Synthesis of Novel Haptens and Development of an Enzyme-Linked

Nov 24, 2014 - Novel haptens were designed and synthesized to prepare antibodies against free histamine, but none resulted in producing suitable antib...
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Synthesis of Novel Haptens and Development of an Enzyme-Linked Immunosorbent Assay for Quantification of Histamine in Foods Lin Luo,†,‡ Zhen-Lin Xu,†,‡ Jin-Yi Yang,† Zhi-Li Xiao,† Yong-Jun Li,§ Ross C. Beier,∥ Yuan-Ming Sun,*,† Hong-Tao Lei,† Hong Wang,† and Yu-Dong Shen*,† †

Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642, China Zhongshan Quality Supervision & Inspection Institute of Agricultural Products, Zhongshan, 528403, China ∥ Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, 2881 F&B Road, College Station, Texas 77845-4988, United States §

S Supporting Information *

ABSTRACT: Novel haptens were designed and synthesized to prepare antibodies against free histamine, but none resulted in producing suitable antibodies for developing an enzyme-linked immunosorbent assay (ELISA). However, an antiserum was obtained having high specificity and affinity to p-nitrobenzoylated histamine (NPHA), which can be easily formed from reaction between histamine and p-nitrobenzoic acid N-hydroxysuccinimide ester (PNBA-OSu) under mild conditions. Based on rabbit polyclonal antibodies, a competitive indirect ELISA (ciELISA) for histamine determination in foods was developed. After ciELISA and derivatization optimization, the assay showed good sensitivity, with limits of detection of 1.8 mg/kg, 93.6 μg/L, and 93.6 μg/kg in fish, red wine, and yoghurt, respectively, with negligible cross-reactivity with related biogenic amines and amino acids. Average recovery of histamine in fortified food samples ranged from 80.9% to 110.1% with coefficients of variation below 16.3%. Good correlation between the ciELISA and liquid chromatography−tandem mass spectrometry was obtained for spiked food samples. KEYWORDS: histamine, haptens, ELISA

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INTRODUCTION Biogenic amines (BAs) are low molecular weight organic bases commonly present in living organisms and often possess essential physiological functions.1,2 Minor quantities of BAs are commonly biosynthesized in plant and animal cells, and larger amounts occur in fermented and deteriorated foods (fermented sausage, fish products, cheeses, vegetables, and beverages) due to microbial amino acid decarboxylation.3 Although moderate levels of BAs are essential for living organisms, BAs can cause food poisoning at high concentrations.4 Histamine (HA), 4-(2aminoethyl)-1H-imidazole, is the most important BA in relation to allergies and food poisonings.5 Histamine is commonly found in the food supply formed through the decarboxylation of the essential amino acid, L-histidine, by bacterial action during food processing and storage.6 Consuming foodstuffs that contain high levels of HA may cause HA poisoning (or scombroid fish poisoning) characterized by headache, nausea, vomiting, diarrhea, itching, an oral burning sensation, red rash, and hypotension.7 Foodborne poisoning resulting from HA ingestion has been reported time and again from all parts of the world.8−10 However, the HA content is also an important indicator of food freshness and quality as HA can be produced during production, storage, or transportation of protein-rich foods by thermal or bacterial enzymatic decarboxylation of histidine.11 Hence, many countries and organizations have set regulatory levels for HA in foods. The European Union (EU) has regulated the HA content in fish and processed fish from the families of Coryphaenidae, Scombridae, Clupeidae, and Eugraulidae, and the following requirements must be fulfilled: © XXXX American Chemical Society

nine different samples must be taken from each batch; the HA level must not exceed 100 mg/kg; two samples may have a value of more than 100 mg/kg but must be less than 200 mg/ kg; and no sample may have a value that exceeds 200 mg/kg.12 This regulation has been obligatory in the EU fish industry and may be extended to fermented foods in the future.13 The United States Food and Drug Administration (FDA) has established regulations for fish products, where 50 mg/kg of HA is the chemical index for fish spoilage and 500 mg/kg is a potential health hazard “danger of death”.14 China has considered 1000 mg/kg HA in mackerel and 300 mg/kg in other marine fish as a permissible limit.15 Several analytical methods have been proposed for HA analysis in foods, such as fluorometric,16 thin-layer chromatography,17 capillary electrophoresis,11,18 high-performance liquid chromatography (HPLC),19,20 gas chromatography,21,22 and electrochemical biosensor methods.23,24 Generally, these methods are limited either by poor specificity or by requiring expensive instrumental equipment and extensive sample cleanup procedures. Therefore, a simple, specific, low-cost HA assay which could routinely be performed for large numbers of samples is required. Antibody based immunoassays, such as the ELISA, have been proven to be rapid, sensitive, and low-cost screening tools for Received: September 28, 2014 Revised: November 24, 2014 Accepted: November 24, 2014

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Figure 1. Synthetic route for haptens.

derivatized HA in foods. Because HA can be efficiently and quantitatively converted to NPHA by PNBA-OSu under mild basic conditions at room temperature, a ciELISA was developed here for the analysis of HA in food samples via a preanalysis HA derivatization step. This work also fully optimized the assay conditions. In addition, the immunoassay results were validated by comparison with LC−MS/MS results.

chemical residues. However, the development of a high affinity antibody against HA is not easy, and has puzzled immunochemists for some time due to the simple structure of HA (MW, 111 Da).25 In general, all reported antibody methods used for immunoassay determination of HA in foods can be classified into three categories. (1) An antibody that specifically recognized the p-benzoquinone−histamine conjugate was initially reported in 1986.26 Based on this conjugate, ELISAs were proposed for HA detection in foodstuffs, using a preanalysis HA derivatization step with p-benzoquinone.27,28 But drawbacks did exist for this strategy. The derivatization reaction between HA and p-benzoquinone was complicated due to side reactions, and the product was unstable. (2) A second antibody was developed against a modified HA by coupling succinylated HA to BSA grafted with a glycyl residue.29 The obtained antibody also showed no binding affinity for free histamine, but it had high binding affinity for modified HA which was formed through the reaction between HA and N-hydroxysuccinimide ester−succinyl-glycinamide (NHS-SGA). However, the synthesis of the derivatization reagent, NHS-SGA, was laborious with multisteps, making this reagent costly. (3) The only antibody developed that was able to recognize intact HA was developed by coupling HA to KLH via glutaraldehyde, and then the conjugate was used as an immunogen to immunize New Zealand white rabbits.30 However, the repeatability of this strategy is poor, and four attempts using this strategy to produce an antihistamine antibody have failed in our group. This study aimed at finding a reliable strategy for producing an antihistamine antibody, as well as the development of a competitive indirect enzyme-linked immunosorbent assay (ciELISA) for HA determination in foods based on the obtained antibody. For this purpose, several haptens were synthesized in attempts to obtain an antibody with specific histamine affinity or derivatized histamine affinity. Although, the development of an antibody against free HA failed, a highly specific polyclonal antibody produced against p-nitrobenzoylated HA (NPHA) was used to develop a ciELISA for

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MATERIALS AND METHODS

Animals and Reagents. Histamine dihydrochloride, L-histidine, Ltryptophan, tryptamine hydrochloride, tyramine hydrochloride, phenethylamine hydrochloride, and cadaverine hydrochloride were obtained from Heowns Biochem Technologies Co. Ltd. (Tianjin, China). Methyl 4-(chlorocarbonyl)benzoate, p-nitrobenzoic acid, Nhydrosuccinimide, dicyclohexylcarbodiimide (DCC), and triethylamine were obtained from Aladdin Chemical Technology Co., Ltd. (Shanghai, China). Histamine, 3,3′,5,5′-tetramethylbenzidine (TMB), glutaraldehyde (50%, v/v), complete and incomplete Freund’s adjuvants, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin (OVA), horseradish peroxidase, and peroxidaselabeled goat anti-rabbit IgG (secondary antibody) were obtained from Sigma-Aldrich (St. Louis, MO, USA). N,N-Dimethylformamide (DMF), acetonitrile (ACN), Tween-20, ethanol, methanol (MeOH), ethyl acetate (EtOAc), tetrahydrofuran (THF), and chloroform (CHCl3) were obtained from Damao Chemical Reagent Co., Ltd. (Tianjin, China). New Zealand white rabbits were raised at the Guangdong Medical Experimental Animal Centre. 96-Well polystyrene microplates were obtained from Shenzhen Jinchanhua Industrial Co. Ltd. (Shenzhen, China). All other reagents were of analytical reagent grade or higher purity. Instruments. ELISA plates were washed in a Multiskan MK2 microplate washer (Thermo Scientific, Hudson, NH, USA). Absorbance was measured at a wavelength of 450 nm using a Multiskan MK3 microplate reader (Thermo Scientific). Ultraviolet spectrometry (UV) was recorded on a UV-3010 spectrophotometer (Hitachi, Tokyo, Japan). LC−MS/MS analysis was carried out by using the 1200 series LC system (Agilent Technologies) equipped with the Agilent 6410 Triple Quad LC−MS system (Agilent Technologies). Nuclear magnetic resonance (NMR) spectra were achieved with either a DRX-400 or DRX-600 NMR spectrometer (Bruker, Rheinstetten, Germany). B

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Figure 2. Synthetic route for the derivatizing reagents, PNBA-OSu and NPHA. analysis (negative ion) m/z 258 [M − H]−; 1H NMR (400 MHz, methanol-d4) δ 8.54 (d, J = 1.4 Hz, 1H), 8.12−8.05 (m, 2H), 7.90− 7.82 (m, 2H), 7.27 (d, J = 1.3 Hz, 1H), 3.72 (t, J = 6.8 Hz, 2H), 3.04 (t, J = 6.8 Hz, 2H). Hapten E, 4-(4-(2-Aminoethyl)-1H-imidazol-1-yl)butanoic Acid (14). Histamine dihydrochloride (3.68 g, 20 mmol) and triethylamine (56 mL, 40 mmol) were mixed in acetontrile (150 mL), then di-tertbutyl dicarbonate (9, 4.36 g, 20 mmol) was added. The mixture was stirred at room temperature overnight and then filtered. The filtrate was evaporated under vacuum, and the residue was washed with petroleum ether (100 mL). The undissolved substance was treated with d.w. (100 mL), and the mixture was filtered. The aqueous solution was saturated with NaCl and extracted with ethyl acetate. The organic layer was dried over anhydrous Na2SO4 and evaporated under vacuum, leaving an oil. The residue was crystallized from ether/ petroleum ether to afford the intermediate 10 (2.3 g, 10.8 mmol). Ethyl 4-bromobutanoate (11, 386 mg, 2 mmol), the intermediate (10, 422 mg, 2 mmol), and DMF (20 mL) were mixed, and then K2CO3 (500 mg, 3.6 mmol) and tetrabutyl ammonium bromide (5 mg, 0.016 mmol) were added. The mixture was stirred under reflux at 80 °C overnight and then was concentrated by rotoevaporation under vacuum. The crude product was purified on a silica gel column, using EtOAc/MeOH (20:1) as eluent to afford 12 (334 mg, 1.02 mmol). Compound 13 was synthesized from 12 by subjection of 12 to the same hydrolysis step as previously used to convert 7 to 8. The resulting mixture was concentrated by rotoevaporation under vacuum and purified on a silica gel column using EtOAc/MeOH (4:1) as eluent to give 13 (280 mg, 0.94 mmol). Compound 13 was dissolved in methanol (20 mL), and concentrated hydrochloric acid (5 mL) was added. The mixture was stirred on an ice bath for 1 h and then concentrated by rotoevaporation under vacuum to give hapten E (14, 262 mg, 0.78 mmol, in its hydrochloride form): ESI analysis (negative ion) m/z 196 [M − H]−; 1H NMR (600 MHz, methanol-d4) δ 8.97 (s, 1H), 7.61 (s, 1H), 4.30−4.26 (m, 2H), 3.30−3.28 (m, 1H), 3.14 (t, J = 7.5 Hz, 2H), 2.45 (t, J = 7.2 Hz, 2H), 2.21−2.18 (m, 2H). Hapten F, 4-((4-(2-Aminoethyl)-1H-imidazol-1-yl)methyl)benzoic Acid (18). The synthetic route of hapten F (18) was similar to hapten E (14), except the initial ester, ethyl 4-bromobutanoate (11), was replaced with 4-((4-(2-aminoethyl)-1H-imidazol-1-yl)methyl)benzoic acid (15) (see Figure 1): ESI analysis (positive ion) m/z 246 [M + H]+; 1H NMR (600 MHz, methanol-d4) δ = 9.05 (s, 1H), 8.03 (t, J = 5.3, 2H), 7.54 (s, 1H), 7.50 (t, J = 6.6, 2H), 5.47 (s, 2H), 3.24 (t, J = 7.6, 2H), 3.07 (t, J = 7.5, 2H). Derivatization Reagent (PNBA-OSu) and N-(2-(1H-Imidazol-4yl)ethyl)-4-nitrobenzamide (NPHA). PNBA-OSu was synthesized as previously described:31 ESI analysis (positive ion) m/z 306 [M + ACN]+; 1H NMR (600 MHz, CDCl3) δ 8.37 (d, J = 9.0 Hz, 2H), 8.33 (d, J = 9.0 Hz, 2H), 2.94 (s, 4H). A solution of PNBA-OSu (700 mg, 2.65 mmol) in ACN (20 mL) was added dropwise (with stirring) to a solution of histamine dihydrochloride (460 mg, 2.5 mmol) in 1 M NaOH (10 mL). The resulting mixture was stirred at room temperature for 2 h, and then the organic solvent was removed by evaporation under vacuum. The yellow precipitate that formed was filtered off and washed with 0.1 M NaOH and dried to give NPHA (588 mg, 2.25 mmol): ESI analysis (positive ion) m/z 261 [M + H]+; 1 H NMR (400 MHz, methanol-d4) δ 8.54 (d, J = 1.1 Hz, 1H), 8.09 (d, J = 8.5 Hz, 2H), 7.86 (d, J = 8.5 Hz, 2H), 7.27 (s, 1H), 3.72 (t, J = 6.8 Hz, 2H), 3.04 (t, J = 6.8 Hz, 2H).

Buffers and Solutions. The following were the buffers and solutions used in this study: (1) Borate buffer (0.2 mol/L, pH 9.0) was used for histamine chemical derivatization; (2) 50 mmol/L carbonate buffer (pH 9.6) was used for coating antibody on plates; (3) PBST1 (10 mmol/L phosphate-buffered saline (PBS) with Tween-20 (0.05%), pH 7.4) was used for washing plates and secondary antibody dilution and was the working buffer used in the nonoptimized ciELISA; (4) PBST2 (20 mmol/L PBS, Tween 20 (0.05%), pH 7.4) was used for the working buffer in the optimized ciELISA; (5) 0.1 mol/L sodium acetate (pH 5.5) was used for the substrate buffer; (6) 2 mol/L H2SO4 was used for the stop reagent; and (7) HA stock standard solution (1 mg/mL HA in distilled water (d.w.)). Synthesis and Verification of Histamine Derivatives. Five histamine derivatives (haptens B−F) were synthesized by introduction of different spacer arms containing a carboxyl group at histamine’s primary amine or by substitution of H on the ring NH group (Figure 1). Figure 2 shows the synthetic route to produce the derivatization reagents PNBA-OSu and NPHA. The following is a detailed description of the synthetic procedure and characterization of these compounds. Hapten A, HA (1). Hapten A is intact HA. Histamine was conjugated to the carrier protein via glutaraldehyde according to previous work.30 Hapten B, 4-((2-(1H-Imidazol-4-yl)ethyl)amino)butanoic Acid (3). Histamine dihydrochloride (1.84 g, 10 mmol) and triethylamine (6 mL) were added to DMF (20 mL) containing 4-bromobutanoic acid (2, 1.65 g, 10 mmol). The mixture was stirred at room temperature for 1 h and then refluxed at 80 °C for 3 h, after which time the solvent was evaporated under vacuum, leaving a gummy residue. The residue was purified on a silica gel column, using CHCl3/MeOH/NH4OH (10:5:1) as the eluent, and then crystallized from ethanol (10 mL) to afford hapten B (3, 832 mg, 4.2 mmol): ESI analysis (positive ion) m/z 198 [M + H]+; 1H NMR (600 MHz, D2O) δ 8.84 (s, 1H), 7.57 (s, 1H), 3.11 (t, J = 5.2 Hz, 2H), 2.94 (t, J = 5.2 Hz, 2H), 2.62 (t, J = 7.7 Hz, 2H), 2.34 (t, J = 8.2 Hz, 2H), 1.86 (t, J = 7.9 Hz, 2H). Hapten C, 4-((2-(1H-Imidazol-4-yl)ethyl)amino)-4-oxobutanoic Acid (5). Succinic anhydride (4, 500 mg, 5 mmol) was added to a solution of histamine dihydrochloride (920 mg, 5 mmol) in methanol (15 mL) and stirred at room temperature for 4 h, and the solvent was removed by evaporation. The residue was redissolved in ethanol (20 mL) and kept at 4 °C overnight, and then hapten C (5, 650 mg, 3.1 mmol) was crystallized: ESI analysis (negative ion) m/z 210 [M − H]−; 1H NMR (600 MHz, D2O) δ 8.54 (s, 1H), 7.26 (s, 1H), 3.45 (t, J = 6.3 Hz, 2H), 2.89 (t, J = 6.4 Hz, 2H), 2.44−2.35 (m, 4H). Hapten D, 4-((2-(1H-Imidazol-4-yl)ethyl)carbamoyl)benzoic Acid (8). A solution of methyl 4-(chlorocarbonyl)benzoate 6 (198 mg, 1 mmol) in 5 mL of ACN was added dropwise to a solution of histamine dihydrochloride (184 mg, 1 mmol) in 1 M NaOH (5 mL). The mixture was stirred at room temperature for 3 h, and then the ACN was removed by rotoevaporation under vacuum. A yellow precipitate was filtered and washed three times with 0.1 M HCl and dried to yield intermediate 7 (232 mg, 0.84 mmol). A solution of intermediate 7 (232 mg, 0.84 mmol) in a mixture of THF/MeOH/H2O (1:1:1, 15 mL) was treated with LiOH·H2O (84 mg, 2.0 mmol) and refluxed at 40 °C overnight. The organic solvent was evaporated under vacuum, and the aqueous layer was acidified with 1 M HCl until a white precipitate formed. The precipitate was filtered off and washed with 0.1 M HCl, and dried to yield hapten D (8, 196 mg, 0.75 mmol): ESI C

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Synthesis of Immunogen and Coating Antigen. Histamine (hapten A) was coupled to KLH via glutaraldehyde for the immunogen and to OVA for the coating antigen.30 Haptens B−F were conjugated to BSA with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and used as the immunogen, and they were conjugated to OVA and used for the coating antigen according to previous work, with modifications.32 Briefly, a hapten (0.1 mmol) and carrier protein (10 mg) were dissolved in the conjugation buffer (PBS, pH 6.0, 5 mL). The resulting solution was cooled on an ice bath, and EDC (32 mg) was added with stirring. The mixture was held at 0 °C overnight with stirring, and then the conjugation mixture was dialyzed against 10 mM PBS (4 × 5 L) at 4 °C for 72 h, and finally stored at −20 °C until used. UV−vis spectral data was used to confirm the structures of final conjugates, and the hapten density was estimated by the TNBS method.33 Immunization and Polyclonal Antibody Production. Twelve New Zealand white rabbits were housed and maintained at the Guangdong Medical Laboratory Animal Center. All animal experiments were performed in compliance with the protective and administrative laws for laboratory animals of China and conducted with the approval of the Institutional Authority for Laboratory Animal Care, South China Agricultural University, Guangzhou, China. The rabbits (10-wk-old, 1.5−2.0 kg) were equally divided into six groups for the six immunogens, and each was intradermally and intramuscularly immunized with 1 mL of an emulsion (1:1) containing 0.5 mg/mL of an immunogen in PBS and complete Freund’s adjuvant. Four booster immunizations using the same amount of immunogen emulsified in incomplete Freund’s adjuvant were given monthly. Ten days after the fourth booster injection, the rabbits were exsanguinated and their blood was collected. The obtained antiserum was stored at −20 °C until purified by saturated ammonium sulfate precipitation.34 Serum was collected from the marginal ear vein from each rabbit prior to the first immunization and used as the negative controls. Antibody Screening and Characterization. A ciELISA was used to evaluate the antiserums for binding ability to the analytes (histamine or its derivatives). Briefly, the coating antigens (1 mg/L, 100 μL/well) in carbonate buffer were added to 96-well polystyrene ELISA plates and incubated at 4 °C overnight, and then the wells were washed twice with PBST1 solution prior to adding 5% skimmed milk in PBST1 (100 μL/well) to block the uncoated sites for 3 h at 37 °C and dried at 37 °C for 1 h. Histamine standards or other competitors in PBS (50 μL) and the diluted antiserum (50 μL) in PBST1 were added to each well and incubated at 37 °C for 40 min, and then the wells were washed five times with PBST1. The secondary antibody diluted 1:10000 in PBST1 (100 μL/well) was then added to the wells and incubated for 40 min at 37 °C. The wells were washed again five times with PBST1 before the TMB solution was added to the wells (100 μL/well) and incubated for 10 min. Finally, 50 μL of 2 M H2SO4 was added to quench the reaction and the optical density was measured at 450 nm. The percent inhibition of antibody binding used to characterized the binding ability of antibodies was expressed as follow: inhibition (%) = [1 − (B/B0)] × 100; B0 was the mean absorbance of the wells in the absence of a competitor; B was the mean absorbance of wells in the presence of a competitor. Optimization of ELISA Conditions. To improve performance of the ELISA, heterology in the chemical structure of the coating hapten was used, and optimization was performed of the physicochemical parameters considered to have significant effects on immunoassay performance,35 such as antibody dilution/coating concentration, and working buffer (concentration of Tween 20, ionic strength, and pH). For each condition, NPHA was used as the competitor analyte to construct inhibition curves (n = 3). The maximum value of the inhibition curve (Amax) and competitor analyte concentration leading to a 50% decrease in Amax (IC50) were calculated from these curves. The optimal conditions were confirmed by evaluating the ratio of Amax to IC50. Higher value of Amax/IC50 equates to better assay performance under such conditions. Preparation of Food Samples. Fish, red wine, and yoghurt samples were purchased from a local supermarket in Guangzhou, China. For fish, an equivalent weight of d.w. was added to the muscles

and homogenized, and then d.w. (8 mL) and the homogenate (2 g) were mixed and shaken vigorously for 1 min. The mixture was centrifuged at 3000g for 10 min, and the supernatant was then diluted 20-fold in d.w. and subjected to the derivatization procedure as follows: 200 μL of diluted supernatant, 400 μL of borate buffer (pH 9.0), and 400 μL of PNBA-OSu solution (2 mg/mL in ACN) were successively added to a 1.5 mL conical tube and vortexed for 10 s. The reaction mixture was kept at room temperature (25 °C) for 20 min, and 100 mg of NaCl was added to separate the organic layer (ACN) from the aqueous layer. The organic layer was diluted (10-fold) in PBST2 (optimized working buffer) for ciELISA analysis, and in a mixture of MeOH/H2O (v/v, 10:90) for analysis by LC−MS/MS. For red wine and yoghourt, d.w. (9 mL) was added to the samples (1 mL or 1 g) and shaken for 1 min, and then centrifuged at 3000g for 10 min. The supernatant was directly subjected to the same derivatization process as were the fish samples above, and then analyzed by ciELISA and LC−MS/MS. All prepared samples were filtered through a 0.22 μm membrane before LC−MS/MS analysis. Histamine Assay. Each prepared sample was split and subjected to histamine analysis by both ciELISA and LC−MS/MS methods. ciELISA. A calibration curve was constructed as follows: 200 μL serial dilutions of HA from the HA stock solution were subjected to the derivatization procedure as described above, 50 μL of the resulting solution was used as competitor in the ciELISA, and then the calibration curve was obtained by plotting the percent binding of antibodies in the wells (B/B0 × 100) against the logarithm of the HA concentration. For sample analysis, 50 μL of the prepared samples, as described above, were used as competitor in the ciELISA, and the determined B/B0 × 100 was used to calculate the HA concentration from the calibration curve. LC−MS/MS. The results from the ciELISA were confirmed by the LC−MS/MS method (completed by the Zhongshan Quality Supervision & Inspection Institute of Agricultural Products of Guangdong Province, Zhongshan, China). A 5 μL portion of the prepared samples was injected into the LC system (Agilent 1200-6410) equipped with an Agilent ZORBAX SB-C18 column (2.1 mm × 150 mm, 3.5 μm) and separated at a 0.4 mL/min flow rate (mobile phase A, 0.1% acetic acid; mobile phase B, ACN) in the following gradient profile: 0−3 min, 8%−15% B; 3−5 min, 15%−30% B; 5−5.01 min, 30%−90% B; 5.01−8 min, 90% B. Analytes were determined by an Agilent 6410 Triple Quad mass spectrometer (Agilent Technologies, Lexington, MA, USA) using ESI-MS/MS in the positive ion mode. The MS parameters were as follows: capillary voltage at 4.0 kV; desolvation temperature at 350 °C; desolvation gas (N2), flow rate at 10 L/min; ion spray voltage at 4000 V. The analytes were identified by parent/ daughter ions as well as peak retention times in comparison to the standards. The [M + H]+ at m/z 261.0 was the parent ion of NPHA. The daughter ion at m/z 95.1 was used for quantitation, and the daughter ion at m/z 68.1 was selected as a qualitative ion (see Figure S1 in the Supporting Information). A calibration curve for HA analysis by LC−MS/MS was constructed as follows: Serial concentrations (10−1000 ng/mL) of HA standard solutions in d.w. were prepared from the HA stock solution, and were subjected to the derivatization and treatment steps as described above. The treated solutions (5 μL) were injected into the instrument and analyzed. The responses of the mass spectrometer were plotted against the concentration of HA, which resulted in the calibration curve.

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RESULTS AND DISSCUSSION Hapten Synthesis and Antibody Production. It cannot be underestimated how big a key role the proper hapten design plays in antibody production and immunoassay development, especially for a compound like HA having an extremely simple structure. As can be seen from the structure of HA (Figure 1), the structure consists of two parts, an imidazole ring and a side chain (amino ethyl) containing a primary amino group. The only previously reported antibody that could specifically recognize free HA was produced by conjugating HA to KLH

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Table 1. Evaluation of Antiserum Binding Ability To Free Histamine under Homologous and Heterologous Coating Format antiserum A coating antigen hapten AOVA hapten BOVA hapten COVA hapten DOVA hapten EOVA hapten FOVA

antiserum B

titera (×103)

inhibnb (%)

titer (×103)

4

2.1