Purification of cellulase monocomponents, Cel7A, Cel6A, Cel7B, and Cel5A
The cellulase monocomponents Cel7A, Cel6A, Cel7B, and Cel5A were purified from Celluclast (Novozyme) using previously described methods [12, 30–32]. The Ninhydrin assay  was then used to determine the concentrations of these purified enzymes as well as the commercial enzyme mixtures. Bovine serum albumin (BSA, Sigma) was used as the protein standard.
Preparation of antibodies and determination of their specificity
MAbs against Cel7A, Cel6A, and Cel7B as well as PAb against Cel7B were a kind gift from Dr. Larry Taylor of the National Renewable Energy Laboratory (NREL). PAbs against Cel7A and Cel6A were prepared commercially by Alpha Diagnostic International, Texas. Briefly, synthetic peptides containing amino acid sequence with high antigenicity from enzymes Cel7A and Cel6A were identified and synthesized The peptide sequence used to raise the Cel7A 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 these peptides. The antibody titers in the rabbit sera and its reactivity to the target peptide were tested using ELISA. Once the test results met the required criteria, the antibody was then purified from the sera by using affinity columns coated with the respective peptide.
The specificity of all MAbs and PAbs were first tested against purified enzymes and enzyme mixtures by using the Western Blot technique following a protocol described by the assay kit producer (Immun-Blot Assay Kit, Bio-Rad). The reactivity and specificity of MAbs against all 3 enzymes (Cel7A, Cel6A, and Cel7B) were tested against purified Cel7A from Celluclast and 3 commercial enzyme mixtures (30 μg each) Accellerase 1000 (Genencor-DuPont), Celluclast, and Cellic CTec 2 (Novozymes). PAbs against Cel7A and Cel6A were tested against purified Cel7A and Cel6A from Celluclast as well as the commercial cellulase mixtures Celluclast and Cellic CTec 2. The specificity and reactivity of PAb against Cel7B were similarly tested against purified Cel7A, Cel6A, and Cel7B from Celluclast as well as the 3 enzyme mixtures Accellerase 1000, Celluclast, and Cellic CTec 2.
Briefly, purified enzymes and enzyme mixtures were separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 4-12% (w/v) Bis-Tris Criterion XT polyacrylamide gels (Bio-Rad). Following electrophoresis, the polyacrylamide gel was equilibrated in the transfer buffer (Towbin buffer containing 25 mM Tris, 192 M glycine, and 20% (v/v) methanol) for 30 minutes. The proteins in the polyacrylamide gel were then transferred to a polyvinylidene difluoride (PVDF) membrane using a Trans-Blot Semi-Dry Electrophoretic Transfer Cell (Bio-Rad) for 60 minutes at 15 V. After washing with Tris-buffered saline containing 0.05% (v/v) Tween 20 (TTBS), the membrane was immersed in Tris-buffered saline (TBS) containing 3% (w/v) gelatin to block any unoccupied sites on the membrane. Antibodies to be tested were then added at a concentration of 5 μg/ml diluted in TTBS containing 1% (w/v) gelatin, and the membrane was incubated for 1 hour. Bound MAbs were detected by immersing the membrane in TTBS-1% (w/v) gelatin containing 1/3000 dilution of goat anti-mouse-IgG antibody conjugated to alkaline phosphatase (GAM-AP, Bio-Rad) for 1 hour whereas bound PAbs were detected by using goat anti-rabbit-IgG antibody conjugated to alkaline phosphatase (GAR-AP, Bio-Rad). After a final wash, the membrane was developed 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. The reaction was stopped by immersing the membrane in nanopure water for 10 minutes.
Optimization of double-antibody sandwich ELISA
A double-antibody sandwich ELISA was developed as it was previously shown to have improved specificity for a target cellulase enzyme present in a cellulase enzyme mixture. MAbs were used as the coating antibodies and PAbs as the detecting antibodies to minimize possible interference from other enzymes, sugars and other materials that may be present in the enzyme mixture . Unless otherwise stated, all reagents were added at a volume of 100 μl, and incubation was carried out at 37°C. Maxisorp plates (Nunc) were coated with MAb diluted in 1× phosphate-buffered saline (PBS) pH 7.5 at 4°C overnight. The wells were then washed with PBS and blocked with 2% (w/v) BSA diluted in 1× PBS for 2 hours. After the wells were washed, enzyme standards and/or samples were added to the wells and incubated for 2 hours. As antibody-antigen interaction is optimum at pH > 7 , the enzyme samples were added to the wells after dilution in PBS pH 7.5 to ensure that the enzyme 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 standard curves. After incubation with each of the enzymes, the plate was washed, and the PAb, diluted in PBS with 1% (w/v) BSA, was added to each well. The plate was then incubated 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 the wells and incubated for another hour. After a final washing step, 35 mg/ml of p-nitrophenylphosphate (Bio-Rad), a substrate for alkaline phosphatase (AP), was added to the wells and the plate was incubated at room temperature for 30 minutes or until sufficient colour had developed. Colour development was stopped by adding 400 mM glycine-NaOH. The amount of enzymes bound to the sandwich ELISA was quantified by measuring the absorbance of p-nitrophenyl at 405 nm.
Determining the concentrations of the MAb, PAb, and the enzyme-antibody conjugate
The concentrations of the MAb, PAb, and GAR-AP were optimized for the Cel7A ELISA. Various concentrations of each antibody were tested against a series of concentrations of purified Cel7A. During each antibody optimization, the concentrations of the other two antibodies were kept constant. MAb’s against Cel7A was tested 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 of 1.75, 3.5, 7, and 14 μg/ml. Similarly, two different dilutions (1/500 and 1/1750 or 1 and 0.3 μg/ml, respectively) of the third antibody, (the GAR-AP conjugate) were assessed.
Optimization of sample treatments
As heat treatment had previously been used successfully to improve the sensitivity of an ELISA system for Cel7A  we investigate the possible influence of heat treatment on the ELISA when 5 μg/ml of each of the purified enzymes were heated at 100°C for 10 minutes. Each enzyme was heated in either Na-acetate buffer (0.05 M pH 5.0) or in PBS pH 7.5. After cooling the samples to room temperature, the enzymes that had been heated in Na-acetate buffer were first diluted with PBS and then added to the ELISA plate. Samples heated in PBS were directly added to the wells at the same final concentration. Unheated samples were added as controls.
Determination of the specificity of ELISA
The specificity of each ELISA was determined by comparing the ELISA signal of the target enzyme in the absence and presence of the 3 other cellulase enzymes (Cel7A, Cel6A, Cel7B, and Cel5A). The reconstituted enzyme mixture consisted of 5 μg/ml of the target enzyme and 2.5 μg/ml of each of the other 3 cellulase enzymes in Na-acetate buffer (0.05M, pH 5.0). For Cel7A and Cel6A ELISA, the reconstituted enzyme mixture was heated at 100°C for 10 minutes, serially diluted in PBS to make a standard curve, and then added to the well. Similarly, 5 μg/ml of the pure enzyme sample was subjected to the same treatment. The standard curve obtained from the purified enzyme sample was then compared with that obtained from the reconstituted enzyme mixture. The specificity of Cel7B ELISA was determined in a similar manner except that the enzyme samples were not heated but directly added to the wells after dilution in PBS. The specificity of ELISA was also tested using commercial enzyme mixtures to determine if a dilution of a commercial enzyme mixture can be used to construct a standard curve, obviating the need to use purified enzymes. Commercial enzyme mixtures were diluted in Na-acetate buffer (0.05M, pH 5.0), subjected to the heat treatment when required (i.e. for Cel7A and Cel6A ELISA), serially diluted in PBS, and then added to the wells.
Lignocellulosic feedstocks and their pretreatment
An agricultural residue (corn stover), softwood (Douglas-fir) and hardwood (hybrid poplar) chips were used as feedstocks and were pretreated by SO2-catalyzed steam pretreatment. The pretreatments were performed at near optimal conditions that had previously been determined to provide maximum hemicellulose recovery while ensuring effective enzymatic hydrolysis of the cellulose component (steam pretreatment: corn stover , Douglas-fir , and poplar ). After pretreatment, the cellulose rich water insoluble components were washed, filtered and refrigerated for long-term storage. The details of the pretreatment conditions and the chemical compositions of the pretreated substrates have been described earlier [36, 37].
Influence of lignocellulosic derived components present in the hydrolysis supernatants on the ELISA
Other than the enzymes, lignocellulosic hydrolyzates can contain various materials derived from the biomass such as soluble phenolic compounds that may interfere with the ELISA. Therefore, to try to determine the possible influence of these substrate materials on the ELISA’s, lignocellulosic supernatants obtained from steam pretreated corn stover (SPCS), steam pretreated poplar (SPP), steam pretreated douglas fir (SPDF), and Avicel PH-101 (Sigma), a pure crystalline cellulose substrate, were incubated in 0.05 M Na-acetate buffer pH 5.0 for 24 hours at 50°C with rotational mixing in an incubator (Combi-D24) in the absence of any enzymes. After centrifugation to remove the solid substrate, a known concentration of the target enzyme was added to these supernatants. The same enzyme concentration diluted in 0.05 M Na-acetate buffer pH 5.0 was used as a control. These samples were subjected to heat treatment when required, diluted in PBS and then added to the well. The influence of sugar was not determined as previous work had shown that sugars did not interfere with the ELISA when a MAb was used as the first antibody . As previous work had suggested that “diluting-out” these substrate-derived materials could minimize their interference of the ELISA  the supernatants were diluted 10 or 100 times with PBS.
Enzymatic hydrolysis of SPCS
The enzymatic hydrolysis of SPCS was carried out in 15 ml tubes (Corning) in four replicates at 50°C with a rotational mixing at 20 rpm. The SPCS was diluted to 2% (w/v) solid loading with Na-acetate buffer (0.05 M, pH 5.0) to a total volume of 5 ml. Accellerase 1000 was added at 51 mg protein/g glucan, which corresponded to 20 FPU/g glucan. Similarly, SPCS hydrolysis was also carried out using Celluclast at 20 FPU/g glucan or 52 mg protein/g glucan. Concurrently, SPCS was also incubated in Na-acetate buffer (0.05 M, pH 5.0), in the absence of enzymes, to serve as a substrate alone control (SPCS SC).
During hydrolysis, samples were taken at different time points over a period of 72 hours. After centrifugation, the unbound proteins in the supernatant were recovered by transferring the supernatant into 15 ml tubes. One ml of the supernatant was collected and heated at 100°C for 10 minutes for subsequent glucose measurement using the glucose oxidase assay . The remaining supernatant was stored at 4°C for subsequent ELISA assay using the optimized conditions to determine any changes in Cel7A, Cel6A, and Cel7B concentrations during hydrolysis.
The development of a double-antibody sandwich ELISA to quantify Cel7A, Cel6A, and Cel7B adsorption during SPCS hydrolysis
ELISA plates were incubated with 10 μg/ml of MAb in PBS at 4°C overnight. The wells were then washed with PBS and blocked with 2% (w/v) BSA diluted in PBS for 2 hours. After the wells were washed, enzyme standards and/or samples were added to the wells and incubated for 2 hours. For the Cel7A and Cel6A ELISA’s, before the addition of samples to the ELISA plate, the purified enzyme samples or the hydrolysate samples were first heated at 100°C for 10 minutes. The heat treatment was always done in Na-acetate buffer (0.05 M pH 4.8). After cooling to room temperature, the samples were diluted in PBS and then added to the ELISA plate. This dilution in PBS not only adjusted the pH of the added samples but also diluted any interfering materials that might be present in lignocellulosic supernatants. After incubation with the enzyme samples for 2 hours, the plate was washed with PBS. A PAb toward the enzyme of interest was added at a concentration of 14 μg/ml diluted in PBS containing 1% (w/v) BSA. The plate was then incubated for 1 hour. Following another washing step, the third antibody, a commercial GAR-AP (Bio-Rad) diluted 1/500 in PBS containing 1% (w/v) BSA, was added and incubated for another hour. After a final washing step, p-nitrophenylphosphate (Bio-Rad) was added, and the plate was incubated until sufficient colour had developed. The colour development was stopped by adding 400 mM glycine-NaOH. The amount of enzymes bound to the sandwich ELISA was quantified by measuring the absorbance of p-nitrophenyl at 405 nm.
By following this protocol, the amount of Cel7A, Cel6A, and Cel7B present in SPCS hydrolysates (unbound proteins) during 72-hour hydrolysis could be quantified. Purified Cel7A, Cel6A, and Cel7B were used to make standard curves. In each of the ELISA assays, the SPCS SC were included and treated in the same way as the hydrolysate samples, to determine the possible influence of any materials in the hydrolysates. The initial enzyme in buffer without any substrate (enzyme control-EC) was also included, to determine the initial concentration of each enzyme. Protein samples for Cel7A ELISA were obtained from SPCS hydrolysis using 20 FPU/ g cellulose of Accellerase 1000. Those for Cel6A and Cel7B ELISA were obtained from SPCS hydrolyzed by 20 FPU/ g Celluclast complemented with 40 CBU/ g cellulose of β-glucosidase.