Development of a fluorescent latex microparticle immunoassay for the detection of staphylococcal enterotoxin B (SEB)

Staphylococcus aureus enterotoxin B (SEB) is a highly heat resistant enteric toxin with a potential as a biothreat agent. A sensitive method for the detection of staphylococcal enterotoxins is needed for food safety and food defense monitoring. The objectives of this research were to develop a competitive fluorescent immunoassay with detection of SEB below toxic levels of 1 ng/mL and to minimize sample preparation. Anti-SEB was immobilized onto carboxylated polystyrene microparticles, and SEB was labeled with fluorescein isothiocyanate (FITC). The concentrations of these reagents were optimized for the detection of SEB below 1 part per billion (1 ng/mL), and other assay conditions (sample volumes and incubation periods) were optimized. Drinking water and milk samples were spiked with 0.125-10 ng/mL SEB and were equilibrated overnight prior to analysis. The water and milk samples were directly analyzed, but heating the milk samples for 10 min at 90 degrees C improved the assay performance. SEB in samples bound with the anti-SEB linked to the latex followed by the competitive binding of SEB-FITC tracer. The excess, unbound tracer was separated by centrifugation, and the fluorescence density of the supernatant was measured. SEB was detected at levels as low as 0.125 ng/mL in drinking water and 0.5 ng/mL in whole milk. This fluorescent latex particle immunoassay will be utilized for the detection of SEB in various foods matrices.     

Development of a novel and automated fluorescent immunoassay for the analysis of beta-lactam antibiotics

An automated immunosensor for the rapid and sensitive analysis of penicillin type beta-lactam antibiotics has been developed and optimized. An immunogen was prepared by coupling the common structure of the penicillanic beta-lactam antibiotics, i.e., 6-aminopenicillanic acid to keyhole limpet hemocyanin. Polyclonal antibodies raised in rabbits after immunization with this conjugate have been applied for the development of a competitive fluoroimmunoassay (FIA), using a novel fluorescent penicillin {[2S,5R,6R]-3,3-dimethyl-7-oxo-6-[(pyren-1ylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxilic acid, PAAP} as the tracer and penicillin G as the reference antibiotic. Protein A/G covalently bound to an azlactone-activated polymeric support was used for the orientated capture of the antibody-antigen immunocomplexes. Upon desorption from the immunosupport, the emission signal generated by the PAAP-Ab complexes is related to the antibiotic concentration in the sample. The 50% binding inhibition concentration of penicillin G standard curves was at 30 ng mL(-)(1) with a detection limit (10% binding inhibition) of 2.4 ng mL(-)(1) and a dynamic range from 6.0 to 191 ng mL(-)(1) (20-80% binding inhibition) penicillin G. The generic nature of the antiserum was shown by good relative cross-reactivities with penicillin type beta-lactam antibiotics such as amoxicillin (50%), ampicillin (47%), and penicillin V (145%) and a lower response to the isoxazolyl penicillins such as oxacillin, cloxacillin, and dicloxacillin. No cross-reactivity was obtained for cephalosporin type beta-lactam antibiotics (cephapirin), cloramphenicol, or fluoroquinolones (enrofloxacin and ciprofloxacin). The total analysis time was 23 min per determination, and the immunoreactor could be reused for more than 200 cycles without significant loss of activity. The immunosensor has been successfully applied to the direct analysis of penicillin G and amoxicillin in spiked influent and effluent sewage treatment plant water samples with excellent recoveries (mean values for penicillin G and amoxicillin, 99 and 105%, respectively). Results displayed by comparative analysis of the immunosensor with a chromatographic procedure for penicillins showed excellent agreement between both methods.     

Development of a lateral flow colloidal gold immunoassay strip for the rapid detection of olaquindox residues

A rapid immunochromatographic lateral flow test strip of competitive format has been developed for the specific determination of olaquindox (OLA) residues in pig urine and muscle tissues. The sensitivity of the test strip was found to be 1.58 ± 0.27 μg/kg and 1.70 ± 0.26 μg/kg of OLA in pig urine and muscle tissues, and the lower detection limit was 0.27 ± 0.08 μg/kg and 0.31 ± 0.07 μg/kg respectively. For negative pig urine and muscle samples spiked with 4, 12, and 36 μg/kg, the recovery range was 83.0-94.0% and 78.8-87.4% and the coefficient of variation scope [CV (%)] was 3.17-7.41% and 4.66-7.64% respectively. Parallel analysis of OLA samples from pig urine and muscle tissue showed comparable results from the test strip and HPLC. Each test requires 5-8 min, and the test strip can provide a useful screening method for quantitative, semiquantitative, or qualitative detection of OLA residues.     

Development and comparison of three diagnostic immunoassay formats for the detection of azoxystrobin

The currently accepted method of detection for azoxystrobin, a strobilurin fungicide, involves a labor-intensive organic solvent extraction and gas chromatography analysis. Three diagnostic assay formats, i.e., enzyme-linked immunosorbent assay (ELISA), fluorescence polarization (FP), and time-resolved fluorescence (TR-FIA), were developed and compared with regard to detection and quantification of azoxystrobin in grape extract and river, lake, and well water samples. These three assay formats require no initial sample extraction and were not affected by any of the environmental matrices tested, and each had a linear working range of 0-400 pg/mL. The polyclonal antibodies used for each of the immunoassays were specific to azoxystrobin; that is, the highest cross-reactivity to other pesticides observed was 5.7%. The limits of detection of the immunoassays were similar at 3 (ELISA), 46 (FP), and 28 (TR-FIA) pg/mL, as were the respective IC50 values of 306, 252, and 244 pg/mL. Each of the three immunoassays developed was less labor-intensive and approximately 100-fold more sensitive than the gas chromatographic method. While the three formats were comparable in terms of performance, the fluorescence polarization assay was the least labor-intensive and required the least time to perform.     

Development of a capillary electrophoresis-based immunoassay with laser-induced fluorescence for the detection of carbaryl in rice samples

A capillary electrophoresis-based competitive immunoassay (CEIA) with a laser-induced fluorescence (LIF) detector for the determination of carbaryl was developed. The method was based on the competitive reactions between fluorescently labeled carbaryl tracer (Ag*) and free carbaryl (Ag) with a limited amount of anticarbaryl antibody (Ab), and the relative amounts of each were separated and determined by capillary electrophoresis (CE) with an LIF detector. Using CEIA, equilibrium was reached in 30 min, and the analytical results were obtained within a further 8 min. The linear range and the detection limit for carbaryl were 0.16-50 ng/mL and 0.05 ng/mL, respectively. The sensitivity of this CEIA with an LIF detector was almost 14 times greater than that of ELISA, which used the same immuno-reagents. The method was also applied to the analysis of carbaryl in rice with rapid and simple sample pretreatment. The method is thus proposed as a fast and sensitive assay for the detection of carbaryl.     

Development of a dual-label time-resolved fluorometric immunoassay for the simultaneous detection of two recombinant proteins in potato

Immunological methods such as ELISA have been traditionally employed to quantify protein levels in plants improved through modern biotechnology. Combined trait products (i.e., plants producing multiple recombinant proteins) created by introducing multiple genetic traits by transformation or traditional breeding methods have prompted the need for the development of analytical assay technologies capable of detecting and quantifying multiple proteins in a single assay. The development of a two-site, sandwich, dual-label, time-resolved fluorometry-based immunoassay (TRFIA) capable of simultaneously quantitating two recombinant proteins (CP4 EPSPS and Cry3A) in plant sample extracts of genetically improved potato cultivars is reported here. The performance characteristics of TRFIA were similar to or exceeded those of current ELISA methods used to detect and quantitate these proteins. TRFIA is a practical and reliable assay for the quantitation of proteins in genetically improved potato plants and offers an alternative approach to conventional ELISA methods with the added benefit of multiple analyte detection.     

Development of a solid-phase cleanup and portable rapid flow-through enzyme immunoassay for the detection of ochratoxin a in roasted coffee

A membrane-based flow-through enzyme immunoassay (patent application pending) for the detection of ochratoxin A (OA) in roasted coffee was developed. First, an extraction and solid-phase cleanup method was developed. A high partition coefficient for OA in the mobile phase was achieved by using methanol/5% aqueous NaHCO(3) as the sample extraction and cleanup solvent. The solid-phase (aminopropyl) cleanup was developed to chromatographically elute OA but retain cross-reacting compounds. Without using aminopropyl cleanup, cross-reacting compounds resulted in 100% false positives for both flow-through enzyme immunoassay and HPLC methods. However, after cleanup with aminopropyl, no false positives were observed. The flow-through results were visually evaluated. The sensitivity achieved for the flow-through was 4 microg kg(-1) in spiked roasted coffee. The assay was used to screen roasted coffee samples. Results were confirmed with HPLC with a detection limit of 1 microg kg(-1).     

玉米赤霉烯酮化学发光免疫分析检测系统设计

为了保障粮食安全,该研究根据玉米赤霉烯酮抗原抗体反应,以及辣根过氧化物酶催化鲁米诺过氧化氢反应产生化学发光,设计一款应用于粮食行业的玉米赤霉烯酮检测系统。采用侧窗型高精度光电倍增管MD983以及16位AD转换芯片,实现化学发光强度信号的准确测量;步进电机驱动旋转精密转台,通过优化步进电机的S型脉冲驱动控制曲线参数,完成转台的高精度定位控制,实现光电倍增管的测试窗口和化学发光孔精确对准;通过精密直线导轨滑台驱动加样器的进给,实现反应液微米量级的准确微量加样。利用竞争性免疫分析方法,使得赤霉烯酮毒素为0 μg/kg情况下,化学发光反应具有最大发光量,解决真菌毒素低浓度情况下的检测精度难题。经过试验验证,系统检出限为0.1 μg/kg,样品加标回收率在90%以上,标准曲线决定系数为0.996 5,系统检测玉米赤霉烯酮的线性范围为0~60 μg/kg。研究表明建立的玉米赤霉烯酮检测系统满足国家粮食行业对于粮食中玉米赤霉烯酮含量检测要求,为真菌毒素检测仪器的国产化提供参考。     

Development of a monoclonal immunoassay selective for chlorinated cyclodiene insecticides

Organochlorine pesticides still generate public health concerns because of their unresolved health impact and their persistence in living beings, which is demanding appropriate analytical techniques for their monitoring. In this study, an enzyme-linked immunosorbent assay based on monoclonal antibodies (MAbs) for the detection of an important group of organochlorine pesticides, the cyclodiene group, has been developed. With this aim, several hapten-protein conjugates, characterized by exposure of the common hexachlorinated bicyclic (norbornene) moiety and differing in the linking structure to the carrier protein, were prepared. From mice immunized with these conjugates, several MAbs with the ability to sensitively bind the majority of cyclodienes were obtained. Among them CCD2.2 MAb displaying the broadest recognition to cyclodiene compounds (endosulfan, dieldrin, endrin, chlordane, heptachlor, aldrin, toxaphene: I(50) values in the 6-25 nM range) was selected for the assay. Interestingly, this MAb showed certain stereospecificity toward other polychlorinated cycloalkanes because the gamma-isomer of hexachlorocyclohexane (lindane) was also very well recognized (I(50) value of 22 nM). This immunoassay is potentially a very valuable analytical tool for the rapid and sensitive determination of cyclodiene insecticides and related compounds, which in turn may contribute to the understanding of the biological activities and of the overall environmental impact of these persistent organic pollutants.     

Nanocolloidal gold-based immunoassay for the detection of the N-methylcarbamate pesticide carbofuran

Nanocolloidal gold particles were prepared and labeled to an anti-carbofuran monoclonal antibody (Mab). This conjugate was dispensed on the conjugated pad of a porous glass fiber. Ovalbumin (OVA)-carbofuran and goat anti-mouse IgG were dispensed on the nitrocellulose (NC) membrane and served as the test line and control line, respectively. The carbofuran-containing sample migrated to the NC membrane and reacted with the anti-carbofuran Mab labeled with the colloidal gold. The mixture diffused along the membrane and passed through the OVA-carbofuran in the test line via capillary action. The more analyte present in the sample, the more effectively it will compete with the carbofuran immobilized on the test line for binding to the limited amount of antibody labeled with colloidal gold. An adequate amount of carbofuran could prevent attachment of the colored conjugate to the test line. The presence or absence of a colored band on the test line could indicate a negative or positive result, respectively. When measured to the water sample spiked with carbofuran, this was obtained at or above 0.25 mg/L of carbofuran. The major advantages of the one-step strip test are that the detection time needed was <10 min and all of the reagents are included in the test device.     

Enzyme immunoassay for detection of Salmonella in foods: collaborative study

A collaborative study was performed in 25 laboratories to validate an enzyme immunoassay (EIA) procedure utilizing 2 specific monoclonal antibodies for rapid detection of Salmonella in foods. The EIA was compared with the standard culture procedure for detection of Salmonella in 6 food types: ground black pepper, soy isolate, dried whole eggs, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed, with the exception of poultry which was naturally contaminated. There was no significant difference in the productivity of the EIA and culture procedures at the 5% level for any of the 6 foods. The enzyme immunoassay screening method has been adopted official first action.     

Visual immunoassay for detection of Salmonella in foods: collaborative study

A collaborative study was performed in 13 laboratories to validate a visual enzyme immunoassay (EIA) procedure, TECRA, for rapid detection of Salmonella in foods. The EIA method was compared with the standard culture procedure for detection of Salmonella in 6 food types: ground black pepper, soy flour, dried whole eggs, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed. There was no significant difference in the productivity of the EIA and culture procedures at the 5% level for any of the 6 foods. The enzyme immunoassay screening method has been approved interim official first action.     

Development and application of solvent-free extraction for the detection of aflatoxin M1 in dairy products by enzyme immunoassay

The official methods for the quantification of aflatoxin M1 in dairy products (cheese and yogurt) include extraction into dichloromethane or chloroform, evaporation of the solvent, partitioning of the reconstituted residue with hexane, and subsequent analysis. To secure a rapid and inexpensive screen for aflatoxin M1 contamination, a sensitive competitive ELISA, using a rabbit polyclonal antibody, was developed for measuring aflatoxin M1 in milk and used in a comparative study for measuring the extraction efficiency of aflatoxin M1 in aqueous or organic solvent buffers using yogurt samples. An aqueous sodium citrate solution was found to be suitable for extracting aflatoxin M1, thus eliminating the need for organic solvents. The citrate extraction proved to be efficient (recovery ranged from 70 to 124%) in fortified samples of very different kinds of dairy products, including yogurt and six types of cheese. Fourteen yogurt and cheese samples were extracted with citrate solution and analyzed by ELISA. A good correlation was observed (y=0.95x-0.59, r2=0.98) when the data were compared with those obtained through the official method, across a wide range of aflatoxin M1 contaminations (10-200 ng/kg).     

Development of a new HPLC method with precolumn fluorescent derivatization for rapid, selective and sensitive detection of triterpenic acids in fruits

Triterpenic acids are widespread in plants and have multiplicity of biological properties. Unfortunately the method for accurate analysis of these compounds remains poorly investigated. This study proposed a highly sensitive and selective precolumn derivatization method for accurate determination of five triterpenic acids (betulinic acid, betulonic acid, maslinic acid, ursolic acid and oleanolic acid) in fruits using acridone-9-ethyl-p-toluenesulfonate (AETS) as fluorescent labeling reagent by HPLC with fluorescence detection (FLD). Response surface methodology was employed to optimize the derivatization reaction, ensuring the sufficient labeling of the analyzed components. The rapid separation of five triterpenic acids could be achieved in as little as 16 min. This developed method offered the exciting detection limits of 1.68-2.04 ng/mL. When applied to several popular fruits in China, it revealed satisfactory applicability and reproducibility. This developed method also exhibits powerful potential for accurate detection of triterpenic acids from other foodstuffs and nature products.     

Development of a magnetic particle-based enzyme immunoassay for the determination of penoxsulam in water

A competitive enzyme-linked immunoassay (ELISA) for the quantitation of Penoxsulam [2-(2,2-difluoroethoxy)-6-(trifluoromethyl-N-(5,8-dimethoxy[1,2,4]triazolo[1,5-c]pyrimidin-2-yl))benzenesulfonamide] in ground and surface waters was developed. This immunoassay utilizes magnetic particles as the solid phase to which polyclonal rabbit anti-Penoxsulam antibodies are attached. The ELISA has an estimated detection limit of 0.17 ppb (microg/mL) of Penoxsulam in water. Specificity studies indicate that the antibody can distinguish Penoxsulam from its major metabolites and structurally similar pesticides. Interference studies indicate that the ELISA has a wide tolerance of sample pH and salinity and for compounds commonly found in surface and ground waters. The ELISA was shown to compare favorably to LC-MS/MS on ground and surface water samples (r(2) = 0.957). The various studies performed demonstrate the usefulness of the ELISA technique as a rapid and high-throughput analytical method for the cost-effective monitoring of water samples.     

Fluorescence polarization immunoassay based on a monoclonal antibody for the detection of the organophosphorus pesticide parathion-methyl

A fluorescence polarization immunoassay (FPIA) based on a monoclonal antibody for the detection of parathion-methyl (PM) was developed and optimized. Fluorescein-labeled PM derivatives (tracers) with different structures were synthesized and purified by thin-layer chromatography. The influence of immunogen and tracer structures on the assay characteristics was investigated. PM concentration determinable by the FPIA ranged from 25 to 10000 ppb. The detection limit was 15 ppb. Methanol extracts of vegetable, fruit, and soil samples were diluted 1/10 for the analysis. Recovery in spiked samples averaged between 85 and 110%. The method developed is characterized by high specificity and reproducibility (CV ranged from 1.5 to 9.1% for interassay and from 1.8 to 14.1% for intra-assay). The FPIA method can be applied to the screening of food and environmental samples for PM residues without complicated cleanup.     

Enzyme immunoassay for detection of Salmonella in low-moisture foods: collaborative study

A collaborative study was performed in 15 laboratories to evaluate a modification of the enzyme immunoassay (EIA) method for detection of Salmonella in foods (46.B21-46.B29). The modified EIA requires 18-24 h pre-enrichment, 6-8 h selective enrichment, and 14-18 h M-broth post-enrichment prior to performing the assay, which requires 1-2 h. Total assay time is 40-52 h. The modified method was compared with the standard culture method for detection of Salmonella in 5 low-moisture foods: nonfat dry milk, milk chocolate, meat and bone meal, dry whole egg, and ground pepper. The modified method has been adopted official first action for use with low-moisture foods.     

Development of an immunoassay for the pyrethroid insecticide esfenvalerate.

A competitive enzyme-linked immunosorbent assay was developed for the detection of the pyrethroid insecticide esfenvalerate. Two haptens containing amine or propanoic acid groups on the terminal aromatic ring of the fenvalerate molecule were synthesized and coupled to carrier proteins as immunogens. Five antisera were produced and screened against eight different coating antigens. The assay that had the least interference and was the most sensitive for esfenvalerate was optimized and characterized. The I(50) for esfenvalerate was 30 +/- 6.2 microg/L, and the lower detection limit (LDL) was 3.0 +/- 1.8 microg/L. The assay was very selective. Other pyrethroid analogues and esfenvalerate metabolites tested did not cross-react significantly in this assay. To increase the sensitivity of the overall method, a C(18) sorbent-based solid-phase extraction (SPE) was used for water matrix. With this SPE step, the LDL of the overall method for esfenvalerate was 0.1 microg/L in water samples.     

A boron-dipyrromethene-based fluorescent probe for colorimetric and ratiometric detection of sulfite

BODIPY-Le, a colorimetric and ratiometric fluorescent probe based on boron-dipyrromethene for selective detection sulfite ion, was investigated. Boron-dipyrromethene levulinyl ester (BODIPY-Le) is composed of an indole-based BODIPY dye and the levulinyl protective group, which could be easily and selectively deprotected by sulfites. As a result, the absorption and emission spectra show a dramatic red shift, and the development of a colorimetric and ratiometric fluorescent sulfite probe could be achieved. Besides, BODIPY-Le also exhibited prominent turn-on or turn-off type fluorogenic signaling toward sulfite ions once excited at 510 and 620 nm, respectively.     

Hapten synthesis and monoclonal antibody-based immunoassay development for detection of the fungicide trifloxystrobin

High-affinity and selective monoclonal antibodies have been produced against the strobilurin fungicide trifloxystrobin. A battery of functionalized haptens has been synthesized, and conjugate-coated enzyme-linked immunosorbent assays following different procedures have been developed. On the one hand, a two-step conjugate-coated immunoassay was optimized using extended or short incubation times, with limits of detection of 0.10 ng/mL for the extended assay and 0.17 ng/mL for the rapid assay. On the other hand, an immunoassay in the conjugate-coated format was optimized following a procedure consisting of just one incubation step. This one-step assay had a limit of detection of 0.21 ng/mL. All of these assays showed detection limits for trifloxystrobin in the low parts per billion range, well below the common maximum residue limits for this pesticide in foodstuffs (50 microg/kg).