Preparation of antibodies and development of an enzyme-linked immunosorbent assay (ELISA) for the determination of doxycycline antibiotic in milk samples

This paper reports the development of an immunoassay for the specific analysis of doxycycline (DC), a congener of the tetracycline antibiotic family (TCs), in milk samples. This is the first time that DC antibody production is reported, based on a rationally designed and well-characterized immunizing hapten. The chemical structure of the immunizing hapten (13-[(2-carboxyethyl)thiol]-5-hydroxy-6-α-deoxytetracycline, TC1) was designed to maximize recognition of the tetracycline characteristic moiety defined as lower periphery of the TCs plus the region of the upper periphery composed by the hydroxyl group at position C(5) (B ring) and the dimethylamino group in ring A. Polyclonal antibodies raised against TC1 coupled to horseshoe crab hemocianyn (HCH) were used to develop a homologous indirect competitive enzyme-linked immunosorbent assay (ELISA). The microplate ELISA can detect DC in buffer down to 0.1 μg L(-1). The ELISA has been proven to tolerate a wide range of ionic strengths and pH values. The assay is very selective for DC with a minor recognition of methacycline (32% of cross-reactivity). Experiments performed with whole milk samples demonstrate that samples can be directly analyzed after a simple treatment method, reaching detectability values below 5 μg L(-1).     

Enzyme-linked immunosorbent assay for the pyrethroid deltamethrin

A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of deltamethrin was developed. Two haptens, cyano[3-(4-aminophenoxy)phenyl]methyl 1R-cis-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropanecarboxylate and 3-[(+/-)-cyano[1R-cis-3-(2,2-dibromoethenyl)-2,2-dimethylcyclopropan ecarbonyloxy]methyl]phenoxyacetic acid, were synthesized and conjugated with thyroglobulin as immunogens. Four antisera were generated and screened against six different coating antigens. The assay that was the most sensitive for deltamethrin was optimized and characterized. The I(50) for deltamethrin was 17.5 +/- 3.6 microg/L, and the lower detection limit was 1.1 +/- 0.5 microg/L. This ELISA assay had relatively low cross-reactivities with other major pyrethroids, such as permethrin, phenothrin, bioresmethrin, cyfluthrin, and cypermethrin. Methanol was found to be the best organic cosolvent for this ELISA, with optimal sensitivity observed at a concentration of 40% (v/v). The assay parameters were unchanged at pH values between 5.0 and 8.0, whereas higher ionic strengths strongly suppressed the absorbances. To increase the sensitivity of the overall method, a C(18) sorbent-based solid-phase extraction was used for river water samples. River water samples fortified with deltamethrin were analyzed according to this method. Good recoveries and correlation with spike levels were observed.     

Development of an enzyme-linked immunosorbent assay for the pyrethroid cypermethrin

A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of cypermethrin was developed. Two haptens, the trans- and cis-isomers of 3-[(+/-)-cyano-3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarbonyloxy]methyl]phenoxyacetic acid, were conjugated with thyroglobulin as immunogens. Four antisera were generated and screened against six different coating antigens. The assay that was the most sensitive for cypermethrin was optimized and characterized. The IC(50) for cypermethrin was 13.5 +/- 4.3 microg/L, and the lower detection limit (LDL) was 1.3 +/- 0.5 microg/L. This ELISA had relatively low cross-reactivities with other major pyrethroids, such as deltamethrin, phenothrin, resmethrin, fluvalinate, and permethrin. Methanol was found to be the best organic cosolvent for this ELISA, with an optimal sensitivity observed at a concentration of 40% (v/v). The assay parameters were unchanged at pH values between 5.0 and 8.0, whereas higher ionic strengths strongly suppressed the absorbances. To increase the sensitivity of the overall method, a C(18) sorbent-based solid-phase extraction was applied to various domestic and environmental water samples. The water samples, fortified with cypermethrin, were analyzed according to this method. Good recoveries and correlation with spike levels were observed.     

抗苯巴比妥单克隆抗体杂交瘤细胞株的筛选及ciELISA试剂盒的研制

用BSA-pAPB免疫Balb/c小鼠，用细胞融合技术制备并用间接ELISA和阻断ELISA筛选抗苯巴比妥单克隆抗体(PB mAb)杂交瘤细胞株，体内诱生腹水法生产PB mAb，应用PB mAb研制PB残留竞争ELISA(ciELISA)快速检测试剂盒(PB-Kit)，并测定其性能。结果表明，筛选出3株杂交瘤细胞，最好的3F6-C4株的PB mAb间接ELISA效价为1∶6.4×105，亲和常数(Ka)为1.96×1010 L/moL，半数抑制浓度(IC50)为5.7 μg/L，与巴比妥的交叉反应率(CR%)12.4%，与其它化合物无CR；PB-Kit的线性检测范围1.0～81 μg/L，灵敏度0.75 μg/L，检测限1 μg/L；饲料样和猪尿样的平均添加回收率85.8%和91.3%，平均批内和批间变异系数均<15%。PB-Kit具有快速、敏感、特异、简便等特点，适合PB残留快速检测的推广应用。     

Poly- and monoclonal antibody-based ELISAs for fipronil

An enzyme-linked immunosorbent assay (ELISA) for fipronil was developed by using polyclonal antibodies (pABs) or monoclonal antibodies (mABs), and its suitability of the determination of this analyte in spiked water samples was studied. The pABs-based assay showed I50 = 17.95 ppb, I90 = 203.40 ppb, and I10 = 0.066 ppb, whereas the mABs-based assay showed I50 = 5.99 ppb, I90 = 485.40 ppb, and I10 = 0.074 ppb. The recoveries of fipronil from tap water samples by pABs-based ELISA were 93.00-124.00% in the range of 0-500 ng/mL, and those obtained from the samples by mABs-based ELISA were 94.70-108.00%. Different types of water from pool, river, and sea were spiked at different levels (ranging form 0.1 to 10 microg/L) and were assayed by the indirect ELISA with mABs. The recoveries of fipronil by this ELISA were in the range of 80-120%. The results demonstrate that this assay is suitable for the quantitative detection of fipronil at trace levels in water samples.     

Competitive enzyme-linked immunoassay for sialoglycoprotein of edible bird's nest in food and cosmetics

The proliferation of fake and inferior edible bird's nest (EBN) products has recently become an increasingly serious concern. To identify and classify EBN products, a competitive enzyme-linked immunoassay (ELISA) was developed to quantitate sialoglycoprotein in EBN used in food and cosmetic applications. The characteristic sialoglycoprotein in EBN was found, extracted, purified, and analyzed. Sialoglycoprotein, considered the main carrier of sialic acid in EBN, consisted of 106 and 128 kDa proteins. A monoclonal antibody that could recognize both proteins was prepared. The heat-treated process did not change the affinity of sialoglycoprotein with the antibody. An optimized ELISA method was established with a cross-reactivity of less than 0.1% and an IC(50) of 3.3 μg/mL. On the basis of different food and cosmetic samples, the limits of detection (LOD) were 10-18 μg/g. Recoveries of fortified samples at levels of 20 and 80 μg/g ranged from 81.5 to 96.5%, respectively. The coefficients of variation were less than 8.0%.     

Development and validation of an indirect competitive enzyme-linked immunosorbent assay for the screening of tylosin and tilmicosin in muscle, liver, milk, honey and eggs

Incorrect use of tylosin and tilmicosin could result in allergy and select resistance. To monitor the illegal use of these antibiotics in animals, a monoclonal-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been established. Several haptens were synthesized and conjugated to carrier protein. Female Balb/c mice were inoculated with the four different conjugates to produce monoclonal antibodies according to the schemes of immunization. Aftercell fusion and culture several times, nine hybridoma cell lines were isolated. Only one, 3C4 that has isotype IgG2a, was selected for detailed study. The cross-reactivity of the monoclonal antibody 3C4 to tylosin and tilmicosin was 100% and 51% respectively. The standard curves based on the tylosin and tilmicosin matrix calibration ranged from 2.5 to 40 μg L(-1), with an IC(50) value of 6.1 μg L(-1) and 12.1 μg L(-1), respectively. The limits of detection of the ic-ELISA ranged from 5.1 μg kg(-1) to 13.8 μg kg(-1) in edible animal tissues. The recoveries were 74.1% to 120.7% with less than 18.6% of the coefficient of variation when tylosin and tilmicosin were spiked in various biological matrices with the concentrations of 25.0-200.0 μg kg(-1). Good correlations between the results of the ic-ELISA and high performance liquid chromatography were observed in the incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for the screening of the residues of tylosin and tilmicosin in muscle, liver, milk, honey and eggs.     

Enzyme-linked immunosorbent assays based on rabbit polyclonal and rat monoclonal antibodies against isoproturon

This work describes the production and characterization of rabbit polyclonal antisera (pAb) and rat monoclonal antibodies (mAb) against isoproturon. Coating antigen and enzyme-tracer formats were developed. Standard curves for isoproturon were conducted either in 40 mM phosphate buffered saline (PBS) or in Milli-Q water. PAb 352 together with the best enzyme tracer revealed in the optimized ELISA (enzyme tracer format) a test midpoint of 1.06 +/- 0.34 microg/L (n = 19, standard set up in Milli-Q water) with a detection limit of about 0.1 microg/L. The comparable ELISA with mAb IOC 7E1 had test midpoints of 0.07 +/- 0.04 microg/L (n = 7, standards in Milli-Q water) and 0.11 +/- 0.08 microg/L (n = 33; standards in 40 mM PBS). The limits of detection were about 0.003 and 0.01 microg/L in Milli-Q water and PBS, respectively. Noticeable cross reactivities (CRs) were seen with the major metabolites, namely 4-isopropylaniline, 4-isopropylphenylurea, and 1-(4-isopropylphenyl)-3-methylurea. With pAb 352, these CRs were 5%, 7%, and 31%, respectively, and with mAb IOC 7E1, they were 3%, 5%, and ca. 19%, respectively. All arylurea herbicides had only minor CRs, which ranged from no CR (e.g., chlorosulfuron) to a maximum of 3.3% (chlortoluron). Influences of organic solvents (methanol, ethanol, acetonitrile, and acetone) were evaluated. Both pAb- and mAb-based immunoassays showed the highest tolerance for methanol, up to 5%. Ethanol and acetonitrile could not be used above 2% without an influence on the assays. The same was true for acetone, although tested only in the mAb-based assay. Water samples of different origins and matrices were spiked and analyzed with these pAb and mAb ELISAs. The results demonstrated that these immunoassays are useful screening tools.     

Development of monoclonal ELISAs for azinphos-methyl. 2. Assay optimization and water sample analysis.

Two enzyme-linked immunosorbent assays (ELISA) for the insecticide azinphos-methyl have been optimized and characterized. Both ELISAs are based on monoclonal antibodies produced from an immunogen with a hapten containing a phthalimido moiety and on protein conjugates of heterologous ligands containing a 1,2,3-benzotriazine group. Assay I was performed in the conjugate-coated ELISA format and assay II in the antibody-coated format. Several physicochemical factors (ionic strength, pH, incubation times, and Tween 20 and BSA concentrations) that influence assay performance were studied and optimized. Regarding specificity, both monoclonal immunoassays highly cross-reacted with azinphos-ethyl and phosmet. Finally, both assays were applied to the analysis of azinphos-methyl in spiked real water samples. For assay I the sensitivity, estimated as the I(50) value, was 0.40 nM, with a practical working range between 0.10 and 1.75 ng/mL and a limit of detection of 0.05 ng/mL. For assay II the sensitivity was 1.01 nM, with a practical working range between 0.32 and 2.54 ng/mL and a limit of detection of 0.08 ng/mL.     

A highly specific enzyme-linked immunosorbent assay for the detection of Cry1Ac insecticidal crystal protein in transgenic WideStrike cotton

A highly selective enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitative detection of the Cry1Ac protein expressed in transgenic cotton. Two Cry1Ac-specific monoclonal antibodies (MAb), Kbt and 158E6, were developed and selected to form a sandwich format ELISA. The MAb Kbt was used as a capture antibody, and 158E6 was conjugated with horseradish peroxidase and served as a detection antibody. The assay was optimized and validated with different cotton matrices. Tissues were extracted with phosphate-buffered saline containing 0.05% Tween 20 and 1% polyvinylpyrrolidone. The extract was then treated with trypsin to truncate full-length Cry1Ac into the core toxin for quantitation. The resulting assay has good accuracy and precision with a validated limit of quantitation ranging from 0.1 to 0.375 mug/g dry weight of cotton tissues. This assay is highly specific for Cry1Ac protein and has no cross-reactivity with the nontarget proteins tested such as Cry1Ab and Cry1F.     

Comparison of enzyme-linked immunosorbent assays with chemiluminescent and colorimetric detection for the determination of ochratoxin A in food

A direct competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for the determination of ochratoxin A (OTA) was developed using soybean peroxidase (SbP) in combination with 3-(10'-phenothiazinyl)propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORPH) as a detection system. By varying the concentrations of the capture monoclonal anti-OTA antibody, a conjugate of OTA with SbP, and the composition of blocking buffers, the conditions of the immunoassay were optimized. Advantages of CL-ELISA were demonstrated by comparison with ELISA with colorimetric detection (COL-ELISA). The values of IC??, IC??, and working range (IC??-IC??) for CL-ELISA and COL-ELISA were 0.01, 0.08, and 0.02-0.3 ng/mL and 0.08, 0.58, and 0.17-2.2 ng/mL, respectively. The recovery values of CL-ELISA from three soybean spiked samples with OTA concentrations of 0.07, 0.1, and 0.15 ng/mL ranged from 72 to 125%. Determination of OTA in 21 various agricultural commodities showed that OTA in 8 examined samples was not detected by COL-ELISA. Furthermore, it was found that in 4 of these 8 samples the developed CL-ELISA determined OTA at levels from 0.96 to 4.64 ng/g.     

兔多杀性巴氏杆菌外膜蛋白A(OmpA)重组蛋白单克隆抗体的制备及潜在应用

制备兔多杀性巴氏杆菌(Pasteurella multocida,Pm)外膜蛋白A(OmpA)重组蛋白单抗,为兔多杀性巴氏杆菌病的诊断提供特异性强,灵敏度高的单克隆抗体。本研究选取并扩增Pm外膜蛋白基因OmpA,原核表达获得了的大小为37.6kD的OmpA重组蛋白,以纯化复性的兔多杀性巴氏杆菌OmpA重组蛋白作为免疫原,按常规方法免疫BALB/c小鼠(Mus musculus),取其脾细胞与SP2/0细胞融合,ELISA筛选出了4株分泌Pm OmpA蛋白单克隆抗体的杂交瘤细胞,分别命名为5D2、BC11、2A2和6A4。其中2A2细胞培养液效价为1∶256,5D2、BC11和6A4效价为1∶128;2A2腹水效价为1∶12800,其余3株达1∶6400。杂交瘤细胞经反复冻存、复苏及多次传代,仍能稳定分泌高效价抗体。Western blot显示,4株单克隆抗体均能与Pm OmpA重组蛋白重组发生特异性反应。用ELISA方法鉴定2A2单克隆抗体亚型为IgG2b,Protein A亲和纯化2A2腹水抗体,获得的单抗浓度为130μg/mL。选取纯化的2A2单克隆抗体进行潜在应用研究,Western blot与间接免疫荧光结果显示,单克隆抗体能与兔多杀性巴氏杆菌分离菌株发生特异性反应。表明利用体外重组表达兔多杀性巴氏杆菌OmpA融合蛋白制成的杂交瘤能够稳定分泌效价高、特异性强的单克隆抗体,可用于兔多杀性巴氏杆菌诊断,本研究结果为兔多杀性巴氏杆菌诊断试剂盒的研制提供技术参数。     

Competitive direct enzyme-linked immunosorbent screening assay for the detection of sulfamethazine contamination of animal feeds.

A sensitive screening method has been developed for detecting sulfamethazine (SMZ) contamination of feeds by using either polyclonal or monoclonal antibodies and a direct competitive enzyme-linked immunosorbent screening assay (ELISA). Feed samples of 25.0 g are extracted with 0.5N HCl and centrifuged. The extract is adjusted to pH 7.0 with 3.0N NaOH and recentrifuged. This pH-adjusted extract is used in the ELISA. Levels as low as 0.004 micrograms SMZ/g feed were detected in supplemented extracts by polyclonal antibodies; levels of 0.4 micrograms SMZ/g feed were detected by a monoclonal antibody.     

Enzyme-linked immunosorbent assay for the pyrethroid permethrin

Permethrin is a predominant pyrethroid widely used in agriculture and public health. A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of permethrin was developed. Two haptens, the trans- and cis-isomers of 3-(4-aminophenoxy)benzyl-3-(2, 2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate, were synthesized and conjugated with thyroglobulin as immunogens. Four antisera were generated and screened against six different coating antigens. The resulting ELISA has an I(50) value of 2.50 microg/L and relatively low cross-reactivities with other major pyrethroids, such as esfenvalerate, cypermethrin, deltamethrin, and cyfluthrin. Methanol was found to be the best solvent for this ELISA, with optimal sensitivity observed at a concentration of 40% (v/v). The assay parameters are unchanged at pH values between 5.0 and 8.0, whereas higher ionic strengths (>0.2 M PBS) strongly suppress the absorbances. River water samples fortified with permethrin were analyzed according to this method and validated by GC-MS. Good recoveries and correlation with spike levels were observed, suggesting this immunoassay is valuable for environmental monitoring and toxicological studies at parts per trillion levels of permethrin.     

Development of a monoclonal antibody against domoic acid and its application in enzyme-linked immunosorbent assay and colloidal gold immunostrip

A monoclonal antibody (mAb) specific to domoic acid was produced from a stable hybridoma cell line, 9F1F11, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with domoic acid--keyhole limpet hemocyanin. The 9F1F11 mAb belongs to the immunoglobulin G1 (kappa-chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. In the cdELISA, the concentration causing 50% inhibition (IC50) of binding of domoic acid-horseradish peroxidase to the antibody by domoic acid was found to be 0.58 ng/mL. A sensitive and rapid mAb-based colloidal gold immunostrip was also developed. The immunostrip assay, which has a detection limit of 5 ng/mL for domoic acid, can be completed in 10 min. Analysis of domoic acid in blue mussel samples revealed that data obtained from immunostrip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunostrip assay established in this study were sensitive and accurate for rapid screening of domoic acid in shellfish samples.     

Development of an enzyme-linked immunosorbent assay for the detection of the organophosphorus insecticide acephate

A competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the organophosphorus insecticide acephate, O,S-dimethyl acetylphosphoramidothioate, was developed using a polyclonal antibody. Five different haptens mimicking the analyte were synthesized and conjugated with the carrier proteins bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) by the N-hydroxysuccinimide active ester and diazotization methods. Polyclonal antibodies raised against hapten-KLH conjugates in rabbits and hapten-BSA conjugates as coating antigens were screened and selected for the assay in the homologous and heterologous ELISA systems. The effects of various assay conditions such as detergent, organic solvents, pH, and preincubation of the mixture of the polyclonal antibody and the analyte on the sensitivity were evaluated. The IC(50) value for acephate was 25 ng/mL in an optimized heterologous system using hapten-4-BSA as a coating antigen and a polyclonal antibody no. 8377 against hapten-1-KLH, showing the detection range of 5-140 ng/mL and the lowest detection limit of 2 ng/mL. The cross-reactivities of the structurally related organophosphorus insecticides, including the major metabolite of the analyte, methamidophos, were less than 1%. Recoveries from the analyte-fortified tap water, mulberry leaves, and lettuce samples in the assay were in the range of 72-121% by simple extraction, concentration, and dilution. These results indicate that the ELISA could be a convenient and supplemental analytical tool for monitoring acephate residues in environmental and agricultural samples.     

Extending the working range of immunoanalysis by exploitation of two monoclonal antibodies

A newly established rat monoclonal antibody (mAb) for isoproturon, namely, IOC 10G7, is described. This mAb shows a standard curve for isoproturon in phosphate-buffered saline with a test midpoint of 5.5 +/- 1.8 microg/L (n = 15). In combination with the formerly developed mAb IOC 7E1, IOC 10G7 can be exploited to extend the working range for the analysis of isoproturon. Both antibodies were formatted into a competitive enzyme-linked immunosorbent assay (ELISA), using the same enzyme-tracer. MAb IOC 7E1 and mAb IOC 10G7 have different affinities for the target compound, but the signal-response curves of the single antibodies overlap. Cross-reactivity (CR) patterns of both antibodies are comparable, showing the highest CR for the metabolite 1-(4-isopropylphenyl)-3-methylurea (IOC 10G7, 9%; IOC 7E1, 19%). The system described here includes the combined, but individual, usage of both assays on one microtiter plate, as well as the strategy for mixing the two antibodies for the utilization in one assay. When standards are performed in Milli-Q water, the working range for isoproturon with the individual ELISAs using mAb IOC 7E1 is from 0.01 to 1 microg/L (test midpoint = 0.11 +/- 0.03 microg/L; n = 17) and with IOC 10G7, it is 1-100 microg/L (test midpoint = 10.3 +/- 1.6 microg/L; n = 32). The working range with mixed antibodies is usually on the order of 0.03-30 microg/L (test midpoint = 0.5 +/- 0.2 microg/L; n = 17). These strategies (mAbs individually and mixed) cover a range of 4 and 3 orders of magnitude, respectively. As a demonstration, water samples of different origins and an extract of mixed sediment were analyzed. The advantages of these strategies are discussed.     

Development of an enhanced chemiluminescence ELISA for the rapid detection of acrylamide in food products

In this work, a polyclonal antibody for acrylamide (AA) was obtained by immunization of rabbits with N-acryloxysuccinimide (NAS) and keyhole limpet hemocyanin (KLH) conjugate. A direct enzyme-linked immunosorbent assay (ELISA) based on this antibody was developed with enhanced chemiluminescent (ECL) detection of AA in food samples. Assay conditions, such as concentrations of antibody and enzyme conjugate and competition time, were optimized. The effects of ionic strength and pH value were investigated. The optimized ECL-ELISA system allowed AA determination in a linear working range of 26.3-221.1 ng mL(-1) with an IC(50) value of 60.6 ng mL(-1) and a limit of detection of 18.6 ng mL(-1). Good recoveries with spiked food samples were obtained with a recovery range from 74.4 to 98.1%, and these results correlated well with those obtained using an HPLC method. This indicates that ECL-ELISA is applicable to the specific detection and routine monitoring of AA in food samples.     

Enzyme-linked immunosorbent assay based on a polyclonal antibody for the detection of the insecticide fenitrothion. Evaluation of antiserum and application to the analysis of water samples.

For development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the organophosphorus insecticide fenitrothion, the specificity of the antiserum R-3 generated with the bifunctional hapten, LysMNPA (2-[[[(3-methyl-4-nitrophenyl)oxy]methylcarbonyl]amino]-6-(2,4-dinitrophenyl)aminohexanoic acid) and the application to the residual analysis of some water samples were evaluated. At optimized ELISA conditions, the quantitative working range was from 1 to 39 ng/mL with a limit of detection of 0.3 ng/mL and an IC(50) value of 6 ng/mL. Cross-reactivity to structurally similar organophosphorus compounds and related chemicals was determined. The antiserum R-3 showed significant cross-reactivity with fenitrooxon and 3-methyl-4-nitrophenol, which have a 3-methyl-4-nitrophenoxy group as common structures, but showed relatively low cross-reactivity with other compounds. Each water sample (river water, tap water, purified water, and bottled water) had a matrix effect and was investigated by adding Tween 20 in the assay buffer. These four kinds of water samples were fortified with fenitrothion at several concentration levels and were directly analyzed with only dilution with an equal volume of antiserum solution. The mean recovery was 105.9%, and the mean coefficient of variation was 10.9%. The results suggested that the developed ELISA would be very suitable for a preliminary screening for fenitrothion in water samples at such low levels.     

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay to quantify soluble beta-glucans in oats and barley

A set of 31 murine monoclonal antibodies was produced against (1-->3,1-->4)beta-d-glucan from oats (Avena sativa L.) chemically cross-linked to keyhole limpet hemocyanin. Monoclonal antibodies were tested for their cross-reactivity to related and unrelated polysaccharides. The antibodies reacted strongly to unmodified beta-glucan from oats and barley (Hordeum vulgare L.) and to lichenan from Icelandic moss, a polysaccharide with a structure similar to that of beta-glucan but which is not encountered in cereals. Cross-reaction to other polysaccharides tested was minimal at physiological levels. An enzyme-linked immunosorbent assay (ELISA) that could routinely detect and quantify nanogram levels of soluble beta-glucan extracted from the flour of oats or barley was designed with one of these monoclonal antibodies. The beta-glucan extraction procedure from ground oat and barley samples and the ELISA were both optimized for reproducibility, accuracy, and throughput, and results were compared to values obtained from an established, commercially available enzyme-based assay. Correlations between the two assays were consistently high (r (2) > 0.9), indicating that the ELISA presented in this paper is a valuable alternative for assaying beta-glucan levels in cereals and cereal products, both routinely and in preparations in which beta-glucans are present in nanogram amounts. Development of the extraction procedure for ELISA is discussed.