Milk Matrix Effects on Antibody Binding Analyzed by Enzyme-Linked

Mar 30, 2015 - Vector Laboratories (Burlingame, CA). Asialofetuin was obtained from. Sigma (St. Louis, MO). The principal buffers and milk samples use...
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Milk Matrix Effects on Antibody Binding Analyzed by Enzyme-Linked Immunosorbent Assay and Biolayer Interferometry David L. Brandon* and Lisa M. Adams Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, United States Department of Agriculture (USDA) Agricultural Research Service, 800 Buchanan Street, Albany, California 94710, United States ABSTRACT: Biolayer interferometry (BLI) was employed to study the impact of the milk matrix on the binding of ricin to asialofetuin (ASF) and to antibodies. This optical sensing platform used ligands immobilized covalently or via biotin− streptavidin linkage, and the results were compared to those obtained by enzyme-linked immunosorbent assay (ELISA). In sandwich ELISA, the binding of ricin to ASF was dramatically decreased when galactose was present during the analyte or detection antibody binding step. Low concentrations of milk (1%, v/v) produced a similar reduction in ricin binding to ASF but not to a high-affinity monoclonal antibody (mAb), increasing the dissociation rate of ASF−ricin complexes up to 100-fold. The effect of milk on the binding of ricin to ASF was ascribable to dialyzable factors, and milk sugar can account for these effects. The use of high-affinity mAbs in ELISA effectively limits the milk matrix effect on ricin analysis. KEYWORDS: ricin, Ricinus communis agglutinin, castor, monoclonal antibody, biolayer interferometry, milk, asialofetuin

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INTRODUCTION To analyze food samples by immunoassay, the analytes must generally be in solution and reasonably free from interfering constituents. Although liquid foods, such as milk, are easily handled matrices, each food or food extract is a mixture of many components, including those that bind and sequester the analyte or the immunological sensor molecule, usually antibody.1 Storage and processing of foods also present challenges to the analytical process, rendering some proteins less soluble, for example, or modified by Maillard browning reactions, with a demonstrable effect on the epitopes recognized by antibodies in immunoassay.2 In the case of protein toxins, food matrix effects have been demonstrated in both immunoassay3,4 and activity assays,5 and inhibition of ricin binding and activity in the presence of milk has been noted.6,7 In immunoassays of ricin in food matrices, detection by electrochemiluminescence overcame the high blank and nonlinear recovery in ground beef,3 suggesting that the enzyme detection step was most sensitive to the matrix. Matrix interference by low-fat milk and liquid chicken egg was eliminated by dilution of samples 100-fold in the cell-free protein translation assay of ricin.5 A more extensive study of the liquid egg matrix indicated that a component of some individual egg yolks produced a high false-positive response in both enzyme-linked immunosorbent assay (ELISA) and electrochemical luminescence (ECL) detection systems.8 Commercial liquid egg used by the food industry is prepared in lots that comprise many thousands of eggs and was the intended matrix modeled in that study. Assays of such samples obtained in marketing channels did not suffer from the occasional high false positives found among liquid egg prepared in our laboratory from one or a few eggs. The fat content of milk samples influenced recovery, with poor recovery of ricin from spiked cream, but a 5-fold dilution produced consistent 80% recoveries for milk.4 There was a minor influence of the protein content on recovery. Milk was This article not subject to U.S. Copyright. Published 2015 by the American Chemical Society

also demonstrated to inhibit the activity of ricin, measured by changes in recombinant green fluorescent protein (GFP) expression in a Vero cell line system,6 consistent with earlier reports that lactose-based carbohydrates could block the binding step essential for ricin toxicity. 9,10 However, interpretation of milk effects is complicated by observations that low concentrations of milk stimulated production of recombinant GFP in these cells and also had a positive effect on cell viability.7 Another technique for conducting immunoanalyses is biolayer interferometry (BLI), an optical sensing technique for observing binding reactions in real time,11 using dippable sensors without microfluidics. As the association and dissociation processes change the biolayer thickness at the sensor tips, the interference pattern is altered to produce a measurable change in the maximal wavelength of reflected light. In the present study, we used BLI and ELISA to study the effects of milk on the interaction of ricin with monoclonal antibodies (mAbs) and asialofetuin (ASF). Ricin is a highly toxic dimeric protein that comprises an active N-glycosidase in its A chain and a binding site for galactosamine, Nacetylglucosamine, and related sugars in its B chain.12 ASF contains 12 terminal galactose residues that serve as models for cellular ricin-binding sites, with an equilibrium dissociation constant of ∼2−3 nM.13 A plate-based assay using ASF was developed for assessing procedures to develop antibodytargeted immunotoxin conjugates based on ricin A-chain activity,14 and the method was adapted to characterize inhibitors of ricin lectin activity.15 Ricin and the closely related and immunologically crossreactive Ricinus communis agglutinin-1 (RCA-1), a lectin with Received: Revised: Accepted: Published: 3593

October 1, 2014 March 20, 2015 March 21, 2015 March 30, 2015 DOI: 10.1021/acs.jafc.5b01136 J. Agric. Food Chem. 2015, 63, 3593−3598

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hemagglutinating activity,16 are both found in the seed meal after extraction of the valuable oil, constraining the development of castor as an alternative to petroleum for energy and as a raw material for a wide variety of industrial and consumer products.17 The immunochemistry of ricin has been extensively studied, particularly to optimize therapeutic immunotoxins containing ricin A chain18 and to define potentially protective and therapeutic antibodies.19 Exploitation of ricin antibodies and the amplification afforded by the polymerase chain reaction (PCR) has contributed new tools for food defense.20,21 In an earlier study, we used BLI to study the interaction of ricin and RCA-1 with antibodies.22 In this report, we describe BLI and ELISA experiments to quantitate milk matrix effects and mitigate their impact on the analysis of a carbohydrate-binding protein, such as ricin.

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MAbs and ELISA. The monoclonal antibodies that bind ricin were described previously.3,23 MAbs were isolated from hybridoma culture supernatants by hydrophobic charge-induction chromatography on MEP-Hypercel (Pall Life Sciences, Port Washington, NY)24 and affinity chromatography on Protein G-agarose (Pierce, Rockford, IL) in accordance with the protocols of the manufacturers. Purified mAbs were stored at 0.5−1 mg/mL in the presence of NaN3 and a mixture of serine, cysteine, and acid protease inhibitors (Sigma). Biotinylated mAbs (b-mAbs) were prepared using N-hydroxysuccinimide (NHS)− biotin ester (Pierce) with an initial ester/antibody molar ratio of 10. ELISA was performed as described previously,4 and data were fit to a logistic equation (y = a0 + a1/(1 + (x/a2)a3) using SlideWrite, version 7.01 (Advanced Graphics, Rancho Santa Fe, CA). Coefficient a2 is the analyte concentration at half-maximal binding (EC50). Briefly summarized, analytical samples and immunoreagents (ricin, mAb dilutions, biotinylated mAbs, and diluted HRP−SA conjugate) were prepared and applied to ELISA wells with 30 min incubations. Spiked milk samples were incubated for 10 min before application to ELISA wells. Ricin-containing analyte and the first wash from ELISA are collected by a pipet and inactivated using sodium hypochlorite solution. After the analyte binding step and subsequent wash, biotinylated mAb (b-mAb 1443 at 100 ng/mL, unless noted otherwise) was added (100 μL/well) and incubated for 30 min. After an additional wash step, ELISAs were developed with HRP−SA and K-Blue tetramethylbenzidine substrate (Neogen, Lexington, KY). The reaction was stopped by the addition of 100 μL of 0.3 M HCl, and A450 nm − A650 nm was determined using a M2 microplate reader (Molecular Devices, Sunnyvale, CA). Two or three well replicates were averaged for each ELISA data point, and experiments were conducted in triplicate, unless otherwise noted. ELISA on Asialofetuin-Coated Microwells. Ricin (0, 1.25, 2.5, 5, 10, 20, 40, 80, and 100 ng/mL or a subset of these standards as shown on graphs) and b-mAb 1443 (100 ng/mL) were added sequentially, with intervening wash steps. Effect of Milk in ELISA on ASF- and mAb 1797-Coated Microwells. Ricin (10 ng/mL) was prepared in neat and diluted 0% fat fresh milk in PBST + BSA. The ELISA was then conducted and developed under standard conditions with b-mAb 1443, HRP−SA, and substrate. ELISA with Ricin-Spiked Milk or Milk Sequentially Applied. For the “spiked” condition, ricin (10 ng/mL) was added to buffer or to milk at various dilutions with a 10 min pre-incubation before application to wells and incubation for 30 min. In the “sequential” condition, ricin in buffer was applied to the wells. After incubation (30 min) and washing, the dilutions of milk were added to the wells for a further incubation of 30 min. For both conditions, plates were washed and b-mAb 1443 (100 ng/mL) was applied to detect bound ricin. Assays were developed with HRP−SA and substrate. To determine the time course for changes in ASF-bound ricin during the incubation with 1% milk in the “sequential” condition, 100 μL of milk was added to the ELISA wells containing ASF-bound ricin but removed at various times from 1 to 30 min and replaced with the standard ELISA buffer, BPT. Sugar Effect on Ricin Binding to mAb 1797. Ricin was used as the analyte (20 ng/mL in control buffer or 0.133 M galactose or lactose). ELISA used mAb 1797-coated ELISA wells and b-mAb 1642 or b-mAb 1443 (100 ng/mL; n = 2, for each condition) as the detection antibody. Experiments were also conducted in kinetics buffer to mimic conditions used for BLI experiments. The dose−response effect of galactose was determined using 0, 14.7, 44.3, and 133 mM galactose. Multiple Sample Comparisons. For multiple sample comparison of binding data under differing conditions, one-way analysis of variance (ANOVA) was used with Tukey’s post hoc honestly significant difference (HSD) procedure to determine which means were significantly different (Statgraphics Centurion, version 15, Statpoint Technologies, Warranton, VA). A p value of 1000 ng/mL). This pattern of binding was expected from the known lectin activity of ricin,25 and the reversibility of ricin binding reflects that ASF is only a moderate-affinity ricin receptor.13 Effect of Milk on Binding of Ricin to mAbs and ASF in ELISA. The effect of milk on the binding of ricin to mAbs and ASF was studied by ELISA using mAb- and ASF-coated microwells. The results of one set of assays are illustrated in Figure 2 and show that nonfat milk inhibits 50% of ricin binding at about 0.3% (v/v) for ASF. Comparable inhibition of binding to mAb 1797 occurred at a milk concentration of 55% (v/v). The effect of milk on ricin binding to ASF was further explored to determine whether the reduction of ricin binding to ASF by milk required that milk be present during initial ricin binding, the “spiked” milk condition. This condition was compared to a “sequential” ELISA in which milk was added to assay wells after the ricin-binding and wash steps were completed. The results are illustrated in Figure 3. The results show that milk does not have to be present during the binding step to have an inhibitory effect. The inset graph shows the time course for removal of previously ASF-bound ricin in the presence of milk. The process is nearly complete within 30 min, under the 1% milk condition. The effect of milk on ricin binding to ASF is consistent with the demonstration that milk could abrogate the effect of ricin on Vero cells.6 Cellular and in vivo ribosome-inactivating activity begins with binding of ricin to cellular receptors.26 We next conducted preliminary experiments that showed dialysis relieved the effect of milk

Figure 3. ELISAs using microplate wells coated with ASF conducted under two conditions: (◆) ricin (10 ng/mL) spiked into buffer or diluted milk applied to wells with 30 min of incubation and (●) ricin in buffer applied to the wells, incubated, and washed, followed sequentially by incubation with dilutions of milk. (Inset) Time course for removal of ASF-bound ricin during the incubation with 1% milk in the “sequential” condition.

on binding to ASF (data not shown). It seemed a reasonable hypothesis that sugars found in milk could account for this observation. Bovine milk contains approximately 48 g/L (0.14 M) lactose, β-D-galactopyranosyl-(1 → 4)-D-glucose,27 the principal milk sugar, and we next tested the hypothesis. Effect of Sugars on Ricin Capture by mAb 1797 in ELISA. Figure 4 shows the effect of sugars on the detection of ricin in ELISA using mAb 1797 for capture. When present in the analyte “capture” incubation step, galactose and lactose inhibited binding, producing about 20 and 40% inhibition, respectively, at 0.133 M. Means for galactose and lactose were significantly different from controls (p < 0.05, paired t test). Similar results were obtained when ELISAs used kinetics buffer (1 mg/mL), instead of PBST + BSA (10 mg/mL BSA), to mimic matrix conditions used in BLI studies described below. The inset graph shows a representative data set from three experiments determining the dose dependence of the galactose effect. Experiments were also performed with other capture/b3595

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Figure 4. Effect of sugars on binding of 20 ng/mL ricin to mAb 1797, with b-mAb 1642 or b-mAb 1443. The cross-hatched bars illustrate results with ricin in KB, and the open bars illustrate results with ricin in BPT. (Inset) Galactose concentration dependence for ELISA in kinetics buffer with b-mAb 1443.

mAb detection sandwich ELISAs, but no significant sugar effects were observed (data not shown), suggesting that the inhibition observed with mAb 1797/b-mAb 1443 was epitopedependent. Studies Using BLI. To identify which steps in ricin binding to ASF and mAbs are affected by components of milk, we next determined rate constants for binding. Ricin Binding to ASF. For these experiments, ASF was immobilized on AR sensor tips as ligand. The ricin analyte (1 μg/mL) was present in buffer or various types of milk (0.5%, v/ v, in kinetics buffer), with or without prior dialysis versus PBS. The upper panel of Figure 5 shows the ka values determined for ricin binding in four milk samples, undialyzed and dialyzed, and the lower panel of Figure 5 shows the dissociation constants. Multiple-sample comparison of the four control and four dialyzed milk samples indicated no significant differences among the means of either group of samples. For NFDM, the control sample ka was significantly higher than the dialyzed (p = 0.012), but the difference was not significant for the other three samples. The values of kd for all dialyzed fresh milk samples were lower than corresponding values for controls, but the difference was not significant for NFDM. Dialysis thus resulted in stronger binding of ricin in the fresh milk samples. Ricin Binding to mAb 1797 in a BLI Sandwich Configuration. We wished to analyze ricin binding in BLI by mimicking ELISA conditions using the Octet biosensor. The first two layers of the “sandwich” were applied in turn as follows. Capture mAb 1797 was immobilized on AR sensors, and then sensors were moved to wells containing ricin in buffer with or without reconstituted NFDM (2%, v/v). After ricin binding, sensors were moved immediately to wells containing b-mAb 1443, for the upper layer of the sandwich, as for ELISA, although no secondary conjugate was used or needed in the BLI experiment that relied on the optical properties of the biolayer to provide a signal. The results are shown in Figure 6. Overall, the results indicate that ricin binds well to mAb 1797 in the presence or absence of milk, whether dialyzed against PBS or not. This is consistent with results that we reported4 on the effectiveness of mAb 1797 as a capture antibody for ELISA

Figure 5. BLI analysis of ricin binding to ASF immobilized on AR sensor tips: (top panel) association constants, ka, and (bottom panel) dissociation rate constants, kd.

of ricin in food matrices. As in the ELISA, b-mAb 1443 is incubated in buffer, without milk, and is able to provide the upper layer of the sandwich on this platform. Once ricin is tightly bound to mAb 1797, it is available to bind b-mAb 1443. Ricin is thought to have three galactose-binding sites28−30 and can therefore interact with ASF with much higher avidity than indicated by the relatively low association constants of individual sites. The higher affinity binding of mAb 1797 compared to ASF, even with lower probability of multiple site binding, overcomes the milk effect on dissociation of ricin from the antibody binding site. Effect of Milk on Dissociation of Ricin from mAb 1797. The ELISA results above (Figure 3) indicated that milk inhibits the binding of ricin even in the “sequential” procedure. The effect of milk on dissociation of the ricin−mAb complex was studied using biotinylated mAb 1797 immobilized on SA sensors. To achieve greater accuracy near the limit of instrument sensitivity, this experiment included varying percentages of reconstituted NFDM in kinetics buffer during the dissociation step. These conditions were expected to minimize re-association of ricin with immobilized mAb, a procedure referred to as a sink method.31 Figure 7 shows the effect of analysis of ricin binding to mAb 1797 (b-mAb 1797 immobilized on SA sensor tips) in the absence or presence of milk. The association constant, ka, was not affected significantly by the presence of milk, but the 3596

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concept seemed relevant to the analysis of binding by mAb 1797. Even so, the contribution of divalent binding may also be significant, and increased dissociation in the presence of milk could result from abrogation of divalent binding. In either event, estimation of the dissociation rate constant was facilitated by the presence of a binder during dissociation. Although sensing techniques, such as BLI and surface plasmon resonance, use sophisticated, bulky instrumentation to measure optical properties of a surface that are influenced by the binding reactions in the “biolayer”, miniaturized implementations of both sensor platforms have appeared in the marketplace. The practical use of these platforms in the field, in addition to the laboratory, will likely be realized in the near future, providing well-characterized, portable systems for detecting analytes that impact the safety and authenticity of the food supply. The results demonstrated that the effects of milk on the binding of ricin to carbohydrate receptors, as modeled by ASF, are mediated principally by dialyzable factors and that the use of high-affinity mAbs in ELISA mitigates the milk matrix effect on ricin analysis.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +1-510-559-5783. Fax: +1-510-559-5880. E-mail: [email protected].

Figure 6. BLI analysis using a mAb sandwich: (A) typical sensorgrams from one set of experiments (ricin in dialyzed milk) and (B) response (biolayer thickness) and association and dissociation rates shown for three conditions.

Funding

This study was undertaken under Project 2030-42000-048 of the National Research Program 108 (Food Safety) and was facilitated by the Western Regional Research Center student internship program (to Lisa M. Adams). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge Lily L. Yang for assistance with initial BLI studies, Anna M. Korn for antibody purification and biotinylation, and Matthew A. Taylor for assistance with ELISA (initially as a Bridge-to-Biosciences intern from City College of San Francisco).

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ABBREVIATIONS USED ANOVA, analysis of variance; AR, amine reactive; ASF, asialofetuin; b-mAb, biotinylated monoclonal antibody; BLI, biolayer interferometry; BPT, phosphate-buffered saline with Tween 20 with 10 mg/mL bovine serum albumin; BSA, bovine serum albumin; GFP, green fluorescent protein; HRP, horseradish peroxidase; HSD, honestly significant difference; NFDM, nonfat dry milk; RCA-1, Ricinus communis agglutinin-1; SA, streptavidin

Figure 7. BLI analysis of ricin binding to b-mAb 1797 on SA sensor tips, with dissociation conducted under various conditions.

dissociation rate increased in the presence of milk. The multisample comparison test showed that the 50 and 100% milk samples were significantly different from the 0% control at the 95% confidence level, and the 25% sample was significantly different only at the 90% confidence level (Tukey’s HSD test). In this study, we demonstrated that the effect of milk on ricin binding to an antibody and a model receptor could be observed using BLI and ELISA, with complementary results. We also tested the use of a binding molecule as a “sink” to prevent the rebinding of analyte to a very high affinity ligand, mAb 1797. In this case, the sink was milk, a food. The “sink concept” in the context of well-based binding measurements refers to the use of a high-affinity ligand in solution that prevents rebinding of dissociated analyte to the sensing ligand.31,32 The system can therefore emulate microfluidic-based systems that remove dissociating ligand from the sensor vicinity by flow. Because the antibody binding is fit well using a 1:1 binding model, this

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