Production and characterization of monoclonal and polyclonal antibodies to forchlorfenuron

The development of immunoassays for the detection of the plant growth regulator forchlorfenuron (CPPU) is described. To achieve that purpose, a set of CPPU derivatives has been obtained by the previous synthesis of the adequate p-aminophenyl alkanoic acid. Protein conjugates of these compounds have been used as immunogens to produce rabbit polyclonal antibodies and a collection of mouse monoclonal antibodies. Additionally, a battery of structural analogues of the target analyte has been synthesized and used for the characterization of antibody binding. This strategy has demonstrated that most antibodies followed Landsteiner's principle, although some monoclonal antibodies showing important deviations from this behavior have also been found. Finally, different assay formats have been developed with a variety of antibodies and conjugates, and a rapid procedure has been optimized for the indirect ELISA format using monoclonal and polyclonal antibodies. In the indirect competitive ELISA, assay IC50 values for CPPU below 0.5 nM were found with LODs as low as 0.013 nM.     

Production and specificity of polyclonal antibodies to hexanal-lysine adducts

Hexanal content is a widely used index of lipid oxidation in foods. The objectives of this study were to develop antibodies to hexanal-lysine adducts, devise an ELISA, and characterize antibody specificity. Hexanal was made immunogenic by covalent attachment to lysine side chains of bovine serum albumin via reductive alkylation. Polyclonal antibodies had antiserum titers as high as 6.15 x 10(5). A competitive indirect ELISA was developed with a detection limit of 0.7 ng of hexanal/mL. Antibodies were carrier-independent, reacting with hexanal conjugates of several proteins but not with the corresponding native proteins. Cross-reactivities with chicken serum albumin conjugates of n-heptanal, n-pentanal, and n-octanal were 86. 3, 11.8, and 2.2%, respectively. Antibodies reacted strongly with hexanal-modified lysine and hexanal-modified epsilon-aminocaproic acid but did not recognize free amino acids or free hexanal. It may be feasible to use this ELISA to monitor lipid oxidation in food provided hexanal is alkylated to a carrier protein prior to analysis.     

Production and characterization of rabbit polyclonal antibodies to almond (Prunus dulcis L.) major storage protein.

Rabbits were immunized with purified almond major protein (AMP), the primary storage protein in almonds. Rabbit anti-AMP polyclonal antibodies (PA) could detect AMP when as little as 1-10 ng/mL were used to coat microtiter plates in a noncompetitive enzyme linked-immunosorbent assay (ELISA). Competitive inhibition ELISA assays detected the AMP down to 300 ng/mL. PA recognized the AMP in protein extracts from all U.S. major marketing cultivars of almonds (Mission, Neplus, Peerless, Carmel, and Nonpareil) with specific reactivity of 52.6-75% as compared to that of the AMP alone. Immunoreactivity of protein extracts prepared from commercial samples of blanched almonds, roasted almonds, and almond paste was respectively reduced by 50.0%, 56.6%, and 68.4% (noncompetitive ELISA) when compared to the immunoreactivity of the AMP. Moist heat (121 degrees C, 15 min) pretreatment of the AMP reduced the PA reactivity by 87% (noncompetitive ELISA). Exposing AMP to pH extremes (12.5 and 1.5-2.5) caused a 53% and 57% reduction in PA reactivity, respectively (noncompetitive ELISA). PA showed some cross-reactivity with the cashew major globulin, and to a lesser extent, the Tepary and Great Northern bean major storage protein (7S or phaseolin). The presence of almonds in a commercial food was detected using PA in a competitive ELISA.     

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.     

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.     

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.     

Properties of polyclonal, monoclonal, and recombinant antibodies recognizing the organophosphorus pesticide chlorpyrifos-ethyl

A rabbit polyclonal antiserum and two murine monoclonal antibodies recognizing the organophosphorus pesticide chlorpyrifos-ethyl were produced. The two hybridoma cell lines were then used as sources of immunoglobulin genes for the generation of recombinant scFv antibodies in Escherichia coli. The two scFvs showed either similar or improved limits of detection in an ELISA when compared with the monoclonal antibodies. Cross-reactivity studies showed that all of the antibodies were specific toward the chlorinated aromatic ring. Furthermore, scFv gene sequences were linked directly to sequences coding for either a c-Myc tag, a His-tag, or alkaline phosphatase. The fusion products generated were functional, and their properties were determined. The problems associated with producing scFvs and scFv derivatives for detection of pesticide residues from hybridoma are addressed and discussed.     

Production and characterization of monoclonal antibodies specific to the strobilurin pesticide pyraclostrobin

Strobilurin fungicides are nowadays among the most important fungicides in the market of active agrochemicals. Pyraclostrobin, which belongs to the last generation of this family of molecules, shows a broader antifungal activity spectrum and higher efficiency and security profiles than previous fungicides. This paper describes the synthesis of functionalized haptens, the production of monoclonal antibodies, and the development of enzyme-linked immunosorbent assays (ELISA) for the detection of pyraclostrobin. A conformational analysis of hapten structure was performed, which provided relevant data concerning the length of the spacer arm. A very useful strategy has been followed for the screening of hybridomas, leading to the selection of a panel of high-affinity monoclonal antibodies to pyraclostrobin. Moreover, different immunoassays have been characterized using the conjugate-coated indirect ELISA format, and limits of detection below 0.1 microg/L have been obtained. Also, a simplified one-step procedure has been carried out with two indirect assays. Finally, these results have been compared with the performance of the same antibodies in the antibody-coated direct ELISA format.     

Production of polyclonal antibodies against territrem B and detection of territrem B in the conidia of Aspergillus terreus 23-1 by immunoelectron microscopy

Territrem B, a fungal metabolite isolated from Aspergillums terreus 23-1, is a tremorgenic mycotoxin. Immunoelectron microscopy using anti-territrem B polyclonal antibody was used to detect territrem B in the fungal body of A. terreus 23-1 at different times of culture without shaking on potato dextrose (PD) agar medium. The anti-territrem B serum was produced by immunization of rabbits with 4beta-hydroxymethyl-4beta-demethylterritrem B-sccinate bound by a linker to keyhole limpet hemocyanin. This antiserum recognized territrems and immunoelectron microscopy using this antiserum, and colloidal gold-conjugated anti-rabbit IgG antibodies showed that territrem B was localized to the fungal body of A. terreus 23-1. Territrem B was first seen in the cytoplasm of the conidia after 4 days' culture on PD agar medium. Maximal territrem B production in the conidia was seen on the 14th day of culture; however, territrem B was not formed in the hyphae at any stage of culture. These results are consistent with the previous finding that the formation of territrems is related to fungal sporulation.     

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.     

Performance and applications of polyclonal antipeptide antibodies specific for the enterococcal bacteriocin enterocin P

Polyclonal antibodies with specificity for enterocin P (EntP) have been generated by immunization of rabbits with two chemically synthesized N-terminal peptides (P1 and P2) and a C-terminal peptide (P3) of this bacteriocin conjugated to the carrier protein KLH. The sensitivity and specificity of the peptide-KLH-generated antibodies were evaluated by a noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) and a competitive indirect (CI)-ELISA. The NCI-ELISA but not the CI-ELISA was valuable for detecting the existence of EntP specific antibodies in the sera of the P2-KLH and P3-KLH immunized animals and to detect and quantify the EntP in the supernatant of producer strains. The anti-P2-KLH sera cross-reacted with the supernatant of a strain producer of sakacin A, a bacteriocin closely related to EntP. Immunoaffinity chromatography columns with anti-P2-KLH or anti-P3-KLH immunoglobulins retained the EntP from the supernatant of the producer strain. Western blotting of EntP with the anti-P2-KLH-generated antibodies suggests that purified EntP tends to the formation of aggregates with no antimicrobial activity. Monitoring the purification of EntP with antipeptide antibodies suggests that while the performance of the evaluated purification procedures would be reasonably acceptable in terms of recovery of the antimicrobial activity of the bacteriocin, their yield is far from attractive in terms of recovery of the initial concentration of enterocin P.     

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.     

Production of the polyclonal antibody against Sudan 3 and Immunoassay of Sudan dyes in food samples

In this study, 4-aminophenylacetic acid was covalently coupled to aniline to synthesize an intermediate hapten and the intermediate hapten was coupled to β-naphthol to synthesize a tentative hapten of Sudan 3. The hapten was coupled to bovine serum albumin as the immunogen to produce the polyclonal antibody. The obtained antibody was highly specific to Sudan 3, Sudan 1, and Para red, but showed relative low binding ability to Sudan 2, Sudan 4, and Sudan red G. After evaluation of different coating antigens, a heterologous indirect competitive immunoassay was developed to multidetermine the six red dyes in food samples. The cross reactivities to the six analytes were in a range of 21-105%, and the limits of detection were in a range of 0.1-0.8 ng/mL depending on the compound. Intra- and interassay recoveries from the standard fortified blank samples were in a range of 74.5-96.3% with coefficients of variation lower than 15.1%.     

Widespread occurrence of low levels of alternariol in apple and tomato products, as determined by comparative immunochemical assessment using monoclonal and polyclonal antibodies

This study investigated the production of polyclonal (pAB) antibodies and the first time production of monoclonal (mAB) antibodies against the mycotoxin alternariol, and their implementation in enzyme immunoassay (EIA) for the rapid determination of alternariol in foods. Both EIAs were highly sensitive, with detection limits (IC??) of 35 ± 6.9 pg/mL (mAb EIA) and 59 ± 16 pg/mL (pAb EIA). Food products (n = 109; apple and tomato products, white wine) from German retail shops were analyzed. At a detection limit of 1-2 μg/kg, alternariol at 1-13 μg/kg was found with high frequency in apple (67%) and tomato (93%) products. Tomatoes with visible signs of Alternaria infection, stored at room temperature for up to 4 weeks, contained alternariol at levels up to 50 mg/kg, as determined by EIA and HPLC-FLD. It is concluded that the alternariol immunoassays present a versatile screening tool which could facilitate food control for Alternaria toxins.     

生物质致密成型产品的生产及应用

生物质是光能循环转化的载体。这里所指的是广义生物质能,包括植物种子、秸秆、树木、牧草、畜禽粪便以及生活有机垃圾等。狭义的生物质能是指各种作物秸秆、树叶、锯屑、蔗渣及农业、林业废弃物等。生物质具有资源丰富、分布比较均匀的特点,加之在其生长过程中吸收大气中的CO2,因此用工业技术开发利用生物质能,不仅有助于减轻温室效应和促进生态良性循环,而且可替代部分石油、煤炭等化石燃料,成为解决能源与环境问题的重要途径。但是生物质的松散性、易燃性、低含碳量、不均匀性又成为工业化大规模利用以及储存和运输时所必须解决的技术问题。     

Production and characterization of films from cotton stalk xylan

Composite film production based on cotton stalk xylan was studied, and the mechanical and physical properties of the films formed were investigated. Xylan and lignin were separated from cellulose by alkali extraction and, then, lignin was removed using ethanol washing. Self-supporting continuous films could not be produced using pure cotton stalk xylan. However, film formation was achieved using 8-14% (w/w) xylan without complete removal of lignin during xylan isolation. Keeping about 1% lignin in xylan (w/w) was determined to be sufficient for film formation. Films were produced by casting the film-forming solutions, followed by solvent evaporation in a temperature (20 degrees C) and relative humidity (40%) controlled environment. The elastic modulus and hypothetical coating strength of the films obtained by using 8% xylan were significantly different from the ones containing 10-14% xylan. The water vapor transfer rates (WVTR) decreased with increasing xylan concentration, which made the films thicker. The glycerol addition as an additional plasticizer resulting in more stretchable films having higher WVTR and lower water solubility values. As a result, film production was successfully achieved from xylan, which was extracted from an agricultural waste (cotton stalk), and the film-forming effect of lignin on pure xylan has been demonstrated.     

Production of monoclonal antibodies to the N-methylcarbamate pesticide propoxur

Monoclonal antibodies (MAbs) to the pesticide propoxur were produced from haptens with carboxylic spacer arms of different lengths introduced at the carbamate group of the analyte structure. MAbs were subsequently characterized in the conjugate-coated format using these immunizing haptens and newly synthesized compounds as homologous and heterologous assay conjugates, respectively. Appropriate combinations of immunoreagents resulted in competitive enzyme immunoassays (ELISA) with I(50) values in the low nanomolar range (6.5-17.9 nM). A modification of the conjugate-coated format consisting of the simultaneous incubation of the MAb and the peroxidase-labeled secondary antibody in the presence of the analyte resulted in an assay with an I(50) value of 4.4 nM. This one-step conjugate-coated ELISA format is as simple and fast as the antibody-coated format but without the need of synthesizing enzyme-hapten conjugates. Major N-methylcarbamate pesticides were not recognized by the MAb. This immunoassay should reasonably allow the rapid, low-cost, and sensitive determination of propoxur in food, soils, and the environment at levels of regulatory and practical importance.     

Nanoparticles synthesized from soy protein: preparation, characterization, and application for nutraceutical encapsulation

Nanoparticles were synthesized from soy protein, one of the most abundant and widely utilized plant proteins, for nutraceutical and drug encapsulation. The preparation process consisted of dispersion, desolvation, drug incorporation, cross-linking, and evaporation. The role of each procedure in the formation of nanoparticles was systematically investigated by means of particle size, size distribution, and zeta potential as well as morphology observation. Curcumin as a model drug was encapsulated successfully into the nanoparticles, evidenced by Fourier transform infrared spectroscopy and X-ray diffraction patterns. The average size of the curcumin-loaded nanoparticles was 220.1 to 286.7 nm, and their zeta potential was around -36 mV. The highest encapsulation efficiency and loading efficiency achieved were 97.2% and 2.7%, respectively. The release of curcumin in phosphate buffer saline followed a biphasic pattern. Possible mechanisms of the formation of soy protein nanoparticles as well as the incorporation of curcumin were discussed based on the data obtained from this study.     

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%.