Generation of class-selective monoclonal antibodies against the penicillin group.

To develop a penicillin-specific ELISA, different attempts were made to obtain monoclonal antibodies specific for the common structure of penicillins. Ampicillin was coupled to different carrier proteins (bovine serum albumin, chicken ovalbumin, and thyroglobulin) to render it immunogenic. Different coupling methods were compared: two methods using a cross-linker (glutaraldehyde or a succinimide ester), one carbodiimide-mediated coupling method, and one method without any cross-linker or mediator molecule (physiological binding). Mice were immunized with the conjugates intraperitoneally or in the footpad. A screening ELISA was developed to detect anti-ampicillin antibodies in sera. Specificity and affinity of the antibodies were demonstrated by inhibiting their binding with a 10 mM solution of ampicillin. No difference could be observed using electrofusion or PEG-mediated fusion. For the production of the monoclonals, an intravenous final boost gave antibodies with better specificity and affinity than an intraperitoneal final booster injection. At least one anti-ampicillin monoclonal antibody (19C9) cross-reacts with penicillin G, oxacillin, dicloxacillin, and carbenicillin, and not with sulfanilamide, chloramphenicol, neomycin, and streptomycin, and is therefore considered interesting for developing a penicillin-specific ELISA.     

Production and characterization of monoclonal antibodies against the antibiotic tilmicosin

Monoclonal antibodies (Mabs) were developed that specifically bind tilmicosin. Keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) conjugates were used for the immunogen and plate coating antigen, respectively. The conjugates were synthesized by different methods, resulting in different linkages. Six hybridoma cell lines were isolated that produced Mabs that competed with tilmicosin, and have IgG1 isotype. The Til-1 and Til-5 Mabs had IC50 values for tilmicosin of 9.6 and 6.4 ng/well (48 and 32 ng/mL), respectively, and limits of detection at IC20 of 1.84 and 0.89 ng/well (9.2 and 4.45 ng/mL), respectively. The Mabs demonstrated high cross-reactivity to the macrolides containing 3,5-dimethylpiperidine at C20 and the amino sugar at C5. No cross-reactivity was observed for tylosin and other macrolides that did not contain 3,5-dimethylpiperidine. A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the antibiotic tilmicosin by use of the developed Mabs. These Mabs may be excellent candidates for the determination and immunolocalization of tilmicosin.     

Development of monoclonal antibodies against pirimiphos-methyl and their application to IC-ELISA

To detect the organophosphorus (OP) pesticide pirimiphos-methyl in grain samples, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed and optimized. By the active esters method, pirimiphos-methyl hapten A was conjugated to keyhole limpet hemocyanin to be used as the immunogen for the production of monoclonal antibodies, and pirimiphos-methyl hapten B was conjugated to ovalbumin to be used as coating antigen. By using the monoclonal antibody and the coating antigen, an IC-ELISA has been developed. Under the established optimized conditions, the IC-ELISA showed an IC50 of 4.2 ng/mL with a detection limit of 0.07 ng/mL. The IC-ELISA showed negligible cross-reactivity with other OP pesticides except with pirimiphos-ethyl. Recoveries of pirimiphos-methyl from spiked grain samples ranged from 83 to 96%.     

Detection of sulfonamides in animal feeds.

The animal feed sample is extracted with ethanol or acetone and the extract is evaporated to dryness. Another portion of the same sample, spiked at the 3 ppm level with 8 sulfonamides, is similarly extracted and the extract is evaporated to dryness. The residue from each solution is dispersed with 5 ml 0.1N NaOH and, following the addition of 1 ml 1N HCl and mixing, the solution is filtered. The filtrate is mixed with Celite, transferred to a column, and eluted with ammoniacal ether. Aliquots of the concentrated sample and control eluates are spotted on a neutral Adsorbosil-1 thin layer chromatographic (TLC) plate. Following development in chloroform-methanol (95+5) and drying, the plate is sprayed with an alcoholic solution of p-dimethylaminobenzaldehyde until the control chromatogram shows 6 yellow spots which are, from top to bottom; sulfadimethoxine (SO), the combined sulfamerazine (SM)-sulfamethazine (SH)-sulfaquinoxoline (SQ) spot, sulfadiazine (SD), sulfapyridine (SP), sulfathiazole (SZ), and sulfaguanidine (SG). A spot on the sample chromatogram can be identified if the Rf is identical to one of the 5 sulfonamides not overlapping with another compound. If the Rf of the sample spot is approximately the same as that of the combined SM-SH-SQ spot, more definite identification can be obtained by using basic Adsorbosil-1 TLC plates, with chloroform-methanol (92+8) as the developing solvent.     

Cross-reactivity of antibodies in some commercial deoxynivalenol test kits against some fusariotoxins

Cross-reactivity of antibodies in AGRAQUANT, DON EIA, VERATOX, ROSA LF-DONQ, and MYCONTROLDON designed for deoxynivalenol (DON) determination in food and feedstuffs was evaluated against nivalenol, 3-acetylDON, 15-acetylDON, de-epoxy metabolite 1 of DON, DON-3β-glucoside, T2-toxin, HT2-toxin, fusarenone X, diacetoxyscirpenol, verrucarol, and zearalenone. Cross-reactivity measurements were run in water using the 50% reduction of absorbance of the blank for ELISA kits or through direct DON determination upon using the standards of mycotoxins via ROSA LF-DONQ or MYCONTROLDON. For the tested toxin concentrations, all DON kits have low cross-reactivity toward diacetoxyscirpenol, T2-toxin, HT2-toxin, verrucarol, and zearalenone and moderate cross-reactivity toward 15-AcetylDON and fusarenone X. AGRAQUANT, DON EIA, and VERATOX kits showed high cross-reactivity in various ranking orders against DON-3-Glc, DOM-1, and 3AcDON. DON EIA showed also high cross-reactivity against nivalenol and fusarenone X. These mycotoxins could coexist in food or feedstuffs, and analytical results can be wrongly interpreted. Cross-reactivity does not allow checking the compliance with the legal norms, but it does allow an overall risk assessment for the consumers. Updating regularly the cross-reactivity evaluation of the produced batches is recommended for 3-acetylDON, nivalenol, DON-3-Glc, de-epoxy metabolite 1, and fusarenone X.     

Spectrophotometric analysis of binary mixtures of sulfonamides.

Different methods for analyzing binary mixtures by using 2 wavelengths are reviewed. The absorbance ratio calculated at 2 wavelengths, not including the isoabsorptive point, was a quadratic function of relative concentration. The curve-fitting process using orthogonal polynomials was applied to obtain the quadratic equation. An absorbance ratio can be used as a rapid purity index for sulfacetamide sodium in the presence of sulfanilamide. Sulfacetamide sodium has been determined in eye drop preparations.     

Identification of monoclonal antibodies against 2,4-D herbicide by ELISA and DNA sequencing

Seven hybridoma clones, E2/G2, E2/B5, E4/C2, G5/E10, F6/C10, B5/C3, and B7, produced within one fusion experiment in 1991 and the clone E4/C2 originated from 1995 were characterized by sequencing of genes coding for variable domains of the antibodies against 2,4-D herbicide. Amino acid sequences of selected antibodies, deduced from DNA analysis, were confirmed by mass spectrometry. Surprisingly, nucleotide sequence analysis of the clones E2/G2 and E2/B5, producing the most sensitive antibodies, proved to have sequence homology of their variable domains, although the IC(50) values determined for these antibodies 9 years prior to the DNA analysis were 2.0 and 8.2 ng/mL, respectively. The same findings arose from the comparison of the immunochemical to DNA data established for G5/E10, F6/C10, and B5/C3 clones producing antibodies with IC(50) values in the range of 26.3-43.1 ng /mL. The clone E4/C2, originating from the later fusion experiment, did not share nucleotide homology with any of the examined clones. Data obtained by ELISA, immunosensor, and DNA analysis within a 9 year period are discussed with respect to hybridoma stability, methodic artifacts, measurement reliability, and other possible factors influencing the result interpretation.     

Production of polyclonal antibodies against ochratoxin A and its detection in chilies by ELISA

Polyclonal antibodies were produced for Ochratoxin A (OA) by injecting OA-bovine serum albumin (BSA) conjugate subcutaneously at multiple sites into a New Zealand White inbred rabbit. Antiserum could be used at a dilution exceeding 1:100 000 in an indirect competitive enzyme-linked immunosorbent assay (ELISA), and detected OA concentrations up to 0.1 ng/mL. The 50% inhibition binding (I(50)) of OA was 5 ng/mL. Antibodies did not react with ochratoxin B, coumarin, 4-hydroxycoumarin, L-phenylalanine, and aflatoxin B1. OA contamination in chilies (Capsicum annum L.) collected from commercial markets and cold storage units was determined. The mean recoveries from OA-free chilies spiked with 1 to100 microg of OA per kg of chili sample were 90-110% with a standard deviation of <10%. Of 100 chili samples tested, 26 were found to contain over 10 microg/kg of OA. In 12 samples the OA concentration varied from 10 to 30 microg/kg, in 10 samples from 30 to 50 microg/kg, in 3 samples from 50 to100 microg/kg, and in one sample it was 120 microg/kg. This is the first record in India of OA in chilies, a major component of cooked foods in this country, and it is noteworthy that OA contamination exceeded the permissible limit for human consumption of less than 20 microg/kg in over 26% of the market samples tested.     

Spectrophotometric determination of some benzene sulfonamides with 7,7,8,8-tetracyanoquinodimethane

A new spectrophotometric method for the determination of some unsubstituted benzene sulfonamides is presented. The method is based on the interaction of these derivatives with 7,7,8,8-tetracyanoquinodimethane at pH 9.0-9.5 to produce intense blue products. The quantitation of the products was carried out at 578 nm. Beer's law was obeyed over a wide range of concentrations for all sulfonamide compounds studied. Optimum analytical conditions were determined, and the color produced was stable for at least 90 min at 25 degrees C. Analytical data for determination of sulfonamide compounds in pure form are presented together with application of the proposed method for analysis of some commercially available pharmaceutical preparations. The results are in good agreement with those obtained by official procedures.     

猪繁殖与呼吸综合征病毒M蛋白的表达及其单克隆抗体的制备

猪繁殖与呼吸综合征(porcine reproductive and resp iratory syndrom e,PRRS)是以妊娠母猪繁殖失败和仔猪出现呼吸困难为特征的传染病,PRRSV是该病的病原体。将PRRSV CH-1a株ORF6基因疏水性较强的区域删除后,克隆于pGEX-6p-1载体中,转化大肠杆菌,并进行诱导表达。经SDS-PAGA分析发现,表达了M r约为37 000的融合蛋白rtM,W estern b lot分析证实,重组蛋白能被PRRSV抗血清所识别。收获融合表达的rtM,按50μg/只的剂量与等量弗氏佐剂乳化后,经腹腔接种BALB/c小鼠,免疫3次后,取其脾细胞与SP2/0骨髓瘤细胞进行融合,用rtM作为包被抗原,通过间接EL ISA方法筛选阳性克隆,结果获得了1株能稳定分泌抗rtM蛋白抗体的杂交瘤细胞株,将其命名为M 2B3。间接免疫荧光检测结果发现,所获得的单克隆抗体能与PRRSV感染细胞产生特异性免疫荧光。亚型鉴定结果显示,单克隆抗体M 2B3为IgG 1型,其轻链均为κ链。研究获得的融合蛋白rtM及单克隆抗体将为今后深入研究PRRSV病毒结构和功能,分析M蛋白的抗原表位等提供有益帮助。     

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.     

Production, characterization, and cross-reactivity studies of monoclonal antibodies against the coccidiostat nicarbazin

A cELISA was developed for the coccidiostat nicarbazin. On the basis of previous computer-assisted molecular modeling studies, p-nitrosuccinanilic acid (PNA-S) was selected as a hapten to produce antibodies to 4,4'-dinitrocarbanilide (DNC), the active component of the coccidiostat nicarbazin. Synthesis is described for the hapten [p-nitro-cis-1,2-cyclohexanedicarboxanilic acid (PNA-C)] used in a BSA conjugate as a plate coating antigen. Monoclonal antibodies (Mabs) were isolated that compete with nicarbazin, having IgM(kappa) isotype. Because of the lack of water solubility of nicarbazin, N,N-dimethylformamide (DMF) (3%, v/v) and acetonitrile (ACN) (10%, v/v) were added to the assay buffer to achieve solubility of nicarbazin and related compounds. The Nic 6 Mabs had an IC(35) value for nicarbazin of 0.92 nmol/mL, with a limit of detection of 0.33 nmol/mL. Nic 6 exhibited high cross-reactivity for PNA-S and PNA-C, and 3-nitrophenol, 4-nitrophenol, and 1-(4-chlorophenyl)-3-(4-nitrophenyl) urea. However, Nic 6 had little or no cross-reactivity with 15 other related compounds.     

A group-specific microbiological test for the detection of tetracycline residues in raw milk

The potentiality of using a luminescent Escherichia coli strain for the specific detection of tetracycline residues in raw bovine milk was investigated. The sensor cells contain a reporter plasmid carrying the bacterial luciferase operon of Photorhabdus luminescens under the control of the tetracycline responsive control region from transposon Tn10. Incubation of the cells with the sample containing tetracyclines increases the light emission of the sensor cells. The most sensitive tetracycline detection was achieved in 120 min and by using CDTA as a chelating agent in the assay. Heat-treatment of milk before the assay decreased the variations in background luminescence signals and in tetracycline-induced luminescence between different milk samples. The detection limits for tetracycline, oxytetracycline, chlortetracycline, doxycycline, methacycline, demeclocycline, and minocycline were between 2 and 35 ng/mL. Nontetracycline antibiotics did not significantly interfere with the detection of tetracyclines.     

Influence of the hapten conjugation site on the characteristics of antibodies generated against metabolites of clostebol acetate

4-chloro-androst-4-ene-3,17-dione (CLAD) and 4-chlorotestosterone (clostebol, beta-CLT or CLT) were made immunogenic by coupling to protein carriers via the 3 and 17 positions, respectively. These immunogens were used to elicit polyclonal and monoclonal antibodies to CLAD and to clostebol. The antibodies were characterized in an enzyme immunoassay for sensitivity and specificity. Polyclonal antisera generated through position 17 reacted preferentially with 4-chlorotestosterone-17-acetate (clostebol acetate, CLTA), 4-chloro-epitestosterone (epi-clostebol, 17alpha-clostebol, 17alpha-CLT), and clostebol, whereas polyclonal antisera generated through the 3 position almost did not react with these derivatives. Interestingly, the monoclonal antibody generated through the 3 position recognized (35%) epi-clostebol. These results suggest that polyclonal antisera generated through the 17 position have a broad specificity profile and can be used to analyze by immunoassay methods urinary metabolites of clostebol acetate and thereby detect the illegal use of clostebol acetate in livestock farming.     

Liquid chromatographic determination of three sulfonamides in animal tissue and egg

Sulfonamides are widely used as a feed additive in animal production in Japan. The present paper is a determination of 3 sulfonamides: sulfamethazine (SMZ), sulfamonomethoxine [SMX, 4-amino-N-(3-methoxypyrazinyl)-benzenesulfonamide], and sulfadimethoxine (SDX) in animal tissue and egg by liquid chromatography (LC). Tissues were extracted with acetonitrile and fat was removed by liquid/liquid partition. The sulfonamides were purified by an ODS cartridge column; then each compound was separated by an ODS LC column and detected at 268 nm. Quantification levels were 0.02 ppm for SMZ and SMX, and 0.04 ppm for SDX; detection limits were 0.01 ppm for SMZ and SMX, and 0.02 ppm for SDX. Calibration curves were linear between 2 and 40 ng for SMZ and SMX, and between 4 and 80 ng for SDX. Recoveries from muscle and egg samples spiked with 1-2 micrograms/10 g were 81-98%.     

A screening method for sulfonamides extracted from animal tissues.

A screening method for the estimation of possible residues of sulfonamides in poultry tissues is described. The method utilizes thin layer chromatography (TLC) to separate Bratton-Marshall positive reactants. In the absence of interference and the identification of 1 sulfonamide by TLC, the colorimetric method is recommended for quantitation. When interferences are present, TLC should be used for both qualitative and quantitative analysis. The screening method has a sensitivity of less than 0.05 ppm and a recovery of greater than 80%.     

Analysis of sulfonamides in animal feeds by liquid chromatography with fluorescence detection

Two analytical methodologies for the simultaneous analysis of eight sulfonamide antibiotics in animal feeds were developed. Analytes were extracted in a simple and rapid procedure by manual shaking with an ethyl acetate/ultrapure water mixture (99:1, v/v) without further sample cleanup. Mean recoveries ranging from 72.7% to 99.4% with relative standard deviations below 9% were achieved from spiked animal feed samples. Determination was carried out by high-performance liquid chromatography using fluorometric detection with precolumn derivatization. The separation of the derivatized compounds was performed using two different chromatographic columns: a conventional C(18) column and a recently available core-shell particle Kinetex C(18) column. Both methods were validated in-house in six different feed matrices, and the two approaches were compared. The experiments showed that the method using the Kinetex column was superior with regard to speed of analysis and precision, both under repeatability and intermediate reproducibility conditions. The limits of detection and quantification were also greatly improved, below 0.10 and 0.34 μg/g, respectively. Finally, this novel approach was successfully applied to the analysis of real feed samples.     

ELISA for sulfonamides and its application for screening in water contamination

Two enzyme-linked immunosorbent assays (ELISAs) were tested for their suitability for detecting sulfonamides in wastewater from various stages in wastewater treatment plants (WWTPs), the river into which the wastewater is discharged, and two swine-rearing facilities. The sulfamethoxazole ELISA cross-reacts with several compounds, achieving detection limits of <0.04 microg/L for sulfamethoxazole (SMX), sulfamethoxypyridine, sulfachloropyridine, and sulfamethoxine, whereas the sulfamethazine (SMZ) ELISA is more compound specific, with a detection limit of <0.03 microg/L. Samples from various stages of wastewater purifications gave 0.6-3.1 microg/L by SMX-ELISA, whereas river samples were approximately 10-fold lower, ranging from below detection to 0.09 microg/L. Swine wastewater samples analyzed by the SMX-ELISA were either at or near detectable limits from one facility, whereas the other facility had concentrations of approximately 0.5 microg/L, although LC-MS/MS did not confirm the presence of SMX. Sulfamethazine ELISA detected no SMZ in either WWTP or river samples. In contrast, wastewater samples from swine facilities analyzed by SMZ-ELISA were found to contain approximately 30 microg/L [piglet (50-100 lb) wastewater] and approximately 7 microg/L (market-weight hog wastewater). Sulfamethazine ELISA analyses of wastewater from another swine facility found concentrations to be near or below detection limits. A solid phase extraction method was used to isolate and concentrate sulfonamides from water samples prior to LC-MS/MS multiresidue confirmatory analysis. The recoveries at 1 microg/L fortification ranged from 42 +/- 4% for SMZ to 88 +/- 4% for SMX ( n = 6). The ELISA results in the WWTPs were confirmed by LC-MS/MS, as sulfonamide multiresidue confirmatory analysis identified SMX, sulfapyridine, and sulfasalazine to be present in the wastewater. Sulfamethazine presence at one swine-rearing facility was also confirmed by LC-MS/MS, demonstrating the usefulness of the ELISA technique as a rapid and high-throughput screening method.