from substrate materials present in lignocellulosicsupernatants could be minimized by dilution.Conclusion: A double-antibody sandwich ELISA was able to detect and quantify individual enzymes when presentin cellulase mixtures. The assay was sensitive over a range of relatively low enzyme concentration (0 – 1 μg/ml),provided the enzymes were first pH adjusted and heat treated to increase their antigenicity. The immunoassay wasemployed to quantitatively monitor the adsorption of cellulase monocomponents, Cel7A, Cel6A, and Cel7B, thatwere present in both Celluclast and Accellerase 1000, during the hydrolysis of steam-pretreated corn stover (SPCS).All three enzymes exhibited different individual adsorption profiles. The specific and quantitative adsorption profilesobserved with the ELISA method were in agreement with earlier work where more labour intensive enzyme assaytechniques were used.Keywords: Cellulose, Cellulase, Enzyme, Adsorption, ELISA* Correspondence: jack.saddler@ubc.caForest Products Biotechnology/Bioenergy Group, University of BritishColumbia, 2424 Main Mall, Vancouver, British Columbia V6T1Z4, Canada© 2013 Pribowo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Pribowo et al. Biotechnology for Biofuels 2013, 6:80http://www.biotechnologyforbiofuels.com/content/6/1/80IntroductionOne of the key steps in a biomass-to-ethanol process isthe enzymatic hydrolysis of the cellulosic component tofermentable sugars. Typically, a mixture of complemen-tary cellulase and other, so-called, accessory enzymes(such as hemicellulases, GH61, etc.) are required to ef-fectively break down the structural cellulose and hemi-cellulose polysaccharides to their component sugars[1,2]. However, various technoeconomic analyses haveindicated that the cost of enzymatic hydrolysis is still un-acceptably high, primarily because of the high enzymeloadings required to achieve effective hydrolysis [3]. As aresult, a considerable amount of research has focussedon ways to try to improve the efficiency of hydrolysiswhile using low protein/enzyme loadings. Various strat-egies have been assessed, such as increasing substratedigestibility through biomass pretreatments [4,5], im-proving the efficiency of enzyme cocktails [6,7], and re-using the enzymes for multiple rounds of hydrolysis[8,9]. The last two strategies, in particular, have benefit-ted from better characterization of the specific roles andactions of individual enzymes and their synergistic inter-action during cellulose hydrolysis.However, getting a better understanding of the individ-ual enzyme’s interaction with the substrate during hy-drolysis of lignocellulosic substrates has been challenging,primarily because of the lack of specific techniques thatcan overcome both the complexity of the enzyme mixtureand the interference caused by the heterogeneous ligno-cellulosic substrates. Many of the biochemical techniquesthat might be used lack the resolution to specifically probeindividual enzymes and proteins. For example, the enzymeCel7A from T. reesei has a very similar molecular weightto that of Cel6A and Cel7B and, consequently, these threeenzymes typically show up as a single band after gelelectrophoresis [1]. Another commonly used technique isto characterize and evaluate distribution of enzymes basedon their activities on model substrates such as carbo-xymethyl cellulose (CMC), filter paper, or a number ofchromophoric substrates such as p-Nitrophenyl-basedsubstrates [10]. Unfortunately, many of these model sub-strates are not specific enough to distinguish individualenzymes. Protein chromatography techniques have alsobeen utilized to fractionate the enzyme mixture down toits individual components [11,12]. However, this approachis laborious and, depending on the purification protocolsused, the enzyme mixture may not always completelyseparate into its individual components [13]. In addition,interference caused by substrate materials such as ligninauto-fluorescence limits the use of traditional proteinchromatography techniques and protein labelling tech-niques using fluorescent dyes [14].Primarily due to the limitations of the assay methodsthat have been employed, most of the previous enzyme-cellulosic substrate interaction studies have used purifiedenzymes or reconstituted mixtures of purified enzymes[15,16] and/ or model substrates such as pure celluloseor substrates with a very low lignin content [17,18] tosimplify the subsequent enzyme assays and analyses.While these studies have advanced our understanding ofenzymes-substrate interaction, they have not looked atthe interactions occurring during the hydrolysis of anindustrially relevant lignocellulosic substrate using acomplete enzyme mixture.In recent work, the distribution of individual enzymespresent in a commercial cellulase mixture (Accellerase1000) was assessed during the hydrolysis of steampretreated corn stover (SPCS) [1]. A combination ofmethods such as, gel electrophoresis, zymograms, activ-ity assays using chromophoric substrates, and massspectrometry were used to define the general distribu-tion patterns of some of the enzymes during SPCShydrolysis [1]. However, although we were able to semi-quantitatively assess enzyme distribution using thesetechniques, we were not able to quantitatively followthe adsorption profiles of individual enzymes.It is well known that antibodies can bind to specificantigens and this ability has been used as the basis formany assays [19-21]. This specific recognition and bind-ing has been utilized in various techniques including theenzyme-linked immunosorbent assay (ELISA). The ELISAmethod uses antibodies linked to a reporter enzyme tospecifically recognize and bind a target compound in amixture of compounds. This specific compound or pro-tein can then be quantified by adding a substrate for thereporter enzyme and measuring the concentration of theproduct [22]. The ELISA method, using monoclonal and/or polyclonal antibodies (MAbs and PAbs, respectively)raised against various cellulase enzymes, has been success-fully used to quantify target enzymes both in culture fil-trates and commercial enzyme preparations [19].A double-antibody sandwich ELISA, which is an ELISA-based technique using a pair of antibodies to sandwich thetarget compound and specifically quantify it among othercompounds in the mixture, has been successfully used toquantify the amount of Cel7A in a crude culture brothwith minimal interference from other enzymes or othermaterials present in the broth [23]. Improved specificity ofthe assay was achieved when MAb was used as the coatingantibody and PAb as the second, detecting antibody [23].In related work, Buhler et al. (1991) optimized a double-antibody sandwich ELISA for Cel7B in a culture brothusing MAb as the coating antibody and PAb as thedetecting antibody. They were able to show that the assaywas both sensitive and specific for Cel7B [24]. However,the feasibility of using ELISA to quantify specific proteinspresent in the supernatant after hydrolysis of a lignocellu-losic substrate has not yet been demonstrated.Pribowo et al. Biotechnology for Biofuels 2013, 6:80 Page 2 of 15http://www.biotechnologyforbiofuels.com/content/6/1/80In the work described here, a double-antibody sand-wich ELISA was developed and used to quantify some ofthe specific cellulase enzymes present in the supernatantduring the hydrolysis of SPCS. A double-antibodysandwich ELISA was used to specifically quantify theamount of cellulase monocomponents Cel7A, Cel6A,and Cel7B present in a commercial enzyme mixture.The sensitivity was improved by subjecting the enzymesamples to a pH adjustment treatment and/or a heattreatment. While lignocellulosic substrate derived mate-rials did interfere with the assay, this interference couldbe minimized by simple dilution.Results and discussionsDetermination of the specificity of the differentmonoclonal antibodies (MAbs) and polyclonal antibodies(PAbs)We initially wanted to ensure that the MAb and PAbthat we had been provided were specific for their targetcellulase monocomponents. The specificity of Cel7A,Cel6A, and Cel7B MAbs were initially assessed usingWestern Blots against Cel7A that had been purifiedfrom a commercial Celluclast mixture as well as againstthe Cel7A component that was known to be present inthe 3 commercial enzyme mixtures. The Cel7A MAbWestern Blot showed a single band corresponding to thepurified Cel7A and a major band at molecular weight(MW) ~ 70 kDa, which is the molecular weight of theCel7A, present in the 3 commercial enzyme mixtures(Figure 1A). Although the Cel6A MAb also showed aband of protein at MW ~70 kDa when assayed againstthe 3 commercial enzyme mixtures (Figure 1B), thisMAb did not react with the purified Cel7A. In additionto the major bands at MW ~70 kDa, Cel7A and Cel6AMAb Western Blots both showed multiple bands withcommercial enzyme mixtures. Although we could not becertain if these bands corresponded to multiple isoformsof the target enzyme or actual unspecific bindings toPurified Cel7ACelluclast Accellerase CTec 2196 kDa1157739 28 19146MW A196 kDa11577392819146Purified Cel7ACelluclastAccellerase CTec 2 MW B196 kDa1157739 2819146Purified Cel7ACelluclastAccellerase CTec 2 MW CFigure 1 Reactivity and specificity of MAbs against Cel7A (A), Cel6A (B), and Cel7B (C) as determined using Western Blots.Pribowo et al. Biotechnology for Biofuels 2013, 6:80 Page 3 of 15http://www.biotechnologyforbiofuels.com/content/6/1/80other proteins without further experiments, we did notexpect these apparent multiple bindings to significantlyinfluence the specificity of the ELISA for 2 reasons.Firstly, the intensity of these other bands was signifi-cantly less compared to the band intensity of theexpected target enzyme. Therefore, given the low proteinconcentration required for ELISA (< 5 μg/ml), thisapparent unspecific binding (if any) would not likely tohave any significant influence to the specificity of theassay. Secondly, the differing banding patterns betweenCel7A and Cel6A Western Blots seemed to suggest a spe-cific rather than an unspecific binding. The Western Blotthat used the Cel7B MAb did not recognize the purifiedCel7A but recognized a band of protein at MW ~60 kDain all of the 3 commercial enzyme mixtures (Figure 1C).Therefore, it appears that all 3 MAbs were reactive andspecific for their target enzymes.The specificity and reactivity of Cel7A and Cel6APAbs were also determined by Western Blots by usingpurified Cel7A and Cel6A from Celluclast as well as 2commercial enzyme mixtures. The PAb against Cel7Awas specific for its target enzyme since it reacted onlywith purified Cel7A and not with the purified Cel6A(Figure 2A). However, the PAb against Cel6A recognizedboth the purified Cel7A and Cel6A (Figure 2B). Possiblecontamination by Cel6A in the purified Cel7A fractiondid not appear to be an issue as the Cel6A MAb did notreact with the purified Cel7A preparation (Figure 1B).196 kDa11577392819146ACelluclast Ctec 2 Cel6A Cel7A MWCelluclast Ctec 2 Cel6A Cel7AMWB196 kDa11577392819146Celluclast Ctec 2 Cel6ACel7AMW Cel7BAccellerase196 kDa11577392819146CFigure 2 Reactivity and specificity of PAb against Cel7a (A), Cel6A (B), and Cel7B (C) as determined using Western Blots.Pribowo et al. Biotechnology for Biofuels 2013, 6:80 Page 4 of 15http://www.biotechnologyforbiofuels.com/content/6/1/80The reactivity and specificity of the Cel7B PAb was nextdetermined using Western Blots against purified Cel7A,Cel6A, and Cel7B as well as against 3 commercial cellu-lase mixtures. It was apparent that the Cel7B PAb recog-nized the purified Cel7B but also cross-reacted with thepurified Cel7A and Cel6A (Figure 2C). However, thiscross-reactivity with the Cel6A and Cel7B PAbs was notexpected to influence the specificity of the double-antibody sandwich ELISA since both the Cel6A andCel7B MAbs were shown to be specific to their respect-ive target enzymes (Figure 1B and C).Optimization of the assay protocols to improve thesensitivity of the double-antibody sandwich ELISAPrevious work had shown that a double-antibody sand-wich ELISA, using a combination of a MAb and a PAbas the capture and detecting antibodies respectively,resulted in improved specificity compared to normalELISA or to a sandwich ELISA using PAb as the captureand MAb as the detecting antibody [19,23]. Thus, wenext used an MAb as the capture antibody and a PAb asthe detecting antibody to assay different concentrationsof each of the 3 antibodies MAb, PAb, and goat-anti00.020.040.060.080.10.120 2 4 6 8 10 12Absorbance (A 405nm )Cel7A (ug/ml)A0.000.050.100.150.200.250.300.350 0.5 1 1.5 2 2.5Absorbance (A 405nm )Cel7A (ug/ml)B0.00.10.20.30.40.50.60.70.80.92 4 6 8 10Absorbance (A 405nm )Cel7A (ug/ml)CFigure 3 Optimization of the concentrations of MAb (A), PAb (B), and the third antibody, GAR-AP (C) over a range of concentration ofpurified Cel7A. (A). Two different concentrations of Cel7A MAb 10 μg/ml (◊) and 50 μg/ml (□) were added to the well. PAb and GAR-APconcentrations were kept constant at 1/400 and 1/1750 dilutions, respectively (B). Using 10 μg/ml Cel7A MAb, the Cel7A PAb was diluted todifferent degrees: 14(◊), 7(□), 3.5(Δ), and 1.75(X) μg/ml. The GAR-AP was diluted at 0.3 μg/ml. (C). The concentration of GAR-AP was varied bydiluting it to 1 (◊) or 0.3 (□)μg/ml in PBS. Cel7A MAb concentration was kept at 10 μg/ml, and Cel7A PAb was diluted to 14 μg/ml in PBS.Pribowo et al. Biotechnology for Biofuels 2013, 6:80 Page 5 of 15http://www.biotechnologyforbiofuels.com/content/6/1/80rabbit IgG conjugated to alkaline phosphatase (GAR-AP). In this way, we hoped to assess the sensitivity ofthe assay in detecting purified Cel7A at concentrationsranging from 0–2.5 μg/ml.Although two concentrations of Cel7A MAb (10 and50 μg/ml diluted in 1x Phosphate-Buffered Saline orPBS) were initially assessed, as both concentrations gavesimilar absorbance values (Figure 3A), a MAb concen-tration of 10 μg/ml was used in subsequent work. Previ-ous work had also determined that a concentration of 10μg/ml was sufficient to coat the bottom surface of a wellin a typical 96-well ELISA plate [25]. The concentrationsof the PAb (detecting antibody) and GAR-AP, the tertiaryantibody, were similarly optimized over the same range ofCel7A concentrations. A concentration of 0.14 μg/ml ofPAb Cel7A and 1/500 dilution of GAR-AP (correspondingto 1 μg/ml of GAR-AP) were found to improve thesensitivity of the assay for all the three enzymes (Figure 3Band C). These concentrations of antibodies were then alsoused for the Cel6A and Cel7B based ELISA’s.Despite the increased sensitivity gained by optimizingthe concentrations of all 3 antibodies, the improved sig-nal was still quite low when compared to previouslyreported values [23]. Therefore, to try to further increasethe sensitivity of the assay, the enzyme samples weresubjected to pH adjustment and heat treatments priorto addition to the well. Although previous work hadshown that the antigen-antibody interactions are typic-ally optimum at pH > 7 [24], fungal derived enzymes aretypically buffered and used at around pH < 5. We there-fore brought the enzyme samples up to pH 7.5 usingPBS buffer prior to their addition to the wells.Previously, Riske et al. (1990) had reported that a heat-sensitive fungal product caused a signal reduction with0.00.20.40.60.81.01.21.40 0.5 1 1.5 2 2.5 3Absorbance (A405nm)Cel7A (ug/ml)A0.00.20.40.60.81.01.20 0.5 1 1.5 2 2.5 3Absorbance (A405nm)Cel6A (ug/ml)B0.00.10.20.30.40.50.60.70 0.5 1 1.5 2 2.5 3Absorbance (A405nm)Cel7B (ug/ml)CFigure 4 The effect of a heat treatment on the sensitivity of ELISA for pure Cel7A (A) and Cel6A (B) and Cel7B (C). Heated enzymesamples were boiled in Na-acetate buffer pH 5.0 at 100°C for 10 minutes and then serially diluted in PBS (□). Non-heated samples were directlydiluted in PBS (◊).Pribowo et al. Biotechnology for Biofuels 2013, 6:80 Page 6 of 15http://www.biotechnologyforbiofuels.com/content/6/1/80Cel7A ELISA and that this interference disappeared afterthe cellulase preparation was boiled, resulting in in-creased ELISA sensitivity [23]. Therefore, to see if wecould also obtain the same beneficial effect, the enzymemonocomponents were also heated at 100°C for 10 mi-nutes to determine if a heat treatment might also im-prove sensitivity. When the Cel7A and Cel6A ELISA’swere subjected to a heat treatment at 100°C for 10 mi-nutes in a pH 5.0 buffer, followed by dilution in PBS buf-fer at pH 7.5, the sensitivity of ELISA increased by about6× and 10× respectively for Cel7A and Cel6A, at an en-zyme concentration of 2.5 μg/ml when compared to theuntreated samples (Figure 4A and B). However, heattreatment decreased the sensitivity for the Cel7B basedELISA (Figure 4C). Therefore, the enzyme samples forthe Cel7B ELISA were not heated but directly diluted inPBS buffer and then added to the wells.As mentioned earlier, the improved signal achieved byheating the enzymes used for the Cel7A and Cel6Abased ELISA’s was likely caused by the removal of inter-fering heat-sensitive materials present in the samples[23] or by protein denaturation which may lead to theopening up of the protein structure, exposing the anti-gen to the antibody. The ineffectiveness of heating theCel7B may indicate that the interfering materials maynot interfere with the Cel7B based ELISA system. Thisdifferential response to the heat treatment highlights theneed to optimize the double-antibody sandwich ELISAfor each specific enzyme-antibody system.How specific is the ELISA to the enzyme of interest?The specificity of each ELISA was next determined bycomparing the absorbance values of each enzyme whenit was added as a single component and when it wasadded as a mixture of 4 purified enzymes (Cel7A, Cel6A,Cel7B, and Cel5A). For all of the enzyme based ELISA’s(Cel7A, Cel6A, and Cel7B ELISA), the standard curvesobtained with the pure enzymes was similar to thoseobtained with the reconstituted mixture especially whenthe target enzyme concentration was less than 1 μg/ml(Figure 5A, B, and C). It was apparent that the ELISAdouble-antibody sandwich assay was able to specificallyquantify a target enzyme when it was present in a mix-ture with 3 other cellulase monocomponents.We next wanted to determine if a whole commercial en-zyme mixture could be used to make a standard curve, thusobviating the need for purified enzymes. A commercial en-zyme mixture was diluted to 200 μg protein/ml in Na-acetate buffer (0.05 M, pH 5.0). When using the Cel7A andCel6A based ELISA’s, the commercial enzyme mixtures0.00.20.40.60.81.01.21.40 1 2Absorbance (A405nm)Cel6A (ug/ml)B0.00.10.20.30.40.50.60.70.80 1 2 3Absorbance (A405nm)Cel7B (ug/ml)C0.00.20.40.60.81.01.20 1 2 3Absorbance (A405nm)Cel7A (ug/ml)AFigure 5 The specificity of ELISA for Cel7A (A), Cel6A (B), and Cel6A (C) as measured using pure enzymes (◊) and reconstitutedmixtures of the 4 purified enzymes Cel7A, Cel6A, Cel7B, and Cel5A (□).Pribowo et al. Biotechnology for Biofuels 2013, 6:80 Page 7 of 15http://www.biotechnologyforbiofuels.com/content/6/1/80were heated, serially diluted 2-fold in PBS and then addedto the wells. By sufficiently diluting the enzyme mixtures, arelatively linear standard curve could be obtained withwhole enzyme mixtures when using the Cel7A and Cel7Bbased ELISA’s (Figure 6A and C). A linear standard curvewas also obtained with the Cel6A ELISA. However, this lin-ear standard curve was only obtained with Celluclast andnot with Accellerase or CTec 2 (Figure 6B).The linear standard curve obtained for all of the targetenzymes highlighted the ability of the double-antibodysandwich ELISA to detect the target enzyme even whenpresent in complex enzyme mixtures. The high specifi-city of the MAbs could also be the reason why Cel6AELISA only worked with Celluclast and not with othercommercial enzyme mixtures as the Cel6A MAb wasdeveloped by colleagues at the National Renewable En-ergy Laboratory (NREL) to detect Cel6A in Celluclastwhereas the PAb was developed commercially by AlphaDiagnostics using a synthesized peptide. Although boththe MAb and PAb’s against Cel6A recognized the Cel6Apresent in Celluclast, Accellerase and CTec 2 (Figure 1Band 2B), the lower ELISA signal observed in the lattertwo commercial enzyme mixtures might be a result of aslight change in antigen recognition by the MAb. Whenthe concentration of the enzymes and antibodies arehigh, as in the case of the Western Blot studies (30 μg ofenzyme samples and 250 μg MAb or PAb), there is likelyenough interaction between the enzymes and antibodies,resulting in a significant band on the membrane. How-ever, when the enzyme concentration is low (< 0.1 μg),as in the case with the ELISA, the lower binding affinitybetween the antibodies and Cel6A in Accellerase andCTec 2 would result in a lower ELISA signal. It was ap-parent that a double-antibody sandwich ELISA was spe-cific for target enzymes providing appropriate MAbs andPAbs were available. Given the recent rapid developmentof enzyme cocktails to which new-and-improved en-zymes have been introduced, (i.e. CTec3) the highly spe-cific nature of the antibody-antigen interaction shown inthis assay will likely require the development of specificMAbs and PAbs that will recognize individual enzymespresent in these new and improved enzyme mixtures.Determining the possible interference of substratederived materials on the ELISAAlthough various ELISA based methods have been usedto quantify cellulase enzymes, these assays have onlybeen applied to commercial enzyme mixtures or to0.00.20.40.60.81.01.21.41.61.80 50 100 150Absorbance (A405nm)Protein Concentration (ug/ml)A0.00.10.20.30.40.50.60.70.80.90 50 100 150Absorbance (A405nm)Protein Concentration (ug/ml)B0.00.10.20.30.40.50.60.70 20 40 60Absorbance (A405nm)Protein Concentration (ug/ml)CFigure 6 The construction of a standard curve for Cel7A ELISA (A) using whole commercial enzyme mixtures Accellerase 1000 (◊), CTec2 (□), Cel6A ELISA (B) using Accellerase 1000 (◊) and Celluclast 1.5L (Δ), and Cel7B ELISA (C) using CTec 2 (□).Pribowo et al. Biotechnology for Biofuels 2013, 6:80 Page 8 of 15http://www.biotechnologyforbiofuels.com/content/6/1/80culture filtrates [19,23,24,26]. The use of an ELISA totry to follow the distribution of cellulase enzymes dur-ing enzymatic hydrolysis of a realistic, lignocellulosicsubstrate has not, so far, been described in the literatureAs a result, there is limited information on the possibleinfluence of interfering materials that will likely bepresent when attempts are made to use an ELISA in thissituation.Previous work on the use of ELISA’s to detect residualagrochemicals in soil samples had shown that humicsubstances in soil may result in an overestimation ofchemical concentrations [20,27,28], and that sample di-lution could be used to minimize interference [20]. As asimilar type of interference might occur with biomass-derived materials such as soluble lignin fragments, su-pernatants derived from steam pretreated corn stover(SPCS), steam pretreated poplar (SPP), steam pretreateddouglas fir (SPDF), and Avicel were assessed for theirpossible influence on the double-antibody ELISA. Thesupernatants were diluted in PBS to varying degrees todetermine if a simple dilution could minimize the inter-ference caused by these materials.It was apparent that the undiluted biomass derived su-pernatants resulted in considerable interference with allof the Cel7A, Cel6A, and Cel7B based ELISAs (Figure 7).The Cel7A ELISA either over or under estimated theamount of enzyme (Figure 7A) with the supernatants de-rived from the SPCS (5× higher) and SPP substratesresulting in an overestimation and the SPDF and Avicelsupernatants in an underestimation (Figure 7A). In con-trast, only the SPP supernatants caused a signal overesti-mation with Cel6A ELISA while the SPCS, SPDF, andAvicel supernatants gave a signal that was lower thanthe PBS control (Figure 7B). Interference with Cel7Bbased ELISA was only assessed with the SPCS super-natant which caused a slight overestimation (Figure 7C).0.00.51.01.52.02.53.0PBS SPCS SPP SPDF Avicel SPCS 1/10PBSSPCS 1/100PBSAbsorbance (A405nm)A0.00.10.20.30.40.50.6PBS SPCS SPP SPDF Avicel SPCS 1/10PBSSPCS 1/100PBSAbsorbance (A405nm)B0.00.20.40.60.81.01.2PBS SPCS SPCS 1/10 PBS SPCS 1/100 PBSAbsorbance  (A405nm)CFigure 7 Effect of substrate supernatants on Cel7A ELISA (A), Cel6A ELISA (B), and Cel7B ELISA (C). Amount of purified enzymes added:1.25 μg/ml.Pribowo et al. Biotechnology for Biofuels 2013, 6:80 Page 9 of 15http://www.biotechnologyforbiofuels.com/content/6/1/80To assess if a simple dilution could minimize interfer-ence, each supernatant was diluted 10× or 100× in PBS.It was apparent that the interference caused by theaddition of the undiluted SPCS supernatant could beminimized at both dilution levels (Figure 7A). Thisdilution strategy was also effective on both the Cel6Aand Cel7B based ELISA’s and a 100-fold dilution in PBSseemed to consistently give an ELISA signals similar tothe PBS control for both Cel6A and Cel7B ELISA(Figure 7B and 7C).Can an ELISA be used to follow enzyme distributionduring SPCS hydrolysis?We next wanted to assess if the double-antibody sand-wich ELISA could be used to quantitatively monitor thetime course of individual enzyme adsorption (Cel7A,Cel6A, and Cel7B) during the hydrolysis of SPCS. It wasapparent that all 3 enzymes exhibited different adsorptionprofiles when incubated with SPCS (Figure 8A, B, and C).Most of Cel7A immediately adsorbed to the SPCS aftermixing, leaving only about 30% of Cel7A in the super-natant. After 3 hours of hydrolysis, Cel7A started to de-sorb back to the supernatant with maximum desorptionoccurring after 6 hours of hydrolysis with about 65% ofthe initial Cel7A detected in the supernatant. Overprolonged hydrolysis, the concentration of Cel7A in thesupernatant decreased progressively (Figure 8A). This par-tially reversible adsorption of Cel7A confirmed previouswork where a combination of techniques, such as zymo-gram, SDS-PAGE, and enzyme activity assays, were usedto semi-quantitatively determine specific Cel7A adsorp-tion/desorption during SPCS hydrolysis [1].In contrast, Cel6A directly adsorbed onto the SPCSwithin the first 3 hours and remained tightly boundthroughout the course of hydrolysis (Figure 8B). Previ-ous work that looked at Cel6A adsorption used purifiedCel6A due to a lack of a specific assay able to monitorCel6A in the presence of other enzymes. The irreversibleadsorption of Cel6A observed in this study using com-mercial enzyme mixtures was in a good agreement withthis previous work [29].Compared to Cel7A and Cel6A, the adsorption ofCel7B was more gradual with the amount of Cel7Bdetected in the supernatant continuously declining overthe 72 h hydrolysis (Figure 8C). Prior to developing theELISA method, we had tried to follow the specific0204060801001200 12 24 36 48 60 72Cel6A (% Initial Loading)Time (h)B0204060801001200 12 24 36 48 60 72Cel7A  (% initial loading)Time (h)A0204060801001200 12 24 36 48 60 72Cel7B (% Initial Loading) Time (h)CFigure 8 Adsorption profiles of Cel7A (A), Cel6A (B), and Cel7B (C) during hydrolysis of SPCS as determined by a double-antibodysandwich ELISA. Samples for Cel7A ELISA were obtained by hydrolyzing SPCS at 2% substrate consistency in 0.05 M Na-acetate buffer pH 5.0with 20 FPU/ g cellulose Accellerase 1000. Samples for Cel6A and Cel7B ELISA were obtained from SPCS hydrolysis using 20 FPU/ g celluloseCelluclast and 40 CBU/ g cellulose β-glucosidase.Pribowo et al. Biotechnology for Biofuels 2013, 6:80 Page 10 of 15http://www.biotechnologyforbiofuels.com/content/6/1/80adsorption profile of Cel7B by monitoring its profile asdetermined by zymograms using CMC and xylan as sub-strates [1]. The quantitative adsorption profiles obtainedusing the ELISA profile were in a good agreement withthe qualitative results obtained previously using zymo-grams during the 72 h hydrolysis [1].ConclusionsA simple, high-throughput assay that can specifically fol-low and quantify individual enzymes present in the com-plex enzyme mixtures that are used to hydrolysepretreated lignocellulosic substrates was developed anddemonstrated. The protocols for an immunoassay usingantibodies against Cel7A, Cel6A, and Cel7B were devel-oped with the hope of using the method to follow thedistribution of individual enzymes during hydrolysis. Acombination of MAb’s and PAb’s, as the respective coat-ing and detecting antibodies, was used to develop adouble-antibody sandwich ELISA. This method was ableto detect and quantify individual enzymes when presentin cellulase mixtures. The assay was sensitive over arange of relatively low enzyme concentration (0 – 1 μg/ml), provided the enzymes were first pH adjusted and/orheat treated to increase their antigenicity. Although lig-nocellulosic hydrolysates resulted in varying degrees ofinterference with the assay, the interference could beminimized by diluting the samples in PBS buffer. Theimmunoassay was employed to quantitatively monitorthe adsorption of cellulase monocomponents, Cel7A,Cel6A, and Cel7B that are present in both Celluclastand Accellerase 1000, during the hydrolysis of SPCS. Allthree enzymes exhibited different individual adsorptionprofiles. The specific and quantitative adsorption profilesobserved with the ELISA method was in agreement withearlier work where more laborious enzyme assay tech-niques were used.Methods and materialsPurification of cellulase monocomponents, Cel7A, Cel6A,Cel7B, and Cel5AThe cellulase monocomponents Cel7A, Cel6A, Cel7B,and Cel5A were purified from Celluclast (Novozyme)using previously described methods [12,30-32]. TheNinhydrin assay [33] was then used to determine theconcentrations of these purified enzymes as well asthe commercial enzyme mixtures. Bovine serum albumin(BSA, Sigma) was used as the protein standard.Preparation of antibodies and determination of theirspecificityMAbs against Cel7A, Cel6A, and Cel7B as well as PAbagainst Cel7B were a kind gift from Dr. Larry Taylor ofthe National Renewable Energy Laboratory (NREL). PAbsagainst Cel7A and Cel6A were prepared commercially byAlpha Diagnostic International, Texas. Briefly, syntheticpeptides containing amino acid sequence with high anti-genicity from enzymes Cel7A and Cel6A were identifiedand synthesized The peptide sequence used to raise theCel7A PAb was R-A-Q-S-A-C-T-L-Q-S-E-T-H-P-P-L-T-W-Q-K, and that for Cel6A PAb was C-D-T-L-D-K-T-P-L-M-E-Q-T-L-A-D-I-R. Following peptide conjugation,antibodies were raised by immunizing rabbits with thesepeptides. The antibody titers in the rabbit sera and its re-activity to the target peptide were tested using ELISA.Once the test results met the required criteria, the anti-body was then purified from the sera by using affinity col-umns coated with the respective peptide.The specificity of all MAbs and PAbs were first testedagainst purified enzymes and enzyme mixtures by usingthe Western Blot technique following a protocol de-scribed by the assay kit producer (Immun-Blot AssayKit, Bio-Rad). The reactivity and specificity of MAbsagainst all 3 enzymes (Cel7A, Cel6A, and Cel7B) weretested against purified Cel7A from Celluclast and 3commercial enzyme mixtures (30 μg each) Accellerase1000 (Genencor-DuPont), Celluclast, and Cellic CTec 2(Novozymes). PAbs against Cel7A and Cel6A weretested against purified Cel7A and Cel6A from Celluclastas well as the commercial cellulase mixtures Celluclastand Cellic CTec 2. The specificity and reactivity of PAbagainst Cel7B were similarly tested against purifiedCel7A, Cel6A, and Cel7B from Celluclast as well as the3 enzyme mixtures Accellerase 1000, Celluclast, andCellic CTec 2.Briefly, purified enzymes and enzyme mixtures wereseparated using sodium dodecyl sulphate polyacrylamidegel electrophoresis (SDS-PAGE) on 4-12% (w/v) Bis-TrisCriterion XT polyacrylamide gels (Bio-Rad). Followingelectrophoresis, the polyacrylamide gel was equilibratedin the transfer buffer (Towbin buffer containing 25 mMTris, 192 M glycine, and 20% (v/v) methanol) for 30 mi-nutes. The proteins in the polyacrylamide gel were thentransferred to a polyvinylidene difluoride (PVDF) mem-brane using a Trans-Blot Semi-Dry ElectrophoreticTransfer Cell (Bio-Rad) for 60 minutes at 15 V. Afterwashing with Tris-buffered saline containing 0.05% (v/v)Tween 20 (TTBS), the membrane was immersed inTris-buffered saline (TBS) containing 3% (w/v) gelatin toblock any unoccupied sites on the membrane. Anti-bodies to be tested were then added at a concentrationof 5 μg/ml diluted in TTBS containing 1% (w/v) gelatin,and the membrane was incubated for 1 hour. BoundMAbs were detected by immersing the membrane inTTBS-1% (w/v) gelatin containing 1/3000 dilution ofgoat anti-mouse-IgG antibody conjugated to alkalinephosphatase (GAM-AP, Bio-Rad) for 1 hour whereasbound PAbs were detected by using goat anti-rabbit-IgGantibody conjugated to alkaline phosphatase (GAR-AP,Pribowo et al. Biotechnology for Biofuels 2013, 6:80 Page 11 of 15http://www.biotechnologyforbiofuels.com/content/6/1/80Bio-Rad). After a final wash, the membrane wasdeveloped by incubation in the color development/substrate solution containing 5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt (BCIP) and nitro-blue tetrazolium chloride (NBT) for 30 minutes. Thereaction was stopped by immersing the membrane innanopure water for 10 minutes.Optimization of double-antibody sandwich ELISAA double-antibody sandwich ELISA was developed as itwas previously shown to have improved specificity for atarget cellulase enzyme present in a cellulase enzymemixture. MAbs were used as the coating antibodies andPAbs as the detecting antibodies to minimize possibleinterference from other enzymes, sugars and other mate-rials that may be present in the enzyme mixture [23].Unless otherwise stated, all reagents were added at a vol-ume of 100 μl, and incubation was carried out at 37°C.Maxisorp plates (Nunc) were coated with MAb dilutedin 1× phosphate-buffered saline (PBS) pH 7.5 at 4°Covernight. The wells were then washed with PBS andblocked with 2% (w/v) BSA diluted in 1× PBS for 2hours. After the wells were washed, enzyme standardsand/or samples were added to the wells and incubatedfor 2 hours. As antibody-antigen interaction is optimumat pH > 7 [24], the enzyme samples were added to thewells after dilution in PBS pH 7.5 to ensure that the en-zyme samples were in a solution at greater that pH > 7.Purified Cel7A, Cel6A, and Cel7B were serially diluted(concentrations 0–2.5 μg/ml) in PBS to develop standardcurves. After incubation with each of the enzymes, theplate was washed, and the PAb, diluted in PBS with 1%(w/v) BSA, was added to each well. The plate was thenincubated for 1 hour. Following another washing step,the third antibody, a commercial GAR-AP (Bio-Rad)diluted in PBS with 1% (w/v) BSA, was added to thewells and incubated for another hour. After a final wash-ing step, 35 mg/ml of p-nitrophenylphosphate (Bio-Rad),a substrate for alkaline phosphatase (AP), was added tothe wells and the plate was incubated at roomtemperature for 30 minutes or until sufficient colourhad developed. Colour development was stopped byadding 400 mM glycine-NaOH. The amount of enzymesbound to the sandwich ELISA was quantified by measur-ing the absorbance of p-nitrophenyl at 405 nm.Determining the concentrations of the MAb, PAb, and theenzyme-antibody conjugateThe concentrations of the MAb, PAb, and GAR-AP wereoptimized for the Cel7A ELISA. Various concentrationsof each antibody were tested against a series of concen-trations of purified Cel7A. During each antibodyoptimization, the concentrations of the other two anti-bodies were kept constant. MAb’s against Cel7A wastested at two different concentrations of 10 and 50 μg/ml. Once the concentration of the MAb was optimized,the PAb against Cel7A was assayed at concentrations of1.75, 3.5, 7, and 14 μg/ml. Similarly, two different dilu-tions (1/500 and 1/1750 or 1 and 0.3 μg/ml, respect-ively) of the third antibody, (the GAR-AP conjugate)were assessed.Optimization of sample treatmentsAs heat treatment had previously been used successfullyto improve the sensitivity of an ELISA system for Cel7A[23] we investigate the possible influence of heat treat-ment on the ELISA when 5 μg/ml of each of the purifiedenzymes were heated at 100°C for 10 minutes. Each en-zyme was heated in either Na-acetate buffer (0.05 M pH5.0) or in PBS pH 7.5. After cooling the samples to roomtemperature, the enzymes that had been heated in Na-acetate buffer were first diluted with PBS and thenadded to the ELISA plate. Samples heated in PBS weredirectly added to the wells at the same final concentra-tion. Unheated samples were added as controls.Determination of the specificity of ELISAThe specificity of each ELISA was determined by com-paring the ELISA signal of the target enzyme in the ab-sence and presence of the 3 other cellulase enzymes(Cel7A, Cel6A, Cel7B, and Cel5A). The reconstitutedenzyme mixture consisted of 5 μg/ml of the target en-zyme and 2.5 μg/ml of each of the other 3 cellulase en-zymes in Na-acetate buffer (0.05M, pH 5.0). For Cel7Aand Cel6A ELISA, the reconstituted enzyme mixturewas heated at 100°C for 10 minutes, serially diluted inPBS to make a standard curve, and then added to thewell. Similarly, 5 μg/ml of the pure enzyme sample wassubjected to the same treatment. The standard curveobtained from the purified enzyme sample was thencompared with that obtained from the reconstituted en-zyme mixture. The specificity of Cel7B ELISA was deter-mined in a similar manner except that the enzymesamples were not heated but directly added to the wellsafter dilution in PBS. The specificity of ELISA was alsotested using commercial enzyme mixtures to determineif a dilution of a commercial enzyme mixture can beused to construct a standard curve, obviating the needto use purified enzymes. Commercial enzyme mixtureswere diluted in Na-acetate buffer (0.05M, pH 5.0),subjected to the heat treatment when required (i.e. forCel7A and Cel6A ELISA), serially diluted in PBS, andthen added to the wells.Lignocellulosic feedstocks and their pretreatmentAn agricultural residue (corn stover), softwood (Douglas-fir) and hardwood (hybrid poplar) chips were used asfeedstocks and were pretreated by SO2-catalyzed steamPribowo et al. Biotechnology for Biofuels 2013, 6:80 Page 12 of 15http://www.biotechnologyforbiofuels.com/content/6/1/80pretreatment. The pretreatments were performed at nearoptimal conditions that had previously been determinedto provide maximum hemicellulose recovery while ensur-ing effective enzymatic hydrolysis of the cellulose compo-nent (steam pretreatment: corn stover [34], Douglas-fir[35], and poplar [36]). After pretreatment, the celluloserich water insoluble components were washed, filteredand refrigerated for long-term storage. The details ofthe pretreatment conditions and the chemical composi-tions of the pretreated substrates have been describedearlier [36,37].Influence of lignocellulosic derived components presentin the hydrolysis supernatants on the ELISAOther than the enzymes, lignocellulosic hydrolyzates cancontain various materials derived from the biomass suchas soluble phenolic compounds that may interfere withthe ELISA. Therefore, to try to determine the possibleinfluence of these substrate materials on the ELISA’s,lignocellulosic supernatants obtained from steam pre-treated corn stover (SPCS), steam pretreated poplar(SPP), steam pretreated douglas fir (SPDF), and AvicelPH-101 (Sigma), a pure crystalline cellulose substrate,were incubated in 0.05 M Na-acetate buffer pH 5.0 for24 hours at 50°C with rotational mixing in an incubator(Combi-D24) in the absence of any enzymes. After cen-trifugation to remove the solid substrate, a known con-centration of the target enzyme was added to thesesupernatants. The same enzyme concentration diluted in0.05 M Na-acetate buffer pH 5.0 was used as a control.These samples were subjected to heat treatment whenrequired, diluted in PBS and then added to the well. Theinfluence of sugar was not determined as previous workhad shown that sugars did not interfere with the ELISAwhen a MAb was used as the first antibody [23]. As pre-vious work had suggested that “diluting-out” thesesubstrate-derived materials could minimize their inter-ference of the ELISA [20] the supernatants were diluted10 or 100 times with PBS.Enzymatic hydrolysis of SPCSThe enzymatic hydrolysis of SPCS was carried out in 15ml tubes (Corning) in four replicates at 50°C with a rota-tional mixing at 20 rpm. The SPCS was diluted to 2%(w/v) solid loading with Na-acetate buffer (0.05 M, pH5.0) to a total volume of 5 ml. Accellerase 1000 wasadded at 51 mg protein/g glucan, which corresponded to20 FPU/g glucan. Similarly, SPCS hydrolysis was alsocarried out using Celluclast at 20 FPU/g glucan or 52mg protein/g glucan. Concurrently, SPCS was also incu-bated in Na-acetate buffer (0.05 M, pH 5.0), in the ab-sence of enzymes, to serve as a substrate alone control(SPCS SC).During hydrolysis, samples were taken at differenttime points over a period of 72 hours. After centrifuga-tion, the unbound proteins in the supernatant wererecovered by transferring the supernatant into 15 mltubes. One ml of the supernatant was collected andheated at 100°C for 10 minutes for subsequent glucosemeasurement using the glucose oxidase assay [38]. Theremaining supernatant was stored at 4°C for subsequentELISA assay using the optimized conditions to deter-mine any changes in Cel7A, Cel6A, and Cel7B concen-trations during hydrolysis.The development of a double-antibody sandwich ELISAto quantify Cel7A, Cel6A, and Cel7B adsorption duringSPCS hydrolysisELISA plates were incubated with 10 μg/ml of MAb inPBS at 4°C overnight. The wells were then washed withPBS and blocked with 2% (w/v) BSA diluted in PBS for 2hours. After the wells were washed, enzyme standardsand/or samples were added to the wells and incubatedfor 2 hours. For the Cel7A and Cel6A ELISA’s, beforethe addition of samples to the ELISA plate, the purifiedenzyme samples or the hydrolysate samples were firstheated at 100°C for 10 minutes. The heat treatment wasalways done in Na-acetate buffer (0.05 M pH 4.8). Aftercooling to room temperature, the samples were dilutedin PBS and then added to the ELISA plate. This dilutionin PBS not only adjusted the pH of the added samplesbut also diluted any interfering materials that might bepresent in lignocellulosic supernatants. After incubationwith the enzyme samples for 2 hours, the plate waswashed with PBS. A PAb toward the enzyme of interestwas added at a concentration of 14 μg/ml diluted in PBScontaining 1% (w/v) BSA. The plate was then incubatedfor 1 hour. Following another washing step, the thirdantibody, a commercial GAR-AP (Bio-Rad) diluted 1/500 in PBS containing 1% (w/v) BSA, was added and in-cubated for another hour. After a final washing step, p-nitrophenylphosphate (Bio-Rad) was added, and theplate was incubated until sufficient colour had devel-oped. The colour development was stopped by adding400 mM glycine-NaOH. The amount of enzymes boundto the sandwich ELISA was quantified by measuring theabsorbance of p-nitrophenyl at 405 nm.By following this protocol, the amount of Cel7A,Cel6A, and Cel7B present in SPCS hydrolysates (unboundproteins) during 72-hour hydrolysis could be quantified.Purified Cel7A, Cel6A, and Cel7B were used to makestandard curves. In each of the ELISA assays, the SPCSSC were included and treated in the same way as the hy-drolysate samples, to determine the possible influence ofany materials in the hydrolysates. The initial enzyme inbuffer without any substrate (enzyme control-EC) wasalso included, to determine the initial concentration ofPribowo et al. Biotechnology for Biofuels 2013, 6:80 Page 13 of 15http://www.biotechnologyforbiofuels.com/content/6/1/80each enzyme. Protein samples for Cel7A ELISA wereobtained from SPCS hydrolysis using 20 FPU/ g cellu-lose of Accellerase 1000. Those for Cel6A and Cel7BELISA were obtained from SPCS hydrolyzed by 20FPU/ g Celluclast complemented with 40 CBU/ g cellu-lose of β-glucosidase.AbbreviationsBSA: Bovine serum albumin; CMC: Carboxymethyl cellulose; ELISA:Enzyme-linked immunosorbent assay; FPLC: Fast Protein LiquidChromatography; GAM-AP: Goat anti-mouse IgG antibody conjugated toalkaline phosphatase; GAR-AP: Goat anti-rabbit IgG antibody conjugated toalkaline phosphatase; IP: Immune-precipitation; MAb: Monoclonal antibody;PAb: Polyclonal antibody; MW: Molecular weight; PBS: Phosphate-bufferedsaline; SPCS: Steam pretreated corn stover; SPP: Steam pretreated poplar;SPDF: Steam pretreated Douglas-fir.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsAll authors contributed jointly to all aspects of the work reported in themanuscript. AP carried out much of the laboratory work, contributed toplanning, interpretation of results and drafting of the paper. JH contributedto the purified enzymes used in the study. VA contributed to the planning,interpretation and drafting. JS contributed to the planning, interpretationand writing of the manuscript. All authors read and approved the finalmanuscript.AcknowledgementThe authors would like to gratefully acknowledge Dr. Larry Taylor from theNational Renewable Energy Laboratory (NREL) for generously providingsome of the antibodies used in this study. The authors also thankGenencor-DuPont and Novozymes for providing the enzymes used in thisstudy. The support of Genome BC Canada and Natural Sciences andEngineering Research Council of Canada (NSERC) are gratefullyacknowledged.Received: 6 February 2013 Accepted: 10 May 2013Published: 20 May 2013References1. 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