Development and Evaluation of a Monoclonal Antibody-Based

Article pubs.acs.org/JAFC

Development and Evaluation of a Monoclonal Antibody-Based Inhibition ELISA for the Quantification of Chymosin in Solution O. Rolet-Répécaud,† C. Arnould,† D. Dupont,‡,§ S .Gavoye,∥ E. Beuvier,† and C. Achilleos*,† †

INRA, UR342 Technologie et Analyses Laitières, F-39800 Poligny, France INRA, UMR1253 Science et Technologie Lait & Oeuf, F-35042 Rennes, France § AGROCAMPUS OUEST, UMR1253 Science & Technologie Lait & Oeuf, F-35042 Rennes, France ∥ ACTALIA, F-25620 Mamirolle, France ‡

ABSTRACT: Chymosin is the major enzyme of natural rennet, traditionally used in cheese making for its high milk-clotting activity. For technical reasons, an accurate characterization of rennet should include its total clotting activity and also its enzymatic composition. Monoclonal antibodies specific to chymosin were obtained from mice immunized with purified bovine chymosin, and an inhibition enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of chymosin in solution. No cross-reactivity was observed with other milk-clotting enzymes commonly used in cheese making. The limit of detection and limit of quantification were 125 and 400 ng/mL, respectively. The values of precision within and among runs were 7.23 and 7.39%, respectively, and satisfying recovery, from 92 to 119%, was found for spiked samples. The inhibition ELISA was successfully applied to commercial rennets, and the results were consistent with those obtained using the standard chromatographic method (IDF 110: A, 1987). KEYWORDS: chymosin, inhibition ELISA, monoclonal antibody, rennet

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INTRODUCTION Chymosin (EC 3.4.23.4) is a neonatal gastric aspartic proteinase and is of commercial importance in cheese making. Chymosin is secreted in the abomasal mucosa of newborn ruminants during the first days of life. Its natural function is the hydrolysis of κ-casein once the milk is in the calf’s stomach, leading to the formation of a coagulum that can be easily digested. Once extracted from gastric tissues, chymosin and pepsin (EC 3.4.23.1) constitute the main clotting enzymes of calf, kid, and lamb rennets. In cheese technology, rennet is traditionally added to the vat during the first stage of cheese making to coagulate the milk. Chymosin hydrolyses the Phe105−Met106 bond of κ-casein which stabilizes the casein micelles and thereby induces an aggregation reaction leading to a gel and a phase separation of the milk into cheese curd and whey. A fraction of the added chymosin is retained in the curd,1 which, in most types of cheese, catalyzes the slower hydrolysis of other peptide bonds in casein.2,3 In the dairy industry, milk-clotting enzymes are characterized by their milk-clotting activity (MCA) and their general proteolytic activity. The ratio between those two properties gives an overview of the essential quality of a milk-clotting enzyme. Only the enzymes with a high MCA/ general proteolytic activity ratio are considered suitable for cheese manufacturing; high levels of nonspecific proteolysis lead to a weak gel structure, high losses of protein and fat in the whey, and reduced cheese yield.4 Depending on technological factors for cheese making, residual active enzymes remain in the curd and can affect cheese maturation and its final quality of flavor/texture.1,4 In comparison to other milk-clotting enzymes, chymosin is superior in combining a high MCA with a low general proteolytic activity. Chymosin represents >90% of the © 2015 American Chemical Society

MCA of a good-quality calf rennet, the remaining activity being due to pepsin.5 Rennets available on the market have different values for cheese making because of their enzymatic composition. The relative proportion of the enzymes varies, depending on the age of the animal and its diet. In rennets extracted from young milk-fed calves, chymosin represents 88−94% of the clotting enzymes and pepsin represents 6−12%, whereas pepsin predominates in extracts from older fodder-eating bovines.6 Rennets are generally prepared from multiple calf stomachs and are heterogeneous in their chymosin content and chymosin/ pepsin ratio. Moreover, chymosin and pepsin have different reaction rates in their specific hydrolysis of κ-casein and show quite different effects in their proteolytic action during cheese ripening.7 Therefore, for economic and quality reasons, the most important parameters of rennet to be analyzed are the MCA and the composition, i.e., the nature and the concentration of the enzymes. To define a rennet accurately, information should be given on its total MCA with its chymosin/pepsin ratio or its composition in active chymosin and pepsin. Today, the total MCA of a bovine rennet is determined according to an international standard method8 and is expressed in international milk-clotting units (IMCU). For commercialization in France, current regulations require the MCA to be expressed in milligrams of active enzyme, chymosin, and pepsin per liter9 to give information on the enzyme composition whereas IMCU expresses a global activity. Received: Revised: Accepted: Published: 4799

February April 17, April 23, April 23,

23, 2015 2015 2015 2015 DOI: 10.1021/acs.jafc.5b00990 J. Agric. Food Chem. 2015, 63, 4799−4804

Article

Journal of Agricultural and Food Chemistry

Millipore, Darmstadt, Germany) was allowed to permeate monolayers of mixed cells adsorbed on membrane filters. The fused cells were propagated in an azaserine/hypoxanthine (AH) selection medium containing 10% horse serum (Life Technologies SAS, Saint-Aubin, France) and plated in 48-well microculture plates incubated at 37 °C with a 6% CO2 atmosphere. Culture supernatants of resulting hybridomas were collected during the log growth phase of cells and screened for the presence of antibodies that recognized chymosin by indirect ELISA. To avoid cross-reactivity, secreting hybridomas were also screened for bovine and porcine pepsins by indirect ELISA. Selected positive hybridomas were subsequently subcloned by the limiting dilution method and then cryopreserved in liquid nitrogen. The ascites from the selected clones were obtained according to Jones et al.21 and purified by a saturated ammonium sulfate (SAS) precipitation followed by affinity chromatography. Briefly, 75 g of ammonium sulfate were dissolved in 100 mL of distilled water at 80 °C and cooled. Four volumes of SAS were added drop to drop with gentle stirring to 6 volumes of ascite. Stirring was continued at 4 °C for at least 60 min; then the mixture was centrifuged at 3220g for 6 min at 4 °C. The supernatant was removed, and the precipitated fraction was suspended in 5 volumes of 100 mM phosphate buffer, 150 mM NaCl, pH 7.4. Then, 5 volumes of SAS were added as previously described, followed by a centrifugation at 3220g for 6 min at 4 °C. The supernatant was removed, and finally the precipitated fraction of immunoglobulins was suspended in 4 volumes of 100 mM phosphate buffer, 150 mM NaCl, pH 7.4. The resultant solution was dialyzed overnight at 4 °C in a dialysis bag (molecular weight cutoff 10 kDa) against 1000 volumes of 10 mM PBS, 150 mM NaCl. To purify mAbs, affinity chromatography was used with a HiTrap NHS-activated HP column (GE Healthcare Bio-Sciences, Uppsala, Sweden) as described by Muller-Renaud et al.22 Briefly, 50 mg of chymosin in 5 mL of coupling buffer (200 mM NaHCO3, 500 mM NaCl, pH 8) were covalently immobilized on the column. Any excess active groups were deactivated by washing with 500 mM ethanolamine, 500 mM NaCl, pH 8.3, and the nonspecifically bound ligands were washed out with 100 mM acetate, 500 mM NaCl pH 4. One milliliter of ascitic fluid prepurified by SAS precipitation, diluted in 7 mL of running buffer (50 mM PBS, pH 7.3), was then injected. The column was washed with running buffer. Antichymosin mAbs were eluted with 10 mM glycine-HCl buffer, pH 2.1, and the eluted fraction was neutralized by the addition of 1:15 (v/v) 1 M Tris-HCl, pH 9. The purified mAbs were then divided into aliquots and stored at −20 °C. The isotypes of the produced mAbs were determined by a mouse monoclonal antibody isotyping kit (RD-Biotech, Besançon, France) according to the manufacturer’s instructions. All animals were maintained in accordance with the French Ministry of Agriculture ethical guidelines for the care and use of laboratory animals (B 21 231 010 EA). Characterization of mAbs. By Indirect ELISA. Culture supernatants were screened by indirect ELISA to assess their specificity for chymosin and their possible cross-reactivity against other milk-clotting enzymes: rennets from different animal sources (calf, kid, and lamb), recombinant chymosin, bovine and porcine pepsins, and microbial coagulants. Microtiter plates (NUNC F96 Maxisorp, 4000 Roskilde, Denmark) coated with 100 μL/well of milk-clotting enzymes (1 μg/ mL in 100 mM sodium bicarbonate buffer, pH 9.6 (CB)) were incubated for 90 min at 37 °C. The remaining binding sites were blocked by incubation with 10 g/L gelatin (Merck Millipore, Darmstadt, Germany) in PBS 0.05% Tween 20 (PBS-T) (250 μL/ well) for 1 h at 37 °C. Culture supernatants (100 μL/well) previously diluted 1:2 in PBS-T were then added. After 1 h of incubation at 37 °C, 100 μL/well of donkey antimouse immunoglobulin alkaline phosphatase conjugate (Jackson Immunoresearch, Interchim, Montluçon, France) diluted 1:3000 in PBS-T were added. Bound immunoglobulins were detected after 1 h of incubation at 37 °C. Between each incubation step, the plate was rinsed four times for 15 s with PBS-T (250 μL/well) using a HydroFlex microplate washer (TECAN, Männedorf, Switzerland). Finally, 100 μL/well of pnitrophenyl phosphate (Interchim, Montluçon, France) containing 1 g/L of a solution of 1 M diethanolamine-HCl, 1 mM MgCl2, and 0.1

The enzyme composition has been determined with different methods based on spectrophotometric measurements of the proteolytic activities,10 chromatographic separation with MCA determination,11 selective inactivation,12 and rocket immunoelectrophoresis.13 More recently, rennets have been characterized by high-performance liquid chromatography (HPLC)14 and by mass spectrometry.15 The conventional method for the enzymatic characterization of rennets is a chromatographic one.9,16,17 First, a desalted sample of rennet is separated by chromatography into two fractions, chymosin and pepsin. Then, the MCA of each of the separated enzymes is measured and converted to weight contents of chymosin and pepsin calculated in percentages16,17 or in mg of active enzyme/L.9 Despite being sensitive, the chromatographic method is timeconsuming with laborious pretreatment procedures. Antibodies have widely been used as analytical tools in various methods developed for the detection and quantification of proteins, enzymes, vitamins, allergenic ingredients, and contaminants such as toxins, pesticides, and drugs in food.18 Immunoassays are easy to perform, rapid, with a large number of samples analyzed at the same time, and give very high sensitivity and specificity, in particular when based on monoclonal antibodies (mAbs). The high specificity of these methods decreases the need for thorough cleanup; therefore, sample-preparation procedures in immunoassays are generally rather simple. Furthermore, mAbs have the advantage of a continuous supply of constant quality. The aim of the present study was to produce and characterize mAbs specific to bovine chymosin. An mAb-based inhibition enzyme-linked immunosorbent assay (ELISA) specific to the quantification of chymosin in solution was developed. The described assay was applied to the analysis of rennet samples and compared to the IDF standard method (chromatography and MCA measurement)16 to assess its applicability to the direct quantification of chymosin. Moreover, inhibition ELISA may allow measurements of low chymosin concentrations in complex matrices such as milk, whey, and cheese curd in order to determine chymosin inactivation according to cheese-making steps and its remaining activity in the cheese curd, provided that an efficient method of enzyme extraction is developed.

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MATERIALS AND METHODS

Chemicals and Samples. Analytical-grade chemicals were purchased from VWR International (Fontenay-sous-Bois, France). Purified chymosin (rennin from calf stomach) and porcine pepsin were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Calf, kid, and lamb rennets, recombinant chymosin, bovine pepsin, and microbial coagulants were obtained from different suppliers (Chr. Hansen, Denmark; Danisco, Denmark; Laboratoires ABIA, France; Laboratoire Central des Présures S.A.S., France; and Ö sterreichische Laberzeugung Hundsbichler GmbH, Austria). Production, Purification, and Isotyping of mAbs. Immunization with chymosin was carried out by the foot pad route according to Jeanson et al.19 Lyophilized chymosin (1.2 mg/mL in 10 mM phosphate-buffered saline pH 7.2 (PBS)) was 1.5-fold diluted in physiological water in order to obtain a 800 μg protein/mL solution. This solution was then emulsified 1:1 (v/v) in Freund adjuvant (Difco Laboratories, Detroit, MI, USA), complete for the first immunization, incomplete for the booster injection 14 days later. Fifty microliters of the emulsion (i.e., 20 μg of proteins) were injected into two female BALB/c mice (Iffa-Credo, St-Germain sur l’Arbresle, France) per foot pad. Three days after the booster injection, popliteal lymph nodes from immunized mice were removed and pooled. Lymphocytes were fused in a 5:1 ratio with Sp2/O-Ag14 myeloma cells using filter fusion according to Buttin et al.20 Poly(ethylene glycol) (PEG 1000, Merck 4800

DOI: 10.1021/acs.jafc.5b00990 J. Agric. Food Chem. 2015, 63, 4799−4804

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Journal of Agricultural and Food Chemistry

Validation of the Assay. Immunoassay validation was performed using the limit of detection (LOD), the limit of quantification (LOQ), the precision, and the accuracy. LOD and LOQ were expressed as the chymosin concentration corresponding to the average measured content of 20 blank samples plus 3 or 10 times the standard deviation (SD) of the average value, respectively.26 The precision was expressed as the relative standard deviation (RSD), measured as the % ratio between the standard deviation of independent results and their mean value. It was estimated with independent results obtained by the same operator in 1 day on 10 successive analyses of the same rennet sample (intra-assay RSD) and on 12 analyses of the same rennet sample in a small time interval (interassay RSD). The interassay RSD was calculated for a low-chymosin rennet (179 mg/L) and a highchymosin rennet (676 mg/L). All measurements were made in duplicate at three different dilutions. The accuracy was measured as the recovery of exogenous amounts of chymosin, 22, 45, 90, 225, and 450 mg/L, added to a rennet sample (179 mg/L). The accuracy was expressed as the % recovery between the ELISA value and the theoretical value ((detected concentration/concentration of added chymosin) × 100). Statistical Analyses. Chymosin concentrations in commercial rennet samples determined by IDF standard chromatography16 and inhibition ELISA were evaluated and compared using Deming regression analysis and the paired samples t test (Analyze-it 3.90.2, Analyze-it Software Ltd., U.K.). For Deming regression, the intercept value reflects the constant bias and the slope reflects the proportional bias. Agreement was considered to be good if the 95% confidence interval (CI) of the intercept and slope included 0 and 1, respectively.

mM zinc acetate were added to the wells. After 30 min, the absorbance at 405 nm (A405 nm) was read against a blank (substrate only) using a NanoQuant Infinite F200 Pro microplate spectrophotometer (TECAN, Männedorf, Switzerland). By Western Blotting. Chymosin and milk-clotting enzymes were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% acrylamide Novex Tris-glycine gel (Life Technologies SAS, Saint-Aubin, France) according to the manufacturer’s instructions. All samples were reduced before analysis using 100 mM DL-dithiothreitol and loaded onto each lane (10 μg/lane). Electrophoresis was performed at 125 V, variable intensity, for 2 h at 10 °C in 25 mM Tris, 192 mM glycine, 0.1% SDS buffer, pH 8.4. Precision Plus Protein Kaleidoscope standards (Life Technologies SAS, Saint Aubin, France) were used as the molecular-weight standard. Immediately after separation, proteins were transferred onto a 0.2-μmpore-size nitrocellulose membrane (Protran BA83 NC, Whatman GE Healthcare Life Sciences − Europe GmbH, Velizy-Villacoublay, France) for immunoblotting using a TE22 Mighty Small Transfer Tank (Hoefer, Inc., Holliston, MA, USA) at 100 V, variable intensity, for 70 min at 10 °C in 25 mM Tris, 200 mM glycine, 0.01% SDS buffer in 200 mL/L ethanol. The immunodetection was performed as described by Dupont et al.23 By Biacore. Real-time binding assays between purified chymosin and purified mAbs were performed using a Biacore 3000 optical biosensor (GE Healthcare Life Sciences − Europe GmbH, VelizyVillacoublay, France) equipped with a carboxymethyl-dextran-coated (CM5) research-grade sensor chip. The experiments were performed at 21 °C in HBS-EP running buffer (GE Healthcare Life Sciences). Standard amine coupling24 was used to covalently immobilize rabbit antimouse antibodies (RAM) (GE Healthcare Life Sciences) at a final density of 10 000 response units (RUs). First, mAbs (20 μL) were injected over the RAM surface, and then 20 μL of purified chymosin, 1 mg/mL in HBS-EP buffer, was injected over the flow cell and were able to bind to mAbs in a continuous, pulse-free, controlled flow at a flow rate of 20 μL/min. Data were collected during the 60 s association and 100 s dissociation phases. Regeneration of the chip was achieved with 5 μL of 10 mM glycine-HCl buffer, pH 1.7. A flow cell saturated with 1 M ethanolamine was used as a reference for background subtraction and to check for nonspecific binding. Data analysis was performed using BIAevaluation v3.2 software (GE Healthcare Life Sciences). The binding ability of an mAb toward chymosin was defined as the percentage of chymosin bound compared to the theoretical maximum binding capacity of the mAb (Rmax).25 Reactivity against Active Chymosin. The specificity of mAbs against active chymosin was evaluated by indirect ELISA against heattreated rennet samples. Two different rennets, A and B, pH adjusted to 6.54, were split into four aliquots (500 μL). One aliquot was used as reference, and the other three aliquots were submitted to a heat treatment by incubation in a block heater (Lab-line, Thermo Fisher Scientific Inc., Ashville, NC, USA): 60 °C/2 min 30 s; 80 °C/2 min, and 100 °C/2 min. For each aliquot, indirect ELISA was performed as described previously, and the MCA was estimated as described in ref 9. Quantitative Inhibition ELISA. ELISA plates NUNC F96 Maxisorp were coated with 1 μg/mL chymosin in CB (100 μL/ well) and incubated for 1 h at 37 °C. Blocking of the remaining binding sites was performed as previously described. Serial dilutions of a rennet of known chymosin concentration (determined according to an IDF standard16) in PBS-T were used as standards (concentrations ranging from 0 to 20 000 ng/mL). The chymosin standards or the samples diluted in PBS-T (150 μL) were incubated in test tubes with 150 μL of purified antichymosin mAb (1:10 000 in PBS-T) for 1 h at 37 °C. The mixture (100 μL/well) was then added to the ELISA plate and further incubated for 1 h at 37 °C. The plate washing steps, the revelation of the reaction, and the measurement of the absorbance were performed as already described. The standard curve was obtained by plotting the A405 nm obtained from each standard against its chymosin concentration (ng/mL). The unknown concentration of chymosin in samples was calculated from the standard curve and adjusted for the dilution factor. Each sample concentration resulted from the mean of three diluted samples analyzed in duplicate.

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RESULTS AND DISCUSSION mAbs Production and Characterization. One fusion experiment yielded 847 hybridomas. Screening by chymosincoated indirect ELISA allowed a selection of 42 clones which secreted chymosin-specific mAbs. These clones were used for further investigations based on mAbs specificity. The mAbs tested were of IgG isotypes: 60% IgG1, 20% IgG2a, and 20% IgG2b. Cross-Reactivity Measurements. Cross-reactivity of mAbs was evaluated by indirect ELISA and immunoblotting against purified chymosin and other milk-clotting enzymes commonly used in cheese making: rennets extracted from different animal sources (calf, kid, and lamb), recombinant chymosin, bovine pepsin, porcine pepsin, and microbial coagulants. Specific mAbs recognized chymosin present in rennets from all of the different animal sources (calf, kid, and lamb) and in recombinant chymosins whereas no cross-reactivity was detected with bovine and porcine pepsins and microbial coagulants with both methods. As observed by immunoblotting (Figure 1), specific mAbs recognized a protein band of 35 kDa, which coincided well with the expected size of chymosin. mAbs also recognized minor bands between 35 and 25 kDa which were identified as degraded chymosin in rennets and recombinant chymosins.27 These results indicated that mAbs were specific to chymosin whatever its source and thus were suitable for use in the immunoassay. Binding Interactions between mAbs and Chymosin. To investigate the binding ability of selected mAbs to chymosin, mAbs were screened with a Biacore 3000 optical biosensor. An overview of the assay is shown in Figure 2. Late binding, at the end of the association phase, gives information about the affinity of chymosin for the bound mAb. Late stability, at the end of the dissociation phase, corresponds to the stability of the interaction between chymosin and the bound mAb. The best mAb for further ELISA development should present a high affinity for chymosin with a slow dissociation rate. The binding 4801

DOI: 10.1021/acs.jafc.5b00990 J. Agric. Food Chem. 2015, 63, 4799−4804

Article

Journal of Agricultural and Food Chemistry

Thermal Inactivation of Chymosin. The MCA was affected by the temperature. Holding the rennet at 60 °C for 2 min 30 s decreased its MCA. The clotting time was higher for heattreated rennet samples: 2.25 and 1.97-times higher for rennet A and B, respectively. A 2 min heating at 80 and 100 °C totally inactivated chymosin: no coagulation was observed after 6 h 30 min for both rennets. The optimum temperature for the coagulation of milk by calf rennet at pH 6.6 is around 45 °C.5 Indirect ELISA performed on heat-treated rennets showed that the different mAbs tested were specific to chymosin in its active form (Figure 3). The absorbance decreased for high temper-

Figure 1. mAbs specificity was characterized by immunoblotting. (A) SDS-PAGE of different milk-clotting preparations. The amount of sample loaded onto each lane of the 12% gel was 10 μg. (B) Western immunoblot of the fractions obtained in (A) incubated with antichymosin mAb (1:1500). Band 1, protein molecular weight marker (kDa); band 2, purified chymosin (highlighted by an arrow); band 3, bovine serum albumin; band 4, calf rennet; band 5, lamb rennet; band 6, kid rennet; bands 7 and 8, recombinant chymosin; band 9, bovine pepsin/chymosin extract; bands 10 and 11, bovine pepsin; band 12, porcine pepsin; bands 13 and 14, microbial coagulants.

Figure 3. Reactivity of antichymosin mAbs evaluated by indirect ELISA against two different rennets submitted to heat treatment. (Light-gray) not heated; (medium-gray) 60 °C/2 min 30 s; (darkgray) 80 °C/2 min; (black) 100 °C/2 min. The pH of rennet samples was adjusted to 6.54.

atures, 80 and 100 °C. At these temperatures, the inactivated chymosin was not detected by the different mAbs in rennet A but was partially detected in rennet B. Finally, one positive clone named mAb 438(11) was selected for the development of the inhibition ELISA. This selected mAb was of the IgG1 isotype. It showed a high specificity for chymosin according to indirect ELISA and immunoblotting and a high binding ability toward chymosin according to Biacore results: 98.84% Rmax. Characteristics of the Assay. The assay was validated by investigating the LOD, the LOQ, the precision, and the accuracy. The standard curve for the quantification of chymosin was obtained from serial dilutions of a rennet of known chymosin concentration (Figure 4). A good linear correlation between A 405 nm and the chymosin concentration was represented by A405 nm = 1.093−0.0005[chymosin], R2 = 0.882, in the range of 200−2000 ng/mL. The LOD and LOQ were 125 and 400 ng chymosin/mL, respectively. The precision was measured by the relative standard deviation (RSD) of samples within and among runs. The RSDs within runs and among runs were 7.23 and 7.39%, respectively, for both low- and high-chymosin rennets, which indicated that there were minimum variations between wells and limited dayto-day variability. The assay had good precision values. Moreover, the level of precision of the immunoassay remained competitive compared to the standard chromatography: 7 vs 5%.17 ELISA recovery values of chymosin from the spiked rennet samples ranged from 92 to 119% (Figure 5) with a mean recovery value of 102% for a chymosin range from 22 to 450 mg/L. Inhibition ELISA gave acceptable recovery efficiency. These results demonstrated the potential application of immunoassay for the quantification of chymosin in solution. Application to Commercial Rennets. To assess the applicability of inhibition ELISA for quantification of chymosin

Figure 2. Association and dissociation curves for purified chymosin (1 mg/mL) binding to different mAbs. mAbs were bound to rabbit antimouse antibodies immobilized on a Biacore CM5 sensor chip. Data were collected during the 60 s association and the 100 s dissociation phases. In the association phase, chymosin interacts with the bound mAbs. In the dissociation phase, the complex resulting from the interaction starts to dissociate.

ability of mAbs was calculated from the binding of purified chymosin in percent of maximum theoretical binding, Rmax. Assuming that the interaction between chymosin and mAb is monovalent, the binding abilities of the tested mAbs varied between 23.03 and 139.80% Rmax. All tested mAbs were shown to bind to chymosin, but some mAbs had low binding abilities. Values >100% Rmax may be attributed to the mass transport effect between the chymosin in the flow and the mAbs immobilized on the surface of the sensor chip which can potentially perturb the 1:1 interaction. Only mAbs with a binding ability of >85% Rmax were kept for further analyses. 4802

DOI: 10.1021/acs.jafc.5b00990 J. Agric. Food Chem. 2015, 63, 4799−4804

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Journal of Agricultural and Food Chemistry

Figure 4. Representative chymosin standard curve obtained with the optimized ELISA. Data are summarized from seven independent curves generated over a 1 month period. The error bar represents the standard deviation of the mean.

Figure 6. Deming regression fit between chymosin concentrations (mg/L) measured by inhibition ELISA and chromatography in different rennet samples. Sixteen commercial rennets over the range of 200 to 840 mg chymosin/L representative of the diversity available on the market were analyzed. The black line represents the fitted regression line ([chymosin]ELISA = 1.004 + 0.984[chymosin]chromatography), and the dotted gray line represents the identity line (methods equal).

Table 1. Correlation between the Developed Immunoassay and the Standard Chromatographic Method by Deming Regression parameter

estimate

jackknife 95% CIa

jackknife SEa

p valueb

slope intercept

0.984 1.004

0.922 to 1.045 −29.25 to 31.26

0.029 14.107

0.575 0.944

a

Confidence intervals (CI) and standard errors of the parameter estimates (SE) were calculated with the jackknife (leave one out resampling) method. bSignificantly equal to 1 (slope) and 0 (intercept): p > 0.05.

Figure 5. Recovery of chymosin (%) from artificially spiked rennet samples determined by ELISA. Five different amounts of chymosin (22, 45, 90, 225, and 450 mg/L) were added to a rennet sample.

pretreatments and that it provided a noticeable advantage over methods requiring laborious pretreatment procedures. In conclusion, a hybridoma cell line secreting mAbs specific to chymosin was obtained, and chymosin-specific mAbs were used to develop an inhibition ELISA for the quantification of chymosin in solution. The immunoassay has been characterized, optimized, and validated using rennet samples. The comparison of the developed inhibition ELISA with the reference chromatographic method showed the potential of the immunoassay. It provides a versatile alternative tool with high sample throughput and with no pretreatment of the sample for a direct quantification of chymosin, unlike the reference method which is a two-step method with timeconsuming pretreatment procedures. Besides, work is underway to quantify (1) bovine pepsin in solution by ELISA in order to completely characterize rennets regarding their enzymatic composition and (2) residual chymosin in cheese by ELISA in order to characterize its remaining activity in the cheese.

in solution, 16 commercial rennets, with low and high chymosin contents representative of the diversity available on the market, were analyzed. The concentration of chymosin in the samples was determined with both inhibition ELISA and standard chromatography.16 Concentrations obtained by ELISA were statistically similar to concentrations obtained with the standard chromatographic method (Figure 6) using the Deming regression analysis, which considers both methods to be subject to measurement error whereas simple regression allows only the immunoassay concentration to be measured with error.28 The Deming regression equation between ELISA and chromatography results was [chymosin]ELISA = 1.004 + 0.984[chymosin]chromatography (R2 = 0.995). The 95% CI for the slope and the intercept contained the values 1 (p = 0.575) and 0 (p = 0.944), respectively (Table 1). In addition to the Deming regression analysis, a comparative evaluation test was carried out to measure how different the immunoassay and the chromatographic method are. A paired-samples t test was performed with the following results: t(15) = 1.34, p = 0.200. These results indicated that there was no significant difference at the 5% significance level between the two methods. These statistical results indicated that the developed inhibition ELISA could be useful in quantifying chymosin in solution without any

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AUTHOR INFORMATION

Corresponding Author

*Tel: 33 3 84 73 63 00. Fax: 33 3 84 37 37 81. E-mail: [email protected]. 4803

DOI: 10.1021/acs.jafc.5b00990 J. Agric. Food Chem. 2015, 63, 4799−4804

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This work was supported by funding from the Direction Régionale de l’Alimentation, de l’Agriculture et de la Forêt Franche-Comté (Besançon, France), and the Centre National Interprofessionnel de l’Economie Laitière (CNIEL, Paris, France). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank M. H. Duployer (INRA UR342, URTAL Poligny, France) for technical assistance and R. Berges and C. Belloir (INRA UMR1324, CSGA, Dijon, France) for providing access to the animal housing facility and the cell culture laboratory.

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ABBREVIATIONS USED A405 nm, absorbance at 405 nm; CB, bicarbonate buffer; CI, confidence interval; ELISA, enzyme-linked immunosorbent assay; IDF, International Dairy Federation; IMCU, international milk-clotting unit; mAb, monoclonal antibody; MCA, milk-clotting activity; PBS, phosphate-buffered saline; RAM, rabbit antimouse; RU, response unit; SAS, saturated ammonium sulfate

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DOI: 10.1021/acs.jafc.5b00990 J. Agric. Food Chem. 2015, 63, 4799−4804