Rapid monoclonal antibody-based enzyme-linked immunosorbent assay for detection of Listeria in food products

A rapid diagnostic test for the detection of Listeria in food products has been created. This test, known as Listeria-Tek, uses 2 monoclonal antibodies specific for Listeria in an enzyme-linked immunosorbent assay (ELISA) format. The test requires only 40 h of broth enrichment with no culturing on solid media. It is extremely simple to perform and easy to interpret, and is at least as sensitive and accurate as the best of the culture methods. The test can be used with dairy products, meat products, and environmental samples. The ELISA test is safely performed on the open bench of the laboratory because no live cultures, no radioactivity, no phage, etc., are necessary. There is no need for special licenses or reserved laboratory space, and no waste disposal problems are encountered. If necessary, one technician could easily perform hundreds of assays per day. A printed data sheet is available for permanent records.     

High-affinity monoclonal antibodies for detection of the microbial metabolite, 2-methylisoborneol

The production of 2-methylisoborneol (MIB) by certain fungi and algae can contribute musty off-flavors to foods and water supplies if uncontrolled. The goal of this research was to develop a nonsensory simple method for the detection of MIB. Anti-MIB monoclonal antibodies were produced by immunizing mice with borneol-conjugated protein and selecting positive clones with an MIB-protein conjugate. An indirect competitive immunoassay developed using this antibody had a detection limit of 0.6 microg L(-)(1) and an I(50) value of 5 microg L(-)(1). Detection was relatively specific for MIB and showed 20% cross-reactivity with borneol or isoborneol and 4-5% cross-reactivity with camphor. No cross-reactivity to geosmin was observed.     

检测食品沙门氏菌的生物传感器持久性研究

为了研究温度对生物传感器检测的持久性的影响,制备了用于检测食品中沙门氏菌的磁致伸缩生物传感器,以磁致伸缩膜片作为物理传感器,多克隆抗体作为生物识别元件,采用Langmuir-Blodgett(LB)技术将多克隆抗体固定在磁致伸缩膜片表面。当食品中沙门氏菌吸附在生物传感器上时,将引起其共振频率漂移。通过测试与分析磁致伸缩生物传感器共振频率漂移值,并利用扫描电子显微镜(scanning electron microscope)观察吸附了沙门氏菌的生物传感器表面,对生物传感器在25(室温)、45及65℃的持久性进行研究。结果表明:多克隆抗体磁致伸缩生物传感器与沙门氏菌的结合能力随着时间的延长逐渐降低;且温度越高,传感器的持久性越差;在25、45及65℃时,多克隆抗体磁致伸缩生物传感器的持久期分别为30、8和5 d,由此获得了多克隆抗体生物传感器在常用温度下的持久性。并结合阿伦尼乌斯方程,计算得到该生物传感器的激活能为13.024 kJ/mol。进一步证实了磁致伸缩生物传感器可用于定量检测实际溶液中沙门氏菌的浓度,表明生物传感器可应用于食品中细菌的实时快速定量检测。     

单克隆抗体在植物病毒学中的应用

单克隆抗体是由杂交瘤细胞分泌的针对单一抗原决定簇的抗体。单克隆抗体在植物病毒学中的应用非常广泛，包括对植物病毒的检测、种下分类级别划分、抗原决定簇鉴定、非结构蛋白功能分析、体内病毒含量测定及病毒纯化等。     

Determination of seven artificial sweeteners in diet food preparations by reverse-phase liquid chromatography with absorbance detection

The artificial sweeteners aspartame, saccharin, cyclamate, alitame, acesulfam-K, sucralose, and dulcin are determined in diet soft drinks and tabletop sweetener preparations. Samples are diluted, filtered, and analyzed directly by liquid chromatography on a C-18 reverse-phase column with a mobile phase gradient ranging from 3% acetonitrile in 0.02M KH2PO4 (pH 5) to 20% acetonitrile in 0.02M KH2PO4 (pH 3.5). Diet puddings and dessert toppings are extracted with ethanol, filtered, and diluted with mobile phase for analysis. The sweeteners, except sucralose and cyclamate, were detected by UV absorbance at either 200 or 210 nm. Sucralose was determined at 200 nm or by refractive index. Cyclamate was determined after post-column ion-pair extraction. The sweeteners stevioside and talin were not detected. Additives such as caffeine, sorbic acid, and benzoic acid did not interfere.     

Haptens and monoclonal antibodies for immunoassay of imidazolinone herbicides

A set of haptens structurally resembling the herbicide imazethapyr (PURSUIT) was synthesized and used to derive monoclonal antibodies (MAbs) and direct and indirect competition enzyme immunoassays (EIAs) which could detect imazethapyr, imazaquin (SCEPTER), imazapic (CADRE), and imazamox (RAPTOR) in the 3-30 ng/mL (parts per billion) range, and imazapyr (ARSENAL) and imazamethabenz-methyl (ASSERT) in the 300-500 ppb range. Two MAbs, 3A2 and 3A5, had affinities of 10-75 nM for imazethapyr. MAbs 1A5, 1D2, and 3A5 were specific for the S isomers of the herbicides. Some MAbs were stable in solutions containing up to 15% methanol and 5% acetonitrile in indirect EIAs. Plates coated with hapten conjugates for indirect EIA could be stored frozen. Selectivity for the imidazolinones by some MAbs varied with different coating conjugates. These MAbs and haptens should prove useful in immunochemical analysis and residue recovery methods for imazethapyr and other imidazolinone herbicides.     

Characterization of monoclonal antibodies for lead-chelate complexes: applications in antibody-based assays

Monoclonal antibodies against lead were generated by immunizing BALB/c mice with lead conjugated to keyhole limpet hemocyanin (KLH) via a bifunctional chelator, S-2-(4-aminobenzyl)diethylenetriamine pentaacetic acid (DTPA). Stable hybridoma cell lines were produced by fusion of murine splenocytes and SP2/0 myeloma cells. One of the hybridomas generated from this fusion (4/7) synthesized and secreted an antibody that bound tightly to Pb2+-DTPA complexes but not to metal-free DTPA. The performance for a competitive inhibition enzyme-linked immunosorbent assay (ELISA) incorporating this antibody was assessed for its sensitivity to changes in pH, ionic strength, and blocking reagents. The cross-reactivities in this ELISA were less than 3% for Fe3+, Cd2+, Hg2+, and Cu2+ and less than 0.3% for Cr3+, Mn2+, Mg2+, In3+, Ag1+, Ni2+, Co2+, Zn2+, Ca2+, Cu1+, and Hg1+. The IC50 value achieved for lead was 2.72 +/- 0.034 microM, showing the detection range of 0.092-87.2 microM and the lowest detection limit of 0.056 +/- 0.005 microM. Recoveries from the analyte-fortified tap water and ultrapure water were in the range of 80-114% . These results indicate that the ELISA could be a convenient analytical tool for monitoring lead residues in drinking water.     

用于食品安全检测的生物传感器的研究进展

生物传感器特异性好、分析速度快、成本低,在食品安全检测领域有着重要的应用价值。该文介绍了电化学、光学、压电和量热生物传感器在食品安全检测中的应用,包括致病菌、抗生素残留、生物毒素和农药残留检测,指出了目前研究中需要解决的问题并展望了未来发展方向,认为高灵敏度、集成化、微型化、多功能化等是未来用于食品安全检测的生物传感器的发展趋势和重点方向。它在食品污染物的快速实时及特异性检测方面有着广阔的应用前景。     

Characterization of anti-irradiation-denatured ovalbumin monoclonal antibodies. Immunochemical and structural analysis of irradiation-denatured ovalbumin

Five monoclonal antibodies (OVA-01, -02, -03, -04, -06) produced against irradiated ovalbumin were investigated in relation to the conformational change in the ovalbumin molecule induced by irradiation with Cobalt-60 gamma-rays. Four antibodies (OVA-01, -02, -04, -06) recognized both native and irradiated ovalbumin, but OVA-03 reacted only with irradiated ovalbumin. These antibodies were classified by modified competitive ELISA, and their K(d) values were determined by the Klotz equation. Epitope analyses were also performed on OVA-03 using CNBr-cleaved peptide fragments from ovalbumin, and it was confirmed that OVA-03 bound to the fragment corresponding to residues Val173-Met196 of the ovalbumin molecule that consists of internal beta-sheet strand 3A and helix F1 containing one open turn. These results demonstrate that dramatic conformational changes in proteins can be induced or that some tertiary or secondary structures can be broken down by gamma-ray irradiation, producing new antigenic sites.     

Development of antibodies for the detection of N-acetyl-glufosinate

Glufosinate is a widely used herbicide, which is difficult to detect by conventional analytical techniques. For many other herbicides, suitable antibodies have been raised for immunoassay development. Unfortunately, glufosinate is a very small molecule and difficult to immunize with. Thus, a derivatization-assisted immunoassay (DAIA) using the target analyte N-acetyl-glufosinate (NAG) was constructed. The activated hapten was synthesized by a new approach, using a homobifunctional cross-linker suberic acid bis(N-hydroxysuccinimide ester). The preparation of a suitable conjugate, the immunization, and the characterization of polyclonal antibodies are shown. The determination of the conjugation density (hapten density) of the immunogens was performed by four different methods (high-performance liquid chromatography with a refractive index detector, total reflection X-ray fluorescence, inductively coupled plasma mass spectrometry, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry), which gave similar results. The limit of detection was 17 mug/L NAG in water for the direct competitive enzyme immunoassay. NAG is also a main metabolite of glufosinate in resistant transgenic plants. The antibodies might be useful for the selective detection of NAG in the presence of the parent compound glufosinate (cross-reactivity 0.13%) and other metabolites.     

Development of a monoclonal antibody-based cELISA for the analysis of sulfadimethoxine. 1. Development and characterization of monoclonal antibodies and molecular modeling studies of antibody recognition

Sulfonamide antibiotics are used to treat a variety of bacterial and protozoan infections in cattle, swine, and poultry. Current residue methods for the analysis of sulfonamides in animal-based food products include bioassays, chromatographic methods (HPLC, GLC), and immunoassays. Most immunoassays have employed highly specific polyclonal antibodies. In this paper, we describe the isolation of monoclonal antibodies against sulfadimethoxine (SDM) that vary in their sensitivities and cross-reactivities against a large number of sulfonamides. The most sensitive monoclonal antibody, designated SDM-18, exhibits an IC(50) value for SDM of 1.53 ppb. Another monoclonal antibody, designated SDM-44, exhibits IC(50) values for six sulfonamides well below the established threshold level of 100 ppb for animal tissues. Molecular modeling studies of the cross-reactive drugs suggest that, depending on the monoclonal antibody, both steric and electronic features govern antibody binding. Due to the diversity of these monoclonal antibodies, it should be possible to design both compound- and class-specific monoclonal antibody-based immunoassays.     

Molecular and immunochemical characteristics of monoclonal and recombinant antibodies selective for the triazine herbicide simetryn and application to environmental analysis

A monoclonal antibody (mab) selective for the thiomethyl-s-triazine herbicide simetryn was obtained and characterized in enzyme-linked immunosorbent assay (ELISA). An IC(50) value for simetryn was 8.5 ng/mL, and the detection range extended from 1.1 to 70 ng/mL in ELISA. The cDNAs encoding variable heavy chain (VH) and variable light chain (VL) of the mab were cloned to produce various recombinant antibodies. Single-chain variable fragment (scFv) antibodies derived from the mab were characterized in ELISA and showed similar reactivities and specificities to the parent mab. A urea denaturation test revealed that the scFv antibodies bound to simetryn were more stable than those in the absence of antigen. A sandwich ELISA based on VH and VL fragments of the mab was successfully developed and showed similar sensitivity to those based on the mab and scFv antibodies in ELISA. In the recovery experiments using spiked environmental samples, the results obtained in ELISA based on the mab were favorably correlated with those by HPLC.     

Molecular quantification of slug density in the soil using monoclonal antibodies

Molecular ecology techniques are increasingly used to study invertebrate foodwebs and trophic interactions in the field. However, the study of subterranean foodwebs is currently constrained by the difficult, laborious and often expensive methods that need to be employed to simply measure invertebrate population densities accurately. Here we describe and field-test a novel monoclonal antibody-based system for tracking slug populations. Proteins were extracted from soil blocks using sodium chloride and a new slug-specific monoclonal antibody was developed, capable of detecting the proteins liberated by the salt. Detection sensitivity and limits, using enzyme-linked immunosorbent assays (ELISA), were measured for a range of soils and the system proved to be effective, whether the soil was heavy clay or sandy. The sensitivity of the assay varied between soils and needed to be calibrated by ELISA. There was a linear relationship between slug biomass in any given soil and slug proteins detected by ELISA. A field experiment was performed comparing ELISA with the most accurate conventional approach. The latter involved taking blocks of soil from the field and flooding them gradually over 10 days to drive slugs to the surface, where they were collected and weighed. Parallel blocks of soil were taken 1 m away and subjected to the salt extraction/ELISA approach. Results using the two systems proved to be very similar, but ELISA produced results more rapidly. The many advantages of using ELISA to measure slug density are discussed.     

In-vitro and in-vivo binding activity of chicken egg yolk immunoglobulin Y (IgY) against gliadin in food matrix

Chicken egg yolk immunoglobulin Y (IgY) is a promising alternative for the prevention of enteric gliadin absorption, the predisposing factor of celiac disease (CD). IgY antibody was produced from the egg yolk of Single Comb White Leghorn chickens during the immunization period for the development of an oral immunotherapeutic agent. Here, we report the potential use of spray dried IgY antibody formulation using sugar protectants (mannitol, sorbitol, or microcrystalline cellulose powder (MCCP)). The long-term stability of the spray dried egg yolk powder formulated with 37.5% mannitol (EYP-M) preserved IgY antibody activity at 99.9%, which was significantly higher than that with other protectants (p < 0.05). In a dissolution test, the EYP-M shows 82.4% IgY activity after 2 h in simulated gastric fluid (SGF). A competitive ELISA at 50% inhibition (IC(50)) shows that 1.6 mg/mL EYP-M bound to 7.6 mg/mL and 10.5 mg/mL gliadin in SGF without and with food matrix conditions, respectively, whereas in simulated intestinal fluid, the formulation bound to 10 mg/mL gliadin, regardless of a food matrix. In-vivo study: BALB/c mice fed with EYP-M and gliadin at a ratio of 1:5 (w/w) demonstrated that gliadin absorption in the gastrointestinal tract was minimal at <1%. Thus, EYP-M containing IgY antibody may be used in CD patients to eliminate the effects of ingested toxic gliadin.     

Collaborative testing of methods for food analysis

The complex composition of foods makes their analysis difficult. Results of collaborative tests with food materials often show greater coefficients of variation than with other matrices. Some critical points in collaborative testing of foods are discussed.     

动物源食品中兽药残留高通量快速分析检测技术

随着养殖业的迅猛发展,动物源食品兽药残留问题日益成为食品安全领域的重要内容。动物源食品基质复杂,而其中残留的兽药含量甚微,传统的样品制备及检测方法大多存在检测样品基质种类单一、检测兽药种类范围小、耗时长、重现性差等问题,缺乏一定的通用性和准确性,已不能满足当前社会发展的需要。近年来,随着QuChERS法和高分辨质谱等先进样品制备和检测技术在兽药残留检测分析领域的应用,一批高通量、自动化乃至可视化的快速高效分析检测方法也随之而起,该文即对这些高通量快速样品制备和检测方法进行综述,同时对动物源食品中兽药残留的检测和监控等工作提出建议并进行了展望。     

Monoclonal antibodies for the analysis of gossypol in cottonseed products

Immunogens, prepared by conjugating either (+)-gossypol or (-)-gossypol to Limulus polyphemushemolymph protein, were used for immunization in the production of monoclonal antibodies. Hybridoma were evaluated for their relative affinity to racemic gossypol, (+)-gossypol, (-)-gossypol, gossypol analogues, and their lysine derivatives. The monoclonal antibody obtained showed higher affinity to gossypol and gossypol analogues as compared to their lysine derivative counterparts. An indirect competitive enzyme-linked immunosorbent assay (ELISA) with this antibody was used to measure gossypol in 15 cottonseed meal products; the results showed good correlation with results obtained using the AOCS (free gossypol) official method (R(2) = 0.89). The direct recognition of both free gossypol and bound gossypol using this antibody will be useful for rapid screening and quality control.     

Production of monoclonal antibody for the detection of meat and bone meal in animal feed

For the detection of prohibited meat and bone meal (MBM) in animal feed, monoclonal antibodies (MAbs) were raised against heat-stable h-caldesmon purified from bovine intestinal smooth muscle. The obtained hybridoma cells were screened against extracts of the bovine MBM and heat-treated smooth muscle, and MAb 5E12 was identified as having the best performance. Antibody 5E12 did not react with animal feed, milk product, plant proteins, and other ingredients used for commercial animal feed except for the gelatin. This antibody diluted to 100-fold was able to detect MBM mixed in animal feed at 0.05% in an ELISA, and it showed strong affinity toward bovine smooth muscle autoclaved at 130 degrees C. Therefore, this antibody can be used in the ELISA system for field testing of the presence of MBM in animal feed.     

Development of polyclonal antibodies for the detection of Tribolium castaneum contamination in wheat grain

Two polyclonal antibodies (Pab) were developed for the detection of Tribolium castaneum, which is a stored product pest of medical and economical importance. Selected Pab anti- T. castaneumK51 showed low cross-reactivity to other stored product arthropods but revealed high reactivity to T. destructor, T. confusum, and partly to Tenebrio molitor. PTA-ELISA was used to detect adults, larva, and feces of T. castaneum in artificially contaminated grain samples. Calibration methods were applied to determine detection limits for each type of contaminants. Anti- T.castaneumK51 enabled detection of T. castaneum in grain samples; detection limits reached 60 and 640 individuals/kg of grain for larvae and adults, respectively, and 4 mg of feces/kg of grain. After recalculation, the detection limit for feces enables detection of 30 larvae after 5 days of feeding in optimal conditions. The main advantage of the developed assay is traceability of T. castaneum contamination, especially when the adults and larvae are removed from contaminated material, based on the detection of feces that persist in the grain.