Production of a Monoclonal Antibody by Simultaneous Immunization of Staphylococcal Enterotoxin A and B

In this paper, a method of simultaneous immunizing BALB/c mice with staphylococcal enterotoxin (SE) A and B (SEA and SEB) to prepare a monoclonal antibody (3F2) for detecting both of SEA and SEB was developed. The results showed that antibody 3F2 had high titers against both SEA and SEB by enzyme-linked immunosorbent assay (ELISA). The sensitivities of 3F2 to SEA and SEB detected by ELISA were 133.2 and 82.5 ng/mL, respectively, and the detection limits for the two enterotoxins were about 1 ng/mL. The antibody 3F2 had high specificities and affinities to both SEA and SEB, and had no cross-reaction with SEC₁, bovine serum albumin, and ovalbumin. SEs-free skimmed milk samples were spiked with different concentrations of SEA, SEB, or both of them, respectively. Average recoveries of SEA and SEB from the spiked samples were all nearly between 82% and 104%. The result suggested that one cell fusion with simultaneous immunization by multiple antigen to prepare monoclonal antibody against them was possible, simple, and economic. The monoclonal antibody could be used in simultaneous detecting multifarious SEs.     

High sensitivity chemiluminescence enzyme immunoassay for detecting staphylococcal enterotoxin A in multi-matrices

In this study, detection of staphylococcal enterotoxin A (SEA) in multi-matrices using a highly sensitive and specific microplate chemiluminescence enzyme immunoassay (CLEIA) has been established. A pair of monoclonal antibodies (mAbs) was selected from 37 anti-SEA mAbs by pairwise analysis, and the experimental conditions of the CLEIA were optimized. This CLEIA exhibited high performance with a wide dynamic range from 6.4 pg mL⁻¹ to 1600 pg mL⁻¹, and the measured low limit of detection (LOD) was 3.2 pg mL⁻¹. No cross-reactivity was observed when this method was applied to test SEB, SEC1, and SED. It has also been successfully applied for analyzing SEA in a variety of environmental, biological, and clinical matrices, such as sewage, tap water, river water, roast beef, peanut butter, cured ham, 10% nonfat dry milk, milk, orange juice, human urine, and serum. Thus, the highly sensitive and SEA-specific CLEIA should make it attractive for quantifying SEA in public health and diagnosis in near future.     

离子液体保护聚苯胺纳米金复合膜传感器的制备及其在检测乳制品中金黄色葡萄球菌毒素B的应用

采用原位合成法制得聚苯胺修饰纳米金的复合膜,并采用紫外、红外及透射电镜对其进行表征,以离子液体1,3-二丁基-咪唑六氟磷酸盐为保护复合膜的溶剂,成功制得离子液体保护的聚苯胺纳米金复合膜免疫传感器,并应用于乳品中金黄色葡萄球菌肠毒素B(SEB)的检测。在Fe(CN)63-/4-溶液中,采用循环伏安法(CV)及交流阻抗法(EIS)对传感器进行表征与测定,建立了SEB检测标准曲线,Y=933.46x+1399.8,线性范围为0.1～8.0μg/L,相关系数R2=0.9932,检出限明显降低(0.033μg/L,S/N=3)。结果表明,本传感器特异性良好,乳品检测回收率在88%～119%之间,稳定性好,可应用于乳品中的SEB快速检测。     

An ultrasensitive immunosensor array for determination of staphylococcal enterotoxin B

Staphylococcal enterotoxin B (SEB) is a potent gastrointestinal toxin and is heat resistant. SEB is also a potential bioterrorism agent. The ability to measure accurately very low amounts of staphylococcal enterotoxin B in food and other samples is very important. A highly sensitive and stable sandwich fluorescence immunoassay based on a pair of monoclonal antibodies against SEB which were produced by us was developed. Classical sandwich immunoassay was adopted and the glass slides were used as the base of the immunologic reaction. The functionalized fluorescent core-shell silica nanoparticles were used as labels. The fluorescence issued from the labels was detected by a laser-induced fluorescence millimeter sensor array detection platform. The fluorescence intensity has a linear relationship with the amount of SEB in the range of 50 pg/mL-5 ng/mL, and the detection limit of SEB was 20 pg/mL (the absolute detection limit was 0.02 pg). The relative standard deviation (RSD) for 5 parallel measurements of SEB (1 ng/mL) was 9.2%.     

Multiresidue determination of zeranol and related compounds in bovine muscle by gas chromatography/mass spectrometry with immunoaffinity cleanup

A gas chromatography/mass spectrometry (GC/MS) method with immunoaffinity cleanup was developed for the determination of zeranol and related compounds, taleranol, zearalanone, and alpha-zearalenol in bovine muscle. Muscle samples were extracted with methanol and cleaned up with immunoaffinity chromatography (IAC) columns containing monoclonal antibodies raised against zeranol coupled to CNBr-activated Sepharose 4B. After derivatization, the compounds were analyzed by GC/MS. The dynamic column capacities for zeranol, taleranol, zearalanone, and alpha-zearalenol were 2639.7, 2840.3, 2731.5, and 2736.3 ng/mL Sepharose gel, respectively. The limits of detection and quantification were 0.5 and 1.0 ng/g, respectively, for all 4 compounds. Mean recoveries were 79.6-110.7% with coefficients of variation of 3.2-11.4% at spiked levels of 1.0-5.0 ng/g. This IAC-GC/MS method may be used for the determination of zeranol, taleranol, zearalanone, and alpha-zearalenol residues in bovine muscle, and possibly other tissues.     

Matrix effects in the determination of β‐receptor agonists in animal‐derived foodstuffs by ultra‐performance liquid chromatography tandem mass spectrometry with immunoaffinity solid‐phase extraction

Matrix effects in determination of three β‐receptor agonists including salbutamol (SAL), clenbuterol, and terbutaline in animal‐derived foodstuffs were studied by ultra‐performance LC‐MS/MS with cleanup of immunoaffinity SPE column (IAC). Some animal tissue samples including pig liver, swine muscle, and fish muscle were hydrolyzed by the mixed enzyme solution or HCl solution, and the cleanup efficiencies with SAL IAC, MCX SPE column, and C₁₈‐SCX tandem columns were examined and compared by using spiked experiments. The results showed that the matrix effects in the determination of SAL and terbutaline can be eliminated with SAL IAC cleanup, and the average recoveries of SAL were 77.4～81.5%, 79.0～80.3%, and 85.0～87.2% in pig liver, swine muscle, and fish muscle, respectively. The decision limit (ccα) and detection capability (ccβ) for SAL in pig liver were 0.02 and 0.05 μg/kg, respectively.     

Establishment of an immunoaffinity chromatography for simultaneously selective extraction of Sudan I, II, III and IV from food samples

The establishment of an immunoaffinity chromatography (IAC) for simultaneously selective extraction of four illegal colorants Sudan dyes (Sudan I, II, II and IV) from food samples was described. The IAC column was constructed by covalently coupling monoclonal antibody (mAb) against Sudan I to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. It was observed that IAC column was able to separately capture Sudan I, II, III and IV with maximum capacity of 295, 156, 184 and 173ng, respectively. The extraction conditions including loading, washing and eluting solutions were carefully optimized. Under optimal conditions, the extraction recoveries of the IAC column for Sudan I-IV at two different spiked concentrations were within 95.3-106.9%. After 50 times repeated usage, 64% of the maximum capacity was still remained. Six food samples randomly collected from local supermarket without spiking Sudan dyes were extracted with IAC column and detected by high performance liquid chromatography (HPLC). It was found that there was no detectable Sudan II, III and IV in all six food samples, but Sudan I with the content of 2.7-134.5ngg(-1) was detected in three food samples. To further verify the extraction efficiency, other three negative samples were spiked with Sudan I-IV at the concentrations of 20ngg(-1) and 50ngg(-1), which were then extracted with IAC column. The extraction recoveries and relative standard deviation (RSD) were 68.6-96.0% and 4.8-15.2%, respectively, demonstrating the feasibility of the prepared IAC column for Sudan dyes extraction.     

Competitive immunoassay for staphylococcal enterotoxin A using capillary electrophoresis with laser-induced fluorescence detection.

Staphylococcal enterotoxins are a family of toxic proteins secreted by S. aureus. Using capillary electrophoresis (CE) linked with laser-induced fluorescence, a highly sensitive and selective assay using antibody-antigen recognition was developed for the determination of Staphylococcal enterotoxin A. Staphylococcal enterotoxin A (SEA) was chemically labeled with fluorescein and the product was used as a fluorescent tracer. A competitive assay was developed to detect SEA at concentrations between 0.3 nM and 6.5 nM with standard deviations of less than 5%. The detection limit was found to be 3 amol with the potential improvement by further optimization of the assay. No cross-reactivity between staphylococcal enterotoxin B and the SEA antibody was found at the concentrations used for the CE immunoassay.     

A capacitive biosensor for detection of staphylococcal enterotoxin B

A sensitive method for the detection of staphylococcal enterotoxin B (SEB) using a flow-injection capacitive biosensor is presented. SEB was purified from a crude culture filtrate of Staphylococcus aureus through three chromatographic steps. The first two steps were based on ion-exchange chromatography, and the last step was carried out on a gel filtration column. The SEB recovery values after the purification stages were 88%, 74%, and 12%, respectively. A horseradish peroxidase labeled antistaphylococcal enterotoxin B was prepared by the periodate method and was further employed in a sandwich-enzyme-linked immunosorbent assay (ELISA) for the determination of SEB concentrations in different samples obtained during the processing of the crude filtrate. The capacitive biosensor could detect SEB concentrations as low as 0.3 pg ml⁻¹ with a linearity ranging from 2.8 pg ml⁻¹ to 2.8 ng ml⁻¹ under optimized conditions. The response time was about 10 min. A good agreement was achieved between the developed capacitive biosensor system and ELISA as a reference method for detection of SEB levels in different purification samples. The newly developed sensor has the benefits of simplicity, high sensitivity, and multiple use capability.     

Production and characterization of polyclonal antibodies to sulfamethazine and their potential use in immunoaffinity chromatography for urine sample pre-treatment

An immunoaffinity chromatographic (IAC) method for isolating sulfamethazine (SMZ) from incurred urine samples was developed. This was achieved by (i) generating polyclonal antibodies that recognize equally well SMZ and its major urinary metabolites, (ii) evaluating in an ELISA procedure the influence of methanol, salt and pH on the antigen-antibody interaction in order to determine the optimum conditions for IAC and (iii) covalent coupling of the IgG fractions of anti-SMZ to CNBr activated Sepharose for the preparation of re-usable immunoaffinity columns, having a high capacity for SMZ (1900 ng SMZ mL-1 gel). For desorbing SMZ from the immunoaffinity column, different elution modes were evaluated, with 40% MeOH-0.1 mol L-1 HOAc-0.5 mol L-1 NaCl being the most efficient combination. Using the IAC column for processing SMZ spiked urine samples resulted in high recoveries, ranging from 92 to 100%. Because of the high cross-reactivity with the major metabolites of SMZ present in urine of treated animals, the antibodies show excellent properties for use in both IAC and ELISA. For the isolation and concentration of the parent drug and its major metabolites, the urine could be applied directly to the IAC column, without the time-consuming step of deconjugation. Moreover, the use of IAC prior to ELISA for the analysis of incurred urine samples showed good efficiency for the elimination of matrix interferences. Owing to the urine-tissue relationship, the urine concentrations can be used to predict the presence of the parent drug in tissues and so possible violations of the maximum residue limit (MRL) can be controlled.     

Preparation and Evaluation of Sol-Gel Glass-Based Immunoaffinity Column and their Potential Use in Determination of Diazinon in Water and Soil Samples with High Performance Liquid Chromatography

Abstract

An immunoaffinity column (IAC), which can bind diazinon primely, was developed to treat the water and soil samples. The IAC, having an appropriate binding capacity for diazinon (200 ng per mg gel), was prepared by entrapping the anti-diazinon antibody into sol-gel, which was made from hydrolyzed Tetramethoxysilane (TMOS). In comparison with the IACs entrapped with others non-special antibodies, the IAC entrapped with anti-diazinon antibody has a special binding capacity to diazinon. Two different elution modes, 40% and 20% MeCN water solution, were evaluated for desorbing diazinon from the IAC. Efficient desorption of bound diazinon was achieved with the 40% MeCN water elution. The water and soil samples spiked with diazinon were treated by the IAC. The reliable recoveries, which ranged from 50% and 100% with RSD from 4.54% to 16.79%, were achieved.     

乳品中金黄色葡萄球菌肠毒素B免疫传感检测方法的研究

本文利用自制的金黄色葡萄球菌肠毒素B(SEB)抗体构建了一种L-半胱氨酸和纳米金的双层自组装免疫传感器,采用循环伏安法及交流阻抗法对传感器进行表征与测定,并对各项相关条件进行优化,最终建立了SEB检测的标准曲线,线性范围分别在2～10 ng/mL和10～100 ng/mL,相关系数分别为0.9939和0.9926,检出限(S/N=3)为0.667 ng/mL,乳品检测回收率在84.3%～93.2%之间。该传感器特异性良好,稳定性好,可再生使用,可应用于乳品中SEB的快速检测。     

Immunoassay methods for paralytic shellfish poisoning toxins

The current status of immunochemical techniques for analysis of paralytic shellfish poisoning (PSP) toxins is summarized. Important aspects regarding production of the biological reagents necessary for immunochemical methods, the characteristics of polyclonal and monoclonal antibodies against saxitoxin and neosaxitoxin, and the importance of test sensitivity and specificity are discussed. Applications of immunochemical techniques for PSP toxins include microtiter plate enzyme immunoasays and enzyme-linked immunofiltration assays for toxin detection, and immunoaffinity chromatography (IAC) for sample extract cleanup. A major advantage of enzyme immunoassay (EIA) is simplicity and rapidity of the test procedure, and higher sensitivity than other methods. However, quantitative agreement between EIA and mouse bioassay is dependent on antibody specificity and the toxin profile in the shellfish; thus, both over- and underestimation of total toxicity may occur. For screening purposes, however, EIAs offer major advantages over the mouse bioassay, which is criticized in Europe because of animal welfare. A major application of antibodies against PSP toxins is their use for extract cleanup by IAC, which gives highly purified extracts, thereby enhancing determination of PSP toxins by conventional physicochemical methods such as liquid chromatography. IAC can also be used to isolate PSP toxins for preparation of analytical standard solutions.     

Preparation and evaluation of a mixed-bed immunoaffinity column for selective purification of sixteen sulfonamides in pork muscle

This paper describes the preparation of a novel mixed-bed immunoaffinity chromatography (IAC) column by coupling four monoclonal antibodies against different sulfonamides (SAs) to Sepharose 4B. The IAC column can be used to simultaneously extract and purify 16 SAs in pork muscle. The dynamic column capacities for all SAs in mixed standard solution were between 312 and 479 ng/mL gel. After simple extraction and IAC cleanup, the sample solution can be directly injected for liquid chromatography-ultraviolet analysis. The recoveries of SAs from spiked samples at levels of 25, 50 and 100 μg/kg ranged from 83.3 to 103.1% with variation coefficient less than 8.6%. The comparison of IAC with liquid-liquid extraction and solid phase extraction indicated that IAC has better purification effect and needs less organic solution than conventional methods, thus it would be an ideal method for selective purification of SAs in pork muscle.     

Determination of Fusarium mycotoxins by liquid chromatography/tandem mass spectrometry coupled with immunoaffinity extraction

A method for the simultaneous quantitative determination of deoxynivalenol (DON), T‐2 toxin (T‐2), HT‐2 toxin (HT‐2) and zearalenone (ZEN) in wheat and biscuit by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) coupled with immunoaffinity extraction is described. A clean‐up was carried out using a DZT MS‐PREP® immunoaffinity column (IAC), and the effect of the sample dilution rate and sample loading was investigated. Furthermore, the effects of ion suppression of a multifunctional column (MFC) and the IAC in the clean‐up were compared. The results with the DZT MS‐PREP® IAC showed that it is possible to make the sample dilution rate low, and indicated a higher solvent‐tolerance than usual with an IAC. Sample loading was optimized at 0.25 g. Ion suppression was lowered by purification of the toxins using the DZT MS‐PREP® IAC. Recoveries of each mycotoxin from wheat and biscuit samples spiked at two levels ranged from 78 to 109%. The limits of detection in wheat and biscuit was in the range of 0.03–0.33 ng·g^?1. From these studies, it is suggested that use of an IAC is effective in the clean‐up of each mycotoxin, and, when combined with LC/ESI‐MS/MS, it is good for the determination of mycotoxins in foodstuffs due to its rapidity and high sensitivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Miniaturized 96-well ELISA chips for staphylococcal enterotoxin B detection using portable colorimetric detector

A previously developed fluorescence sensing platform, combining spatial illumination using electroluminescence (EL) semiconductor strips with charge coupled device (CCD)-based detection (EL-CCD), was adapted to a new 96-well chip for colorimetric immunological assays, enhancing the capabilities of the EL-CCD platform. The modified system was demonstrated using a colorimetric-based enzyme linked immunosorbent assay (ELISA) for detection of staphylococcal enterotoxin B (SEB). Limits of detection (LODs) of 3.9 ng/mL (±2.4 ng/mL) SEB were determined with the ELISA chip measured using the EL-CCD platform, following a standard 4-h ELISA protocol. The LODs were comparable to those obtained using standard 96-well ELISA plates measured using a standard laboratory 96-well plate reader. The miniature 96-well ELISA chip however required as little as 5-μL samples, representing a tenfold reduction in sample volume compared to a standard 96-well ELISA plates. The ELISA chip also demonstrated detection of SEB spiked into various food matrices (milk, mushrooms, and mayonnaise) using limited-to-no sample preparation, with LODs ranging from 3.9 to 18.5 ng/mL depending on the matrix. The EL-CCD platform is versatile, capable of multi-mode detection (e.g., fluorescent and colorimetric along with solution and solid phase assays), and could readily be applied to other field portable or point-of-care applications. Figure Detection of SEB using miniature ELISA chips coupled with a portable electroluminiscent-charge couple device (EL-CCD) detection system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A novel antibody immobilization and its application in immunoaffinity chromatography

A novel antibody immobilization and its application in immunoaffinity chromatography (IAC) were presented. Using acrylamide (AM) as monomer, ethylene glycoldimethacrylate (EGDMA) as cross-linker and bulk polymerization as synthetic method, we prepared a polymer in which the Cu(II) was embedded. The Cu(II)-embedded polymer was tested for its binding with protein. It was found that Cu(II)-embedded polymer displayed a strong binding with bovine serum albumin (BSA). At 80% of methanol, no BSA was released from Cu(II)-embedded polymer. The Cu(II)-embedded polymer was then used as a novel solid support for antibody immobilization. IAC column was prepared by immobilizing polyclonal antibody (pAb) against clenbuterol (CL) on Cu(II)-embedded polymer and packing the Cu(II)-embedded polymer-pAb into a common solid phase extraction (SPE) cartridge. Under optimal extraction conditions, the IAC column was characterized in terms of maximum binding capacity for target analyte, extraction efficiency and reusability. It was revealed that, for IAC column packed with 0.1 g of solid support immobilized with antibody, the maximum capacity for CL was 616 ng; the extraction recoveries of the column for CL from three spiked food samples were 84.4-95.2% with relative standard deviation (RSD) of 9.3-15.5%; after more than 30 times repeated usage, there was not significant loss of specific recognition. The results demonstrated the feasibility of the prepared IAC column for CL extraction. The proposed antibody immobilization method exhibiting the properties of simplicity, low cost, strong binding for target analyte, no leaching of antibody, etc., would be a very useful tool applied in the field of IAC.     

Determination of zearalenone in cereal grains, animal feed, and feed ingredients using immunoaffinity column chromatography and liquid chromatography: interlaboratory study

A method using immunoaffinity column chromatography (IAC) and liquid chromatography (LC) for determination of zearalenone in cereal grains, animal feed, and feed ingredients was collaboratively studied. The test portion is extracted by shaking with acetonitrile-water (90 + 10, v/v) and sodium chloride. The extract is diluted and applied to an immunoaffinity column, the column is washed with water or phosphate-buffered saline or methanol-water (30 + 70, v/v), and zearalenone is eluted with methanol. The eluate is evaporated, the residue is dissolved in mobile phase and analyzed by reversed-phase LC with fluorescence detection. The presence of zearalenone can be confirmed using an alternate excitation wavelength or diode array detection. Twenty samples were sent to 13 collaborators (8 in Europe, 2 in the United States, one in Japan, one in Uruguay, and one in Canada). Eighteen samples of naturally contaminated corn, barley, wheat, dried distillers grains, swine feed, and dairy feed were analyzed as blind duplicates, along with blank corn and wheat samples. The analyses were done in 2 sample sets with inclusion of a spiked wheat control sample (0.1 mg/kg) in each set. Spiked samples recoveries were 89-116%, and for the 18 naturally contaminated samples, RSDr values (within-laboratory repeatability) ranged from 6.67 to 12.1%, RSDR values (among-laboratory reproducibility) ranged from 12.5 to 19.7%, and HorRat values ranged from 0.61 to 0.90.     

Preparation and characterization of an immunoaffinity column for the selective extraction of salbutamol from pork sample

A rapid, simple, and reliable determination method for salbutamol in pork was developed with immunoaffinity column (IAC) extraction followed by HPLC analysis. The salbutamol immunoaffinity column was prepared by coupling CNBr-activated Sepharose-4B with the anti-salbutamol polyclonal antibody which was purified by caprylic acid-ammonium sulfate. The coupling rate of the antibody and Sepharose-4B was 98.6%, and the dynamic column capacity of IAC was 400 ng/mL gel. The average recoveries of salbutamol from spiked pork samples at levels of 2, 10, 20, and 50 ng/g ranged from 83.3% to 92.2%, with the relative standard deviations of 2.8-7.0% (n=5), and the limits of detection and qualification were 0.25 ng/g and 0.5 ng/g, respectively.     

Determination of zeranol and beta-zearalanol in calf urine by immunoaffinity extraction and gas chromatography-mass spectrometry after repeated administration of zeranol

A method for the determination of zeranol and its metabolite beta-zearalanol in bovine urine is described. It has been applied to samples from calves given multiple subcutaneous doses of zeranol. Samples were extracted with immunoaffinity columns containing antibodies raised against zeranol and were analysed by gas chromatography-mass spectrometry. The immunoaffinity columns were prepared by coupling immunoglobulin G fractions obtained from rabbit antisera with a Sepharose matrix. The immunizing agent was carboxybutylzeranol coupled to bovine serum albumin. Gas chromatography-mass spectrometry was performed in the negative-ion chemical ionization mode, after derivatization of the compounds to their pentafluorobenzyl ethers, and allowed detection of analytes with a sensitivity of 0.01 ppb in spiked urine. The derivatization method and the gas chromatographic determination were also applied to the similar compounds zearalanone, zearalenone and beta-zearalenol. A synthesis of dideuterated zeranol and beta-zearalanol by isotopic exchange is described. These deuterated analogues had an isotopic purity of more than 99% and were used for quantitation of zeranol and beta-zearalanol by isotope dilution mass spectrometry. The recoveries of zeranol and beta-zearalanol, using the immunoaffinity columns, were determined after extraction from spiked urine and were 84 and 64%, respectively. The urines of treated calves were collected for several days after treatments and were analysed after hydrolysis with beta-glucuronidase and arylsulphatase. The samples showed variable but generally decreasing concentrations of zeranol and beta-zearalanol. The levels of beta-zearalanol ranged from less than 0.01 to 98 ppb and were 1.2-3.2 times higher than those of zeranol.