Monoclonal Antibodies with Group Specificity Toward Sulfonamides: Selection of Hapten and Antibody Selectivity

Anal Bioanal Chem (2013) 405:4027–4037 DOI 10.1007/s00216-013-6785-5

ORIGINAL PAPER

Monoclonal antibodies with group specificity toward sulfonamides: selection of hapten and antibody selectivity

Zhanhui Wang & Ross C. Beier & Yajie Sheng & Suxia Zhang & Wenxiao Jiang & Zhaopeng Wang & Jin Wang & Jianzhong Shen

Received: 19 October 2012 /Revised: 10 December 2012 /Accepted: 24 January 2013 /Published online: 16 February 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Immunoassays based on the current available anti- Keywords Sulfonamides . Group specificity . Monoclonal bodies for large multi-sulfonamide screening programs have antibody . Hapten suffered from high selectivity for individual sulfonamides and a wide range of selectivities for different sulfonamides. In this study, five synthesized haptens, HS, BS, CS, SA10, and TS Introduction and two sulfonamides, SG and SMX were used as haptens, which may or may not contain a ring structure at the N1 Sulfonamides, a group of synthetic chemotherapeutics, are position of the sulfonamides, were selected to evaluate the widely used in human medicine as well as in veterinary effectiveness for producing group-specific monoclonal anti- medicine for treatment of infectious diseases and as bodies (MAbs). Mice immunized with three different two-ring growth-promoting feed additives [1]. Foodstuffs derived haptens were used for hybridoma production, which resulted from animals treated with sulfonamides may be contaminat- in three unique MAbs recognizing 10, 13, and 15 sulfona- ed with sulfonamide residues. To protect consumers from mides showing 50 % inhibition (IC50) at concentrations below the risk related to these residues, maximum residue limits 100 ngmL–1. MAb 4D11 derived from one novel immunizing (MRLs) have been established throughout the world. The hapten could recognize 12 sulfonamides with IC50 values European Union and China have established MRLs for total ranging from 1.2 to 12.4 ngmL–1, almost within 1 order of sulfonamides in edible animal tissues and milk at 100 μgkg–1 magnitude. These produced MAbs show lower IC50 values in [2, 3]. Currently, more than 20 different sulfonamides are addition to significantly improved group specificity compared used in human and animal medicine (Fig. 1). For example, with previously generated MAbs. This study clearly indicates at least nine and six sulfonamides are permitted to be used in that the careful selection of the immunizing hapten has an veterinary medicine in the Netherlands and Czech Republic, important effect on the specificity of the generated antibodies. respectively [4, 5]. The use of sulfonamides in China was estimated to be around 20,000 tons in 2003, and one third of this quantity was used in animal production. The five sulfo- Electronic supplementary material The online version of this article namides, sulfamethazine (SMZ), sulfadimethoxine (SDM), (doi:10.1007/s00216-013-6785-5) contains supplementary material, sulfaquinoxaline (SQX), sulfamonomethoxine (SMM), and which is available to authorized users. sulfamethoxazole (SMX), have been approved for use in Z. Wang : Y. Sheng : S. Zhang : W. Jiang : Z. Wang : J. Wang : veterinary medicine and are required to be monitored in J. Shen (*) meat, milk, and eggs. Department of Veterinary Pharmacology and Toxicology, The most frequently used methods for detection of sulfo- College of Veterinary Medicine, China Agricultural University, namides are based on chromatography and mass spectrom- Beijing 100193, China e-mail: [email protected] etry techniques [1, 6, 7]. These methods are sensitive and specific, but cleanup and preconcentration steps consisting R. C. Beier of liquid–liquid extraction followed by solid-phase extrac- Food and Feed Safety Research Unit, Southern Plains Agricultural tion procedures are required, which make screening large Research Center, Agricultural Research Service, U.S. Department of Agriculture, 2881 F&B Road, numbers of samples in a timely way very difficult. An College Station, TX 77845-4988, USA effective alternative for screening large numbers of samples 4028 Z. Wang et al.

Sulfonamides with single-ring

Sulfonamides with two-ring containing a five-atom-ring at N1position

b Sulfonamides with two-ring containing a six-atom-ring at N1 position

Other analytes related to sulfonamides

Fig. 1 Chemical structures of sulfonamides used for cross-reactivity listed in alphabetical order according to the abbreviations of the sulfo- studies. The sulfonamides were sorted by the number of rings and then namides in each group the number of atoms in the ring at the N1 position. The haptens were Monoclonal antibodies with group specificity toward sulfonamides 4029 is the enzyme-linked immunosorbent assay (ELISA) which follows: (1) compare and evaluate the suitability of seven provides some advantages, such as the ability to process haptens including one new immunizing hapten for inducing many samples in a timely way, simplified protocols that may the formation of group-specific antibodies; (2) generate a be conducted in a routine laboratory, and is cost effective. MAb with group specificity and high sensitivity to be used For efficient surveillance purposes, an immunoassay that in a screening test for at least the five sulfonamides ap- can detect multi-sulfonamides rather than a specific sulfon- proved for use in China; and (3) study the selectivity of amide is preferable. To achieve this goal, the generation of the generated MAbs to explain the effect of hapten structure antibodies with group specificity is necessary. Much effort on the produced antibodies, which will help design a more has been made by different researchers to develop group- effective hapten that can be used to produce a MAb with the specific antibodies to sulfonamides; however, the reported best group-specificity for the sulfonamides. antibodies are usually too specific and generally recognize only a small group of sulfonamides. The polyclonal antibodies and recombinant antibody produced by Franek et al. Materials and methods [4] and Korpimaki et al. [8], respectively, were capable of simultaneously detecting 17 sulfonamides at the regulatory Materials and apparatus levels. However, these antibodies had a diverse set of IC50 values [4, 8] and therefore, a large range of detection N-acetylsulfanilyl chloride, 4-aminobenzoic acid methyl ester, concentrations. 4-aminobenzenebutyric acid methyl ester, ethyl 2-amino-4- A monoclonal antibody (MAb) does provide some thiazoleacetate, 6-aminohexanoic acid, and 4-aminobutanoic advantages since it has high reproducibility with unlimited acid were purchased from ChemBridge Corp. (San Diego, supply. Only three research groups have reported the produc- CA). Bovine serum albumin (BSA), ovalbumin (OVA), N- tion of sulfonamide-specific MAbs. Muldoon et al. produced a hydroxysuccinimide (NHS), and 1-(3-dimethylaminopropyl)- MAb, Sulfa-1, that could recognize eight sulfonamides at 3-ethyl carbodiimide (EDC) were purchased from Sigma- concentration values below 10 μgmL–1 [9]. The best MAb Aldrich (St. Louis, MO). Dulbecco’smodifiedEagle’s media prepared by Haasnoot et al., MAb 27 G3, could recognize used for cell culture was obtained from Huamei (Beijing, eight sulfonamides showing 50 % inhibition (IC50) values People’s Republic of China). Incomplete Freund’s adjuvant, below 100 ngmL–1 [10]. The MAb developed by Cliquet et complete Freund’s adjuvant, and fetal calf serum were al. could detect five sulfonamides with IC50 values ranging obtained from Gibco BRL (Carlsbad, CA). All other chem- from 9 to 100 ngmL–1 [5]. To the authors’ knowledge, there icals and solvents were of analytical grade or better and were has been no further report on producing a broad-specificity obtained from Beijing Chemical Reagent Co. (Beijing, MAb that was group specific for sulfonamides since Cliquet et People’s Republic of China). Polystyrene microplates were al. [5]. Unlike polyclonal antibodies that can recognize multi- purchased from Corning Inc. (Costar 2592, Corning, NY). epitopes of an antigen, a MAb only recognizes a single epi- The ELISA plate reader was obtained from TECAN Inc. tope in a very defined and specific manner; therefore, the (Durham, NC). Deionized water was prepared using a Milli- generation of a true group-specific MAb is a large challenge. Q water purification system (Millipore, Bedford, MA). All In a previous study, we demonstrated that careful hapten sulfonamides were obtained from Sigma-Aldrich (St. Louis, design combined with extensive hybridoma screening can MO) or Dr. Ehrenstorfer (Augsburg, Germany), and the abbre- result in MAbs with high sensitivity and relatively mean viations used in the text are shown with the sulfonamide (similar) selectivity for a set of fluoroquinolones [11]. In structures in Fig. 1. N4-acetyl-sulfamethazine (acetyl-SMZ) previous reports designed to produce an antibody that could was previously synthesized in our lab [12]. recognize a broad-range of sulfonamide structures, the structure of the hapten has been regarded as the most important Buffers and solutions factor in the generation of antibody selectivity [4, 5]. Al- though structurally different haptens have been synthesized The following buffers were used in the ELISA: (1) coating and used for immunization [5, 8, 10], the effectiveness of buffer was 0.05 M carbonate buffer, pH 9.6; (2) blocking these haptens to induce a group-specific antibody response buffer consisted of 0.01 M phosphate-buffered saline (PBS), is questionable, and the influence of the hapten structure on 0.5 % BSA, 0.01 % NaN3, and 0.05 % Tween 20, pH 7.4; antibody selectivity for a broad group of sulfonamide struc- (3) washing buffer was PBS with 0.05 % Tween 20; (4) tures is not yet completely clear. To clarify the important antibody dilution buffer was PBS containing 0.2 % albumin; influences of hapten structure required to obtain true broad- (5) enzyme-labeled secondary antibody dilution buffer was specificity antibodies to sulfonamides would help design antibody dilution buffer containing 1.0 % OVA; (6) sub- better haptens and ultimately, a better broad-specificity an- strate was 0.1 % TMB and H2O2 in 0.05 M citrate buffer, tibody. Therefore, the main aims of the present work are as pH 4.5; and (7) the stopping reagent was 2 N H2SO4. 4030 Z. Wang et al.

CH –1 O 3 Standard stock solutions (1 mgmL ) of each sulfonamide H H2N S N were prepared by dissolving an appropriate amount of each O O N standard in DMF. The individual stock solutions of sulfona- 4-amino-N-(5-methylisoxazol-3-yl)benzenesulfonamide mides were stored at −20 °C in amber glass bottles. Working Sulfamethoxazole (SMX) standards (0.01, 0.1, 1, 10, 100, 1,000, 10,000, 100,000, and 500,000 ngmL–1) of each sulfonamide were prepared by diluting the stock solution in PBS. The sulfonamide mixture containing 12 sulfonamides was prepared from aliquots of each stock solution (1 mgmL–1). The total final concentra- –1 O tion was diluted with PBS (pH 7.4) to 100 or 500 ngmL . S H2NSNH O N COOH Synthesis of haptens and conjugates 2-(2-(4-aminophenylsulfonamido)thiazol-5-yl)acetic acid (TS)

Chemical structures of the haptens used in this study are O H2 C COOH NH2 S NH C C shown in Fig. 2. All haptens were synthesized according to H2 O H2 previous reports with some modification [5, 9]. Acid or ester 4-(4-(4-aminophenylsulfonamido)phenyl)butanoic acid (SA10) haptens containing an amino group (3.1 mM) were dissolved in THF (10 mL) and stirred under N2 gas. Sodium hydride O H2 (60 % in oil, 15 mM) was added. The mixture was heated to C COOH H2N S NH CH2 C N H2 70 °C for 2 h and then cooled to room temperature. -acetyl- O sulfanilyl chloride (4.5 mM) was added as a solution in THF 4-(4-aminophenylsulfonamido)butanoic acid (BS) (4 mL). The reaction was stirred at room temperature overnight and then poured into 2 N NaOH (20 mL), and THF was removed under reduced pressure. The solution was refluxed at 70 °C for 2 h, and the aqueous solution was acidified to a pH<

2 with concentrated HCl and then extracted with CH2Cl2 (6× 10 mL). The organic extracts were combined, washed with saturated NaCl (20 mL), dried over MgSO4, and concentrated in vacuo to give a solid. The crude solid was purified using silica-gel chromatography to obtain the following haptens: 4-(4-aminophenylsulfonamido)butanoic acid Fig. 2 Chemical structures of the haptens used for immunization and (BS), 4-(4-aminophenylsulfonamido)benzoic acid (CS), coating conjugates 6-(4-aminophenylsulfonamido)hexanoic acid (HS), 4-(4-(4- aminophenylsulfonamido)phenyl)butanoic acid (SA10), and Hapten HS 2-(2-(4-aminophenylsulfonamido)thiazol-5-yl)acetic acid 1 1 (TS) (Fig. 2). H NMR spectra were obtained with a Bruker HNMR (300 MHz, DMSO-d6), δ (ppm) 11.96 (br, s, 1H, 300 spectrometer. COOH), 7.39 (d,2H,J=8.67 Hz, CHar), 7.03 (t, H, J=11.88,

SO2–NH), 6.60 (d,2H,J=8.70Hz,CHar), 5.89 (s,2H,NH2), Hapten BS 2.65–2.49 (m,2H,NH–CH2), 2.14 (t,2H,J=14.64, CH2– COOH), and 1.45–1.18 (br, m,6H, CH2–CH2–CH2). 1 HNMR (300 MHz, DMSO-d6), δ (ppm) 12.13 (br, s, 1H, COOH), 7.41 (d, 2H, J=8.77 Hz, CHar), 7.11 (t, H, J=9.68, Hapten SA10

SO2–NH), 6.62 (d,2H,J=7.50 Hz, CHar), 5.91 (s, 2H, 1 NH2), 2.67–2.51 (m, 2H, NH–CH2), 2.20 (t, 2H, J=8.67, HNMR (300 MHz, DMSO-d6), δ (ppm) 7.83 (s,1H,SO2NH), CH2–COOH), and 1.60 (m, 2H, CH2–CH2–CH2). 7.37 (d,2H,J=2.51 Hz, CHar), 7.03 (d×d,4H,CHar), 6.53 (d,2H,J=8.59 Hz, CHar), 6.66 (d,2H,J=8.5Hz,CHar), 5.99

Hapten CS (br, s,2H,NH2), 2.42 (t, J=8.79, 2H, –CH2Ph), 2.14 (t, J=14.5 Hz, 2H, –CH2COOH), and 1.71 (m,2H,–CH2−). 1 HNMR (300 MHz, DMSO-d6), δ (ppm) 12.61 (br, s, 1H, COOH), 10.40 (s, 1H, SO2NH), 7.74 (d, 2H, J=8.61 Hz, Hapten TS CHar), 7.48 (d,2H,J=8.5 Hz, CHar), 7.15 (d,2H,J= 1 8.5 Hz, CHar), 6.66 (d, 2H, J=8.5 Hz, CHar), and 6.03 (s, HNMR (300 MHz, DMSO-d6), δ (ppm) 7.42 (d,2H, 2H, NH2). J=8.51 Hz, CHar), 6.55 (d,2H,J=8.52, CHar), 6.27 Monoclonal antibodies with group specificity toward sulfonamides 4031

(s,H,SO2–NH), 5.81 (s,2H,NH2), 3.51 (br, s,H, Cross-reactivity values were calculated according to the fol- CH–S), 2.05 (s,2H,CH2-COOH). lowing equation [15]: The above haptens containing a carboxyl group in their side ÀÁÀÁÀÁ 1 1 chain were covalently attached to BSA for use as an immuno- CRðÞ¼ % IC50 molar SMX; nmol mL =IC50 molar analogs; nmol mL gen and to OVA for use as a coating antigen by the NHS ester 100 method as follows: The hapten (40 μM), EDC (20 μM), and NHS (20 μM) were dissolved in 2 mL of DMF and the mixture Also, the comparison of IC50 values between two different was held at room temperature for 4 h. The activated haptens compounds utilized molar IC50 (IC50 molar)valueswithunits –1 were added drop-wise with mixing to a 5-mL solution of 1 μM of nmolmL [15]. IC50 values in units of both nanograms per BSA or OVA in PBS (pH 7.4). The mixtures were kept milliliter and nanomoles per milliliter can be found in Table overnight at room temperature, dialyzed against PBS S5 (see Electronic supplementary material (ESM)). (pH 7.4) for 3 days, and then stored at −20 °C until needed. Conjugates of SMX-BSA were synthesized by the diazo Matrix effect of milk method according to our previous report [13]. An amount of 40 μM of SMX was reacted with 1 μM BSA or OVA. Sulfonamide-free skimmed milk was supplied by the National Sulfadiazine (SDZ), sulfathiazole (STZ), sulfamethizole Reference Laboratory for Veterinary Drug Residues (Beijing, (SMT), and sulfamethoxypyridazine (SMP) conjugates were People’s Republic of China). Nonspecific interferences pro- prepared by the same procedure. Sulfaguanidine or 4-amino- duced by the skimmed milk were accessed by preparing the N-(diaminomethylene)benzenesulfonamide (SG) was bound SMZ standard curves in skimmed milk at several dilution to BSA using glutaraldehyde as the coupling reagent accord- factors and comparing the data with the SMZ standard curve ing to a published procedure [14]. In brief, an amount of prepared in PBS. Measurements were completed in triplicate 40 μM of SG was mixed with 1 μM BSA or OVA in 1 mL using the MAb 4D11/CS-OVA ELISA. of a 2:1 solution of PBS (pH 7.4) and dioxane. Glutaraldehyde (1 mL, 0.5 % solution) was added drop-wise to the mixture, which was stirred for 3 h. The reaction mixtures were dialyzed Results and discussion against three changes of PBS over 3 days. Selection and synthesis of haptens ELISA methods The generation of antibodies exhibiting the desired group- Procedures for the generation of MAbs against hapten-BSA specificity requires maximizing the steric, hydrophobic, and conjugates, immunization, cell fusion, hybridoma selection, electronic similarities of a hapten to the parent molecules and cloning were similar to those previously described by [16, 17]. The design of a desirable hapten is the key step in Zhang et al. [13]. antibody production. In the last two decades, many inves- Polystyrene 96-well microtiter plates were coated with tigators have put forth much effort to design and synthesize coating antigen (100 μLwell–1)andincubatedat4°C haptens and use them to produce broad-specificity antibod- overnight. The plates were washed three times with washing ies to the sulfonamides [18–20]. These excellent reports buffer and then blocked with blocking buffer (300 μLwell–1) have demonstrated that careful selection of a hapten may at 37 °C for 2 h. Antibody (50 μLwell–1), various concen- improve the selectivity of an antibody, and that the structure trations of analytes or buffer (50 μLwell–1), and goat anti- of a hapten plays the most important role in the formation of mouse IgG-HRP (1:3,000 in PBS, 100 μLwell–1)weresi- antibody selectivity to the sulfonamides. multaneously added and incubated at 37 °C for 1 h. Following In general, the reported generic haptens can be divided incubation, the plates were washed three times with washing into two groups: single-ring haptens and two-ring haptens. buffer. The substrate solution (100 μLwell–1) was added and The data concerning the effectiveness of the single-ring incubated at 37 °C for 30 min before adding 2 N H2SO4 haptens are limited, especially for MAb production. For this (100 μL well–1). The absorbance of each well was measured reason, the single-ring haptens HS and BS (Fig. 2) were at 450 nm with the ELISA plate reader. employed in this study to evaluate the ability to induce a The strength of competition in the antisera screening pro- group-specific antibody response. In the case of two-ring cedures was calculated using the formula: competition (%)= haptens, prior to the report of Adrian et al. [21], most group-

B/B0×100, where B=absorbance of a tested sample solution specific antibodies showed relatively low sensitivity to and B0=the absorbance of buffer without sulfonamide. A four SMZ, the most important individual sulfonamide. Upon parameter logistic equation was used to fit the immunoassay inspection of the immunizing hapten, we concluded that a data. Calculations were performed using OriginPro7.5 two-ring immunizing hapten that contained a six-atom-ring software (OriginLab Corporation, Northampton, MA). structure at the N1 position may be more promising to 4032 Z. Wang et al. generate an antibody that could recognize SMZ than a five- antisera derived from the two-ring haptens CS, TS, SA10, atom-ring hapten like thiazole (i.e., TS). Our research group and SMX gave remarkably higher absorbance values than proposed SA10 as a new immunizing hapten for the pro- the antisera from the single-ring haptens BS, HS, and SG. duction of a broad-specificity MAb (Fig. 2). Hapten SA10 The second strategy was employed to compare the dilution incorporates a four-carbon atom alkyl chain in the para of coating antigen and antisera when generating the maxi- position on the benzene ring at N1. Hopefully, this will keep mum absorbance values ranging from 1.5 to 1.8 in the the six membered benzene ring of the hapten from being absence of a sulfonamide. The summarized data are shown hidden by the carrier protein and allow the formation of in Table S2 (see ESM). Most mouse antisera from the two- antibodies recognizing sulfonamides that contain a six- ring hapten antigens provided titers above 1:1,000, whereas atom-ring at the N1 position, particularly SMZ. Unexpect- antisera from single-ring haptens resulted in titers below edly, hapten SA10 had previously been synthesized by 1:1,000. In general, the results of mouse antisera titers using Adrian et al. [21], but only for use as a competitor, not as both strategies indicated that the two-ring hapten conjugates a immunizing hapten. Therefore, we followed Adrian et al. induced stronger antibody responses in mice than the single- [21] for the naming of SA10 in our work. ring haptens did. In addition, Spinks et al. suggested that group-specific Antisera having antibody titers higher than 1:500 were antibodies should be obtained using individual sulfona- tested for their competitive capability in ELISAs with either mides, like sulfachloropyridazine (SCP) or sulfacetamide homologous or heterologous haptens using the following (SA) as a hapten, and their suggestion was based on molec- mixture of 12 sulfonamide analogs at 500 ngmL–1: SCP, ular modeling [17]. But both of the haptens used by Spinks SDM, SDZ, SG, SMM, SMP, SMR (sulfamerazine), SMX, et al. failed to produce useful broad-specificity recognition SMZ, SQX, STZ, and SXL (sulfamoxole). The competition of the sulfonamides [17]. of all antisera and coating antigen combinations resulting in The synthesis of haptens CS, SA10, and TS was simpli- absorbance values between 1.5 and 1.8 were tested. The fied due to the commercial availability of 4-aminobenzoic competitive response (B/B0<80 %) to the sulfonamide mix- acid methyl ester, ethyl 2-amino-4-thiazoleacetate, and 4- ture combinations of coating antigens and antisera in over aminobenzenebutyric acid methyl ester. The use of sodium 200 tests were carried out. However, out of all tests con- cyanide as catalyst increased the yields of the haptens above ducted, only 22 combinations showed a relatively high

50 % and particularly increased the yields of hapten TS. The competitive response (B/B0<50 %) to the sulfonamide mix- carboxyl groups of these haptens were linked to BSA via the ture at 500 ngmL–1 when the antisera and antigen dilutions NHS/carbodiimide method. SG and SMX were linked to were at 1:500 or higher. BSA using glutaraldehyde as cross-linker and the diazotiza- Table S3 (see ESM) shows the best competitive combi- tion reaction, respectively. However, the coupling method nation for each mouse with optimum dilution of antisera and used for SG was not selective and the amino group on the coating antigen. The results of antisera screening demon- aromatic ring may possibly be linked to the carrier protein. strated that the presence of a second ring (benzyl or hetero- All seven haptens were also linked to OVA by the homog- cyclic) at the N1 position of the hapten molecule plays an enous coupling method and used as coating antigens. The important role in the generation of antibody titer and selec- conjugation ratio of each hapten to carrier protein was tivity to sulfonamides. estimated by measuring the hapten/protein molar ratios by To assess the specific selectivity of antisera to each matrix assisted laser desorption ionization time of flight- sulfonamide, the antisera were tested after the fifth immu- mass spectrometry (MALDI-TOF-MS). Hapten/protein ra- nization. All antisera having a titer above 1:500 in the first tios ranged from 9 to 15 and 5 to 9 for BSA and OVA test were measured for titer again. The results showed that conjugates, respectively (see Table S1 in the ESM). the titer of most antisera from the two-ring haptens improved, however, the antisera from single-ring haptens Screening of antisera remained constant or even decreased in titer, in the case of the BS and HS conjugate immunized mice. The results of Two different strategies were used to evaluate the ability of the best mouse immunized with each hapten conjugate are the seven immunogens to induce an antibody response in summarizedinTableS4 (see Electronic Supplementary the mouse. Using the first strategy, the titer of the antisera Material). was screened against the homologous hapten coating anti- Table S4 shows that antisera from haptens containing a gen at a dilution of 1:1,000 for both coating antigen and carboxyl group in their side chain shows group-specificity antisera of all mice following the fourth immunization. The for the set of sulfonamides tested including antisera from the relationship between the absorbance of antisera from each single-ring hapten HS and BS, which exhibit relatively high mouse in the ELISA and the immunizing haptens are sum- sensitivity for sulfonamides like SCP, SMP, and SMX. marized in Fig. S1 (see ESM). The results indicated that However, no fusion experiments were performed with Monoclonal antibodies with group specificity toward sulfonamides 4033 splenocytes from these mice because of low antibody titers. One main feature of all three MAbs (Table 1) is that they The antisera from CS, TS, and SA10 could recognize sev- could not or only weakly recognize the sulfonamides SA, eral sulfonamides, and the selectivity of these antisera is SAU, SG, SN, and TMP. The non-recognition can easily be different. The antisera of TS-BSA-4 showed high affinity to explained by the dissimilarity of the sulfonamide structures SCP, SMP, SMX, and STZ, while the antisera from SA10- (Fig. 1); however, the low-recognition of SA, SG and SN, BSA-3 had acceptable affinity to all the sulfonamides tested which all have a common portion of the general sulfon- except for SG and SXL. The antisera of CS-BSA-6 also amide structure, is surprising. One reasonable explanation recognized SCP, SMP, SMX, and ST, similar to TS-BSA-4; may lie in the lack of ring substitution at the N1 position in however, with relatively lower sensitivity. these three sulfonamides, which may contribute to poor The results for the antisera from SMX-BSA-1 were un- binding since the immunogens were two-ring haptens with expected, since they demonstrated a high specific selectivity a ring at the N1 position. to SMX and STZ and recognized SCP, SDZ, SMM, and SBA is another sulfonamide that was not recognized well –1 SMR with relatively high affinity, suggesting that hapten by any of the three MAbs (IC50 molar>29nmolmL )(Table1). SMX may have induced group-specific antibodies to the SBA structurally differs from SPY and SDZ only by the sulfonamides. The hapten SG was expected to produce anti- presence of an additional ketone group between N1 and the bodies with group specificity; however, only barely accept- benzene ring (R-group). The additional ketone group able antibody titers from SG were obtained. Also, those increases the flexibility of SBA, allowing the R-group to bend antibodies could only be slightly inhibited in the homolo- down toward the plane of the benzene ring. In addition, gous antigen-coating ELISA by free SG, suggesting that the comparison of the MAb 4D11 affinity for SBA (IC50 molar= –1 antisera were specific for the SG-BSA conjugate only and 31.75 nmolmL ) and the affinities for SPA (IC50 molar= –1 –1 SG was not an appropriate hapten for generating group- 1.051 nmolmL ), SIZ (IC50 molar=5.039 nmolmL ), and –1 specific antibodies. Considering all of the antisera titer data, SDM′ (IC50 molar=1.173 nmolmL )(Table1)suggeststhat only the mice immunized with SMX-BSA-1, TS-BSA-4, keeping the structure of the central portion of the sulfonamide and SA10-BSA-3 were used to produce MAbs. unchanged is more important for antibody binding to the sulfonamides than the substitution on the six-carbon-ring at Production of MAbs position N1. SMX is the exclusive individual sulfonamide having an –1 Eight clones producing MAbs were obtained: one clone IC50 value below 10 ngmL in all three ELISAs (Table S5 in from SMX-BSA-1 (1D10), four clones from TS-BSA-4 the ESM), and the typical calibration curves for SMX in the (4C7, 5E10, 3A10, and 2G5) and three clones from SA10- competitive ELISAs using the three different MAbs are BSA-3 (4D11, 3B12 and 3E1). The MAbs from these shown in Fig. S2 (see ESM). The MAb 1D10 can recognize clones were prepared as ascites and used in the following SMX at least 10 and 25 times better than MAb 4C7 and MAb experiments without further purification. All ascites from 4D11, respectively (Table 1). The IC50 values for SDZ, SMD, the clones showed similar selectivity profiles as did their SMM and SPYare all below 100 ngmL–1 in the three ELISAs, –1 corresponding mouse antisera, while the titer and sensitiv- and the IC50 values for SMR and SMP are below 100 ngmL ity (IC50) of the ascites were generally improved by five to for two of the ELISAs (Fig. 3; Table S1 in the ESM). How- ten times. The ascites with the highest sensitivity from ever, the three MAbs present much diverse selectivity toward each immunogen were chosen to investigate in detail. the other sulfonamides. The comparison between the MAbs Table S5 shows the concentration of three coating anti- may provide insight into the design and selection of a more gens, SDZ-OVA, TS-OVA, and CS-OVA, as 0.5, 1.2, and effective sulfonamide haptens (Table 1). 0.1 μgmL–1, respectively, with the optimal concentration of the corresponding MAbs, MAb 1D10, MAb 4C7, and MAb 1D10 MAb 4D11, of 1, 0.2, and 0.6 μgmL–1, respectively, and used to investigate the characteristics of each sulfonamide To the authors’ knowledge, there are no reports of the in the ELISA. production of MAbs to SMX. Endoh et al. raised antibodies against SMX which were highly specific, but only showed Selectivity of MAbs cross-reactivity to STZ (1.5 %) [22]. The commercial polyclonal antibody to SMX reported by Shelver et al. could

The selectivity of the MAbs was measured by using 28 recognize at least nine sulfonamides with IC50 values below structurally similar sulfonamides, related derivatives, and 100 ngmL–1 [23]. MAb 1D10 that we developed against five haptens. The IC20,IC50, and cross-reactivity values SMX showed acceptable affinity toward most sulfonamides for each sulfonamide are summarized in Table 1 and Table S5 tested (Table 1; Table S5 in the ESM). At least ten sulfona- –1 in the ESM. mides (including PST) had IC50 values less than 100 ngmL . 4034 Z. Wang et al.

Table 1 IC50 values of sulfonamides produced by ELISA in this study and in the literatures (in nanomoles per milliliter)

Sulfonamidesa 1D10 Shelver 4C7 27G3 Cliquet 4D11 Franek Sulfa-1 Zhang Adrian Ermolenko [23] [10] [5] [4] [9] [25] [21] [26]

Single-ring sulfonamides and haptensa Hapten BS 91.25 –c >193.6 –c –c 1.292 –c –c –c –c –c Hapten HS 174.6 –c >174.6 –c –c 0.107 –c –c –c –c –c SA 40.14 1.167 7.422 8.402 –c 20.45 3.538 66.98 –c –c –c SG >233.4b 4.714 57.65 2.334 –c 35.79 0.277 –c –c –c 0.2941 SN >290.4 –c >290.4 –c –c 46.87 0.331 –c –c >0.5807 0.6272 Two-ring sulfonamides containing a five-atom ring PST 0.0003 –c 1.874 –c –c 28.30 –c –c –c –c –c SIZ 4.856 –c 20.29 0.935 1.309 5.039 1.459 748.2 >0.7482 –c –c STX –c –c –c 26.19 –c –c –c –c –c –c –c SMT 0.008 0.009 0.002 0.002 0.033 2.113 0.079 259.0 0.047 –c 1.476 SMX 0.001 0.001 0.011 0.592 –c 0.026 0.005 236.9 0.533 >0.3948 –c SPA 7.816 –c 16.57 –c –c 1.051 0.090 –c –c –c –c STZ 0.0005 –c 0.004 0.039 0.117 1.851 0.032 1.261 0.008 0.005 0.094 SXL 0.486 –c 0.056 –c –c 8.825 –c –c 0.561 –c 0.3292 Two-ring sulfonamides containing a six-atom ring SBA 37.09 6.333 29.68 –c –c 31.75 –c 361.9 –c –c –c SCP 0.011 0.0006 0.011 0.014 0.070 0.077 0.071 15.31 0.049 0.2171 0.081 SCY –c –c –c 21.56 –c –c 0.244 –c –c –c SDM 4.040 0.0013 0.136 0.806 0.322 0.004 0.034 50.67 0.612 0.1328 0.441 SDM′ 22.09 –c >161.1 0.097 –c 1.173 0.3351 –c 0.1000 >0.3222 –c SDT 2.292 –c 0.176 –c –c 0.004 –c –c –c –c –c SDZ 0.010 0.479 0.366 0.320 0.120 0.147 0.118 39.01 0.128 0.056 0.144 SIM 16.85 –c 4.714 –c –c 0.021 –c 11.27 0.005 0.0076 –c SMD 0.122 0.042 0.041 0.107 –c 0.014 0.009 –c 0.011 –c –c SMM 0.004 –c 0.151 –c –c 0.020 –c –c –c –c –c SMP 0.831 0.0005 0.006 0.054 0.010 0.005 –c 0.030 0.0177 0.006 SMR 0.062 2.194 0.583 1.892 2.270 0.047 0.144 14.16 0.072 0.014 0.071 SMZ 5.655 27.31 51.13 28.74 3.773 0.038 0.3773 251.5 0.467 0.0064 0.4240 Acetyl-SMZ 2.987 –c >156.1 –c –c 0.075 –c 218.50 –c >0.3121 –c SPY 0.008 0.030 0.211 0.120 1.404 0.017 0.002 0.091 0.040 0.0090 –c SNT 58.62 –c >149.1 –c –c 0.005 –c 0.004 –c –c –c SQX 13.66 0.088 0.142 3.995 –c 0.031 0.007 83.24 0.300 0.2635 1.515 SSA –c 0.0198 –c 0.007 –c –c –c 10.81 –c –c –c Hapten CS 75.76 –c 29.22 –c –c 0.002 –c 0.2316 –c –c –c Hapten TS 0.073 –c 0.006 –c –c 0.154 –c –c –c –c –c Hapten SA10 76.66 –c 13.01 –c –c 0.001 –c –c –c –c –c Other analytes related to sulfonamides SAU >241.4 –c >241.4 –c 241.4 –c –c –c –c –c TMP >172.2 –c >172.2 –c 172.2 –c –c –c –c –c

SA sulfacetamide, SG sulfaguanidine, SN sulfanilamide, PST, N4 -phtalylsulfathiazole, SIZ sulfisoxazole, STX sulfatroxazole, SMT sulfamethizole, SMX sulfamethoxazole, SPA sulfaphenazole, STZ sulfathiazole, SXL sulfamoxole, SBA sulfabenzamide, SCP sulfachloropyridazine, SCY sulfa- chloropyrazine, SDM sulfadimethoxine, SDM’ sulfadoxine, SDT sulfadimethoxypyrimidine, SDZ sulfadiazine, SIM sulfisomidine, SMD sulfame- thoxydiazine, SMM sulfamonomethoxine, SMP sulfamethoxypyridazine, SMR sulfamerazine, SMZ sulfamethazine, acetyl-SMZ N4 -acetyl- sulfamethazine, SPY sulfapyridine, SNT sulfanitran, SQX sulfaquinoxaline, SSA sulfasalazine, SAU 5-sulfaminouracil, TMP trimethoprim a The sulfonamides were sorted by the number of ring and then the number of atom of ring at the N1 position. The haptens were listed in alphabetical order according to the abbreviations of sulfonamides in each group b Means that sulfonamide decreased in absorbance value by 50 % at a concentration of more than 50,000 ngmL−1 c Not detected Monoclonal antibodies with group specificity toward sulfonamides 4035

MAb 1D10 had the highest affinity for PST, SMX, and STZ (Table 1). The MAb 4C7 has the highest sensitivity for SMT –1 –1 (IC50 values of <0.5 ngmL ), a high affinity for SCP, SDZ, and STZ (IC50 molar values below 0.004 nmolmL ), and then SMD, SMM, SMR, SMT, and SPY (IC50 values ranged from for SCP, SMP, and SMX (IC50 molar values below 0.011 nmol 1.9 to 34.2 ngmL–1) (Table S5 in the ESM), and it also mL–1). The selectivity of MAb 4C7 is in basic agreement with recognized other sulfonamides (Table 1). However, the MAb the MAbs 3B5B10E3 (Cliquet) and 27G3 (Table 1)[5, 10], 1D10 did not recognize SDM, SIM, SIZ, or SMZ at accept- which were both produced by hapten TS. Our MAb 4C7 –1 able IC50 molar values (>4 nmolmL ). MAb 1D10 was toler- shows at least 50 times higher affinity to SDM, SMX and ant to the number of ring atoms of the R-group (five-atom ring SQX than the previously reported MAbs 3B5B10E3 (Cliquet) or six-atom ring) (Table 1), and yet it was sensitive to the bulk and 27G3 [5, 10]. Generally, MAb 4C7 has more sensitivity substitution of the ring. Most sulfonamides recognized by than 3B5B10E3 (Cliquet) and 27G3 and also provides

MAb1D10withhighaffinity(IC50 molar value below greater group specificity for sulfonamides. However, all 0.15 nmolmL–1)(Table1) have one (methyl or methoxy) or the MAbs produced from hapten TS do not recognize no substitution in the R-ring. Any sulfonamides that have two SMZ well. All sulfonamides that produced IC50 values groups substituted on the R-ring, particularly in the ortho- below 100 ngmL–1 in the MAb 1D10-based ELISA except –1 position like SIZ, have decreased affinity to MAb 1D10. It is PST also resulted in IC50 values less than 100 ngmL in not surprising that substitution at the N4 position of the the MAb 4C7-based ELISA except for SMR (IC50 of –1 –1 sulfonamides like PST (IC50 molar of 0.0003 nmolmL )has 156 ngmL )(Fig.3). Both MAbs do not sufficiently no adverse effect on antibody affinity since the amino group of recognize SG, SIM, SIZ, SMZ, SN, and SNT. However, SMX was used in the linkage to BSA, and this portion of the the MAb 4C7 recognizes SDM, SDT, SMP, SQX, and molecule was not exposed to the immune system. Immuniza- SXL almost 10–150 times better than MAb 1D10, dem- tion with an individual sulfonamide most often leads to an onstrating that MAb 4C7 is more tolerant to the substitu- antibody with high specificity to either a single or even several tion of two groups on the meta- and para-positions of the sulfonamides. However, the same approach can lead to anti- R-ring. The negative effect on MAb 4C7 binding caused bodies with group specificity as well, as seen in this study. by N4 substitution can be readily seen by comparing the –1 Although SMX is a useful example of a hapten to use for IC50 molar values of STZ (0.004 nmolmL )andPST producing group-specific antibodies other than SCP, which (1.874 nmolmL–1). Approximately a 468-fold decrease was proposed by Spinks et al. [17], the antibodies produced to in the IC50 molar value occurs as a result of the phthalyl SMX in our laboratory and also by Shelver et al. [23]show group being substituted at the N4 position. This can easily diverse cross-reactivities for the sulfonamides. This suggests be explained based on the structure of hapten TS contain- that haptens of these types should not be used for generating ing an aromatic amino group distal to the carrier protein group-specific antibodies to sulfonamides [23]. and maximally exposed to the antibody. Obviously, the mode of recognition for MAbs 1D10 and 4C7 is distinct. MAb 4C7 Therefore, just as SMX is not the best hapten for generating group-specific antibodies, hapten TS is also not an In the case of MAb 4C7 obtained from Hapten TS, 13 sulfo- appropriate hapten for generating group-specific antibod- –1 namides show IC50 molar values of less than 0.4 nmolmL ies for the sulfonamides.

Fig. 3 The IC50 values of three 120 MAbs for sulfonamides that are – 110 below 100 ngmL 1. When the top of a column is lower than 100 MRL upper horizontal dash line the , 90 1D10 it means that the IC50 of the 4C7 MAb for sulfonamides is below 80

– ) 100 ngmL 1 70 4D11 -1 60 50 (ng mL 50 40 IC 30 20 10 1/10 MRL 0 SMZSMX SMTSPY SPYSMD SMXSMMSXL SMM STZ SMRSDZ SDZ SCP SQX SQXSDM SDMSMP SDT SMZ SM2 PSTSNT SIM Sulfonamides 4036 Z. Wang et al.

MAb 4D11 molecule, not just specifically binding the aromatic amine group, which is common to most sulfonamides. The high MAb 4D11 was produced from the hapten SA10, and it recognition of MAb 4D11 for SNT is difficult to explain, but recognizes at least 15 sulfonamides, including acetyl-SMZ, is in agreement with that observed for the Sulfa-1 MAb –1 with IC50 values less than 100 ngmL (Fig. 3). More produced by Muldoon et al. [9]. The MAb 4D11 recognizes importantly, 12 sulfonamides have IC50 values from 1.2 to SMZ and acetyl-SMZ with similar affinity; however, bulk 12.4 (Table S5 in the ESM)orIC50 molar values from 0.004 substitution at the N4 position has an obvious negative to 0.047 nmolmL–1 (Table 1; Table S5 in the ESM), almost effect on antibody binding as seen by the comparison of –1 within 1 order of magnitude, which is favorable to develop a IC50 molar values of STZ and PST, 1.851 and 28.3 nmolmL , semi-quantitative immunoassay for sulfonamides. Although respectively (Table 1). Upon comparison of the selectivity of MAbs 1D10 and 4C7 show higher sensitivity for some sulfo- MAb 4D11 with polyclonal antibodies or MAbs derived from –1 namides with IC50 molar values even below 0.001 nmolmL , CS or similar haptens like SA1 reported by Adrian et al. for in our opinion, MAb 4D11 is the best MAb produced in several sulfonamides [21], MAb 4D11 is obviously unique respect to this work and also compared with sulfonamide and shows a fairly uniform selectivity for many of the sulfo- antibodies in previous reports. This is due to the high- namides tested. MAb 4D11 is unique compared with all uniform selectivity for many of the sulfonamides tested. Our previously reported MAbs, even the previously reported poly- initial purpose was to develop an immunoassay for screening clonal antibodies used to detect sulfonamides (Table 1). five sulfonamides as required by the Agricultural Ministry of Antibodies from a hapten containing a six-atom-ring China and MAb 4D11 can meet and does exceed the required substitution at the N1 position can recognize SMZ and show criterion. It can be seen in Table 1 (or Table S5 in the ESM) good group-specificity for an array of sulfonamides. There- where SDM, SMM, SMX, SMZ, and SQX have IC50 molar fore, a more effective hapten for the production of a real values that range from 0.004 to 0.038 nmolmL–1. group-specific antibody for the sulfonamides may be Of all the sulfonamides that are well recognized by MAbs designed based on the structure of SA10; for example, by 1D10 and 4C7, MAb 4D11 had low affinity for SMT, STZ, reducing the immunogenicity of the benzene-ring at the N1 and SXL, which all contain a five-atom ring at the N1 position by introducing extra or different ring substitutions. position. In contrast, MAb 4D11 had high affinity for SIM, SMZ (including acetyl-SMZ), and SNT that are not Analysis of milk samples recognized by MAbs 1D10 and 4C7. These results imply that MAb 4D11 has higher affinity for the sulfonamides A preliminary study was performed to assess the matrix containing a six-atom-ring substitution rather than a five- effectofskimmedmilkbyusingSMZasthereference atom-ring substitution at the N1 position. This hypothesis molecule, which is used worldwide in cows [8]. For a was confirmed, since MAb 4D11 demonstrated high- screening assay, the sample preparation should be kept as sensitivity to all six-carbon-ring substituted sulfonamides simple as possible. For this reason, the SMZ standard curve tested, except SBA and SDM, and low-sensitivity for all was developed in PBS and compared with curves prepared five-carbon-ring substituted sulfonamides except for SMX. in skimmed milk after the following dilutions: 0, 2, 5, 10, The recognition pattern of MAb 4D11 may be explained by 1.8 the structure of hapten SA10. In a previous work, the poly- PBS (IC50=10.5) 1.6 Skimmed milk (IC =9.6) clonal antibodies derived from hapten CS, which has a 50 1/2 Skimmed milk (IC =9.3) similar chemical structure to hapten SA10 but differs by a 1.4 50 1/5 Skimmed milk (IC50=10.2) two-carbon chain at the para position of the benzene ring 1.2 1/10 Skimmed milk (IC50=11.3) 1/50 Skimmed milk (IC =10.9) instead of a four-carbon chain in the case of hapten SA10, 1.0 50 could not distinguish between six-atom-ring substituted sul- 0.8 fonamides and five-atom-ring substituted counterparts. The

OD Values 0.6 longer spacer arm of hapten SA10 in the linkage to the 0.4 carrier protein allows the substituted benzene ring of the hapten to be more strongly recognized by the animal im- 0.2 mune system, which allows the antibody to differentiate 0.0 between sulfonamides substituted with a six-atom ring or a 1E-3 0.01 0.1 1 10 100 1000 10000 five-atom ring. Sulfamethazine (ng mL-1) In addition, the sulfonamides that have an extra group at Fig. 4 Matrix effects caused by PBS diluted skimmed milk in the the N4 position like acetyl-SMZ and SNT do not show a – – MAb 4D11 (0.6 μgmL 1)/CS-OVA (0.1 μgmL 1) ELISA using sulfa- decrease in antibody binding affinity, illustrating that MAb methazine as the reference molecule. Vertical bars denote the SE for 4D11 binds to a more central part of the sulfonamide triple measurements Monoclonal antibodies with group specificity toward sulfonamides 4037 and 50 times. As shown in Fig. 4, the maximum OD values Doctoral Dissertation Fund (YB20081001902). We thank Professor of undiluted skimmed milk decreased to 1.0 from 1.6 when Jing Shen of Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Core Laboratory, Peking University the standard curve was obtained in PBS but maintained School of Oncology, Beijing Cancer Hospital and Institute for similar IC50 values, which means that the matrix of MALDI-TOF-MS analysis. skimmed milk can be tolerated in the developed MAb 4D11-based ELISA. Also, with the increase in dilution, the maximum OD values increase while the IC values remain 50 References stable. The results in Fig. 4 show that skimmed milk can be accurately measured with only a 1:5 or 1:10 sample dilution. 1. Cháfer-Pericás C, Maquieira Á, Puchades R, Miralles J, Moreno A (2011) Food Control 22:993–999 2. European Commission (1999) Off J Eur Union 60:16–52 Conclusions 3. Regulation No. 235 Ministry of Agriculture of the People’sRepublic of China (2002). 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