Carbodiimide Method Research Articles

Synthesis of carboxymethyl-β-cyclodextrin conjugated magnetic nano-adsorbent for removal of methylene blue

A novel nano-adsorbent is fabricated by the surface modification of Fe 3O 4 nanoparticles (MNP) with carboxymethyl-β-cyclodextrin (CM-β-CD) and the feasibility of employing these nano-adsorbents for removal of methylene blue (MB) from aqueous solutions are investigated. CMCD–MNP(C) and CMCD–MNP(P) are fabricated by grafting CM-β-CD on the surface of magnetite by covalent bonding via carbodiimide method (two-steps method) and by co-precipitation method (one-step method), respectively. Analyses by TEM and VSM reveal that both CMCD–MNP(C) and CMCD–MNP(P) are superparamagnetic with mean diameter of about 12 nm. The CM-β-CD grafting is confirmed by FTIR, TGA and zeta potential measurement. The TGA results show that CM-β-CD grafted onto CMCD–MNP(C) and CMCD–MNP(P) is 4.7 and 12 wt%, respectively. The adsorption of MB on both adsorbents is found to be dependant on pH, and initial MB concentration. Almost constant MB removal is found in the pH range of 4–10 and more MB is adsorbed at pH ∼ 12. The kinetic data for MB adsorption is found to follow a pseudo-second-order kinetic model. The equilibrium data in aqueous solutions are well represented by the Langmuir isotherm model. The adsorption capacities of methylene blue onto CMCD–MNP(P) and CMCD–MNP(C) are found to be 277.8 and 140.8 mg/g, respectively and the negative value of free energy changes indicates the spontaneous nature of adsorption. The adsorbed MB can be desorbed by using methanol solution containing acetic acid. In addition, adsorption mechanism of MB is studied through FTIR analysis.

Preparation of Monoclonal Antibody for Melamine and Development of an Indirect Competitive ELISA for Melamine Detection in Raw Milk, Milk Powder, and Animal Feeds

Melamine (MEL) has been involved in several food recalls after the discovery of severe kidney damages in children and pets poisoned by melamine-adulterated food. To detect MEL residue in foods and animal feeds, an indirect competitive ELISA (cELISA) method was developed in this study based on preparation of monoclonal antibodies (MAbs) to MEL. The immunogen was prepared by linking MEL hapten with carrier protein via carbodiimide method. The method is applicable in the range of 5.0-135.0 microg L(-1) MEL in buffer solution, with an IC(50) value of 22.6 +/- 1.9 microg L(-1). The MAbs showed high specificity with low cross-reactivity (< or =1%) toward cyanurate, ammelide, and ammeline. The method was utilized in the detection of MEL in raw milk, milk powder, and animal feeds, with detection limits of 0.1 mg L(-1) for milk, 0.2 mg kg(-1) for milk powder, and 0.5 mg L(-1) for feeds. The recovery ratio was 79-110% for all matrices. The intra-assay and interassay coefficients of variation were <12.0 and <13.0%, respectively. Finally, the application of the cELISA in quantity evaluation of MEL in various feeds from local markets was evaluated and discussed.

Labeling of Fluorescent Probes to Albumin Microspheres and B16 Melanoma Extra-Cellular Antigen

Fluorescent coupling of bovine serum albumin (BSA) and extracellular antigen of melanoma (ECA) using modified carbodiimide method was evaluated enabling BSA to serve as model protein to evaluate the release profiles of different microsphere formulations and uptake in B16 melanoma cells. Properties of the fluorescent labeled BSA (FBSA) including stability, transition temperature and fluorescent intensity were evaluated. It was found that FBSA produced using this method showed comparable transition temperature and fluorescent intensity compared to the commercially available FITC-labeled BSA and demonstrated stability of the fluorescent-protein linkage after overnight treatment with both trypsin and human plasma using fluorescent microscope. This study showed that the modified carbodiimide labeling method can serve as an alternative method for fluorescent labeling of target protein at reasonable cost particularly when sufficient amount of target protein is required for microsphere formulation screening.

Synthesis of novel derivatives of camptothecin as potential insecticides

In an attempt to find the biorational pesticides, a series of novel camptothecin derivatives were synthesized via a simple modification of the carbodiimide method using the combination of scandium triflate (Sc(OTf) 3) and 4-dimethylaminopyridine (DMAP), and their structures were identified by IR, ESR, HRMS and 1H NMR. Their antifeedant effect and insecticidal activity against third-instar larva of Mythimna separate were examined. The results of preliminary bioassays showed that these compounds exhibited less potent antifeedant activity than camptothecin. Also, all of these derivatives of camptothecin showed delayed insecticidal activity, which differs from traditional neurotoxic insecticides. The results suggested that 20-hydroxyl group in the camptothecin derivatives is essential to keep the insecticidal activity and nature of the substitutes has a major impact on insecticidal activity of these compounds.

Synthesis of a tetratriacontapeptide corresponding to sequence 112-145 of the β-subunit of hCG and production of antiserum against this synthetic peptide

A tetratriacontapeptide(P-34) corresponding to sequence 112–145 of the β-subunit of hCG(hCG-β) was synthesized by the fragment condensation method. The immunogen was prepared by coupling P-34 to bovine serum albumin by the carbodiimide or glutaraldehyde method. Antiserum was produced by injecting the immunogen into rabbits at multiple intradermal and intrasplenal sites with Freund's complete adjuvant. Eight weeks after the first immunization, antiserum was obtained that bound 30% of 125I-hCG or 125I-hCG-β at a dilution of 1:1,000. This binding was inhibited dose-dependently by hCG. In this radioimmunoassay system, the minimal detectable dose of hCG was 500 mIU/ml and, as expected theoretically, no binding to 125I-hCG-α or 125I-hLH was observed.

Targeted PLGA microparticles as a novel concept for treatment of lactose intolerance

Background Peroral β-galactosidase preparations for the management of lactose intolerance need to be administered in large doses (1500 to 6000 U) immediately before or together with a lactose-containing meal. Aim Therefore, this work aimed at developing an innovative long-acting formulation. For this purpose, biodegradable polymeric microcarriers were functionalized with β-galactosidase and targeted with wheat germ agglutinin (WGA) for bioadhesion and thus prolonged residence time in the small intestine. Methods Spray-dried poly( d, l-lactide-co-glycolide) (PLGA) particles with 2.78 ± 1.05 µm in diameter were functionalized with β-galactosidase from Kluyveromyces lactis and WGA using different types of spacers (polyethyleneimine, hexamethylene diamine, 6-aminocaproic acid) and coupling methods (carbodiimide and glutaraldehyde). The particle-bound enzyme activity was determined, and the bioadhesive characteristics were assessed by interaction with mucin coatings and Caco-2 cell monolayers. Results Up to 1470 U β-galactosidase per gram PLGA were immobilized. The best results were obtained with hexamethylene diamine as a spacer applying the carbodiimide method. Thereby, a nearly 6-fold increase in enzyme activity was obtained as compared to particles without spacer. Upon targeting with WGA, binding to artificial human intestinal epithelium was increased considerably. Conclusions For the delivery of β-galactosidase WGA-targeted PLGA microparticles were prepared, which represent promising candidates for a convenient biomimetic treatment regimen of lactose intolerance.

Abstract 2772: Biodistribution and pharmacokinetics of RGD grafted PLGA nanoparticles after radiolabling with 99mTC on tumor bearing rat model

Abstract Aim of the studies was to investigate influence of targeting (RGD) moiety on biodistribution of Docetaxel (DC) encapsulated PLGA-NPs in tumor bearing rats. Nanoparticles (Nps) were prepared by emulsification technique. RGD-conjugation was done by carbodiimide method. NPs were characterized for size, zeta potential, drug encapsulation efficiency, RGDfk content and drug interaction with PLGA and RGDfk. Radiolabeling of DC, DC loaded nanoparticles (PLGA-DC-NPs) and DC loaded and RGD grafted nanoparticles (PLGA-DC-RGDfk-NPs) formulations were achieved using stannous chloride as reducing agent (Babbar et al., 1991). The quality control of labeling efficiency was performed by ascending TLC using silica gel coated fiber sheets using acetone as a mobile phase. Stability of the Tc-99m labeled DC and formulations was determined in vitro in rabbit serum, normal saline by ascending TLC technique. Tc-99m labeled DC solution; PLGA-DC-NPs &amp; PLGA-DC-RGDfk-NPs were administered i.v. via the tail vein at a dose of 20mg/kg body weight into the tumor bearing rats weighing about 250-300 gm. The blood samples were collected at 0.5, 1,2,4,6, 12 and 24 hrs post injection into anticoagulant. The different organs were isolated and homogenized at 1, 2, 4 and 24hrs. Radioactivities in the samples were measured using gamma counter. Pharmacokinetic parameters were calculated using Kinetica 4.4. Gamma scintigraphy study was done at 4, 24 and 48hrs. The drug loaded Nps were found to be in nano size with 72.5% drug entrapment efficiency and negative zeta potential. 99m Tc labeling efficiency was found to be high with stability up to 24hr. Calculated plasma AUC(0→24), AUC(0→∞), MRT, and t1/2 of DC solution and NPs were found to be in increasing order of PLGA-DC-RGDfk-NPs&amp;gt;PLGA-DC-NPs &amp;gt; DC solution. Which reveals the long circulation and slow clearance of drug loaded targeted NPs compared to non RGD grafted NPs and drug solution. Gamma scintigraphy studies reveals that the drug solution was rapidly eliminates from the tumor site as well as from the body. The DC-PLGA-RGDfk-NPs accumulate in the tumor very fast and show significantly high radioactivity than the PLGA-DC-NPs and drug solution alone for longer period of time. DC loaded Nps were prepared successfully exhibited in nano size with excellent resdispersibility. Labeling of DC and DC Nps with 99mTc resulted in stable complexes. The improved Pharmacokinetic and biodistribution data suggest the slow clearance of PLGA-DC-RGDfk with compared to PLGA-DC-NPs and drug solution from the body. Gamma schintigraphy studies demonstrated the high accumulation of RGD grafted Nps in the tumor for longer period of time. These findings of the studies are suggestive of drug localization in breast cancer site after grafting of Nps with RGD in animals and more extensive studies are necessary to develop a product suited for clinical use for better outcome then present chemotherapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2772.

Facile synthesis of a chitosan hybrid of a laminin-related peptide and its antimetastatic effect in mice.

Laminin, a cell adhesion protein, consists of three peptide chains (alpha-1, beta-1 and gamma-1). The beta-1 chain contains a Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence that has been found to inhibit experimental metastasis in mice. We have prepared a hybrid of a water-soluble chitosan and a laminin-related peptide, and have examined its inhibitory effect on experimental metastasis in mice. A laminin-related peptide, acetyl-Tyr-Ile-Gly-Ser-Arg-betaAla-OH (Ac-YIGSRbetaA-OH), was prepared by a solid-phase method. Ac-YIGSRbetaA-OH was then reacted with a water-soluble chitosan. BetaAla is a spacer and was placed to avoid racemization of the Arg residue when the peptide was coupled with chitosan. Although chitosan has amino groups, they did not react with the peptide. Four methods were tried to achieve a coupling reaction, the diphenylphosphoryl azide method, the diisopropylcarbodiimide/1-hydroxybenzotriazole method, the water-soluble carbodiimide (WSC), and the 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) method, but all four methods were unsuccessful. Therefore, a small spacer, tert-butyloxycarbonyl-Gly, was intercalated in chitosan, by the TBTU method, to facilitate its coupling with the peptide. After removal of the protecting group, the Gly-chitosan was coupled with Ac-YIGSRbetaA-OH by the water-soluble carbodiimide method to give Ac-YIGSRbetaAG-chitosan. Conjugation of the peptide with the larger chitosan molecule did not reduce the inhibitory effect of the peptide on experimental metastasis in mice, it actually potentiated the antimetastatic effect, demonstrating that chitosan may be effective as a drug carrier for peptides.

Poly(vinyl chloride) tubing with covalently bound alkaline phosphatase and alternative approach for investigations of open-tubular bioreactors

A one-step carbodiimide method was found to allow covalent binding of enzymes to the inner wall of poly(vinyl chloride) (PVC) tubing. The immobilization is performed under mild conditions without laborious pretreatment or activation of reactor surface. In these preliminary studies, alkaline phosphatase (ALP, EC 3.1.3.1) and p-nitrophenyl phosphate (NPP) were applied as a model enzyme and substrate, respectively. The resulting open-tubular bioreactor exhibits satisfactory operational and storage stability. In addition, a novel and very simple instrumental concept for optical monitoring of the biocatalytic process directly inside the microbioreactor using a system of paired emitter–detector diodes is presented.

Preparation and Protein Detection of Zn−Ferrite Film with Magnetic and Photoluminescence Properties

Zn−ferrite film with hierachical structure and organic composite was prepared by the coprecipitation method, which has good ferromagnetism and photoluminescence (PL) properties. DMSA-modified Fe3O4 nanoparticles (NPs) with saturation magnetization of 55.6 emu/g were prepared by the one-step process, and bovine serum albumin (BSA) with different concentrations was bound on NPs by the carbodiimide method. After Fe3O4−BSA NPs had been adsorbed onto Zn−ferrite film by magnetic forces, the PL intensity of the films was seen to increase linearly with BSA concentration in the range of 0.25−1.5 mg/mL, suggesting that the films could be used for protein detection.

Use of magnetic field for addressing, grafting onto support and actuating permanent magnetic filaments applied to enhanced biodetection

A simple method combining magnetic field and microparticles has been designed for assembling, locating and grafting permanent microfilaments onto a silanised glass surface. Filaments orthogonal to a glass support are implemented by assembling magnetic carboxylatex particles under magnetic field. They exhibit an aspect ratio up to 14. Carboxyl moieties on beads and silanised glass are activated using the carbodiimide method. A further reaction with long chain diamines allows the formation of permanent filaments covalently anchored onto the glass support. Grafted microfilaments can withstand intense washing with detergent solutions under ultrasound. 500 μm diameter filament spots are formed using ferromagnetic tips connected to an electromagnet. After their formation, filaments can be actuated by an external magnetic field: their orientation is tunable in real time within a 2π solid angle. Filaments can be biofunctionalised in order to perform biomolecular recognition on the support. We show that nucleic acid probes immobilised onto these filaments hybridize with labelled oligonucleotide targets. Due to an increase of the specific surface, the fluorescence signal of targets is 3-fold higher with filaments than with a planar glass support biofunctionalised with the same oligonucleotides. Our methodology thus provides an easy way to create filaments usable for both actuation and biodetection onto a planar support. We expect our strategy to be compatible with the formation of nanofilaments using nanoparticles.

Immunotargeting of collagenase on thrombus.

In this study, we aimed to develop a thrombus-targeting delivery system of collagenase bound to a monoclonal antibody, and to investigate the thrombolysis of an immune-conjugate in vitro and in vivo as well as the targeting effect. We prepared the immunizing conjugation of collagenase by the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI) method. In order to conjugate collagenase and a monoclonal antibody, bovine serum albumin was used as a linker, increasing the number of collagenase molecules carried and keeping collagenase and the monoclonal antibody active. In vitro thrombolysis experiments showed that collagenase had a strong dissolving effect on collagen-embolus within 24 hours. We established a rabbit pulmonary embolism model to investigate the thrombolysis effect of collagenase and collagenase immunizing conjugation in vivo. Our results revealed a significant difference between collagenase and collagenase immunizing conjugation (P < 0.05). We also established a rabbit ear edge vein model to investigate the active target of collagenase immunizing conjugation. We found that collagenase immunizing conjugation had active targets, and had a strong ability to dissolve organized thrombi. In conclusion, the thrombus-targeting delivery system of collagenase we developed has active targeting effects on thrombi.

Preparation of Acetochlor Antibody and Its Application on Immunoaffinity Chromatography Cleanup for Residue Determination in Peanuts

The preparation of an antibody against acetochlor and its application on immunoaffinity chromatography (IAC) cleanup was investigated. The hapten was synthesized by reacting acetochlor with 3-mercaptopropionic acid and then linked with a carrier protein by the carbodiimide method. After immunizing a rabbit with hapten-bovine serum albumin (BSA) conjugate, the obtained antiserum showed an IC(50) value of 0.2 ng/mL. By coupling the purified polyclonal antibody with CNBr-activated Sepharose 4B, an IAC column was prepared. The dynamic column capacity was 5560 ng/mL gel. The IAC was then used to clean up a peanut sample solution, followed by high-performance liquid chromatography (HPLC) separation and ultraviolet (UV) detection. The recoveries of acetochlor from spiked peanut samples at levels of 5-100 microg/kg ranged from 94.7 to 102.5%, with a variation coefficient of 2.6-8.0%, and the limits of detection and quantification were 1.5 and 5 microg/kg, respectively.

New C-protective group for peptide synthesis. Application of 2-(1-adamantyl)-propanol-2 esters.

To investigate the possibility of using the 2-(1-adamantyl)-2-propyl (Adp) ester group for carboxyl protection during peptide synthesis, the tetrapeptide Boc-Phe-Arg(NO2)-Phe-Pro-OAdp (IV) was prepared by the carbodiimide method. In this synthesis the Z group was removed by transfer hydrogenation and the Nps group by treatment with 2-thiopyridone. The Adp group was then cleaved with 3% TFA/DCM, yielding Boc-Phe-Arg(NO2)-Phe-Pro-OH. For subsequent kinetics studies the decapeptide Boc-Leu--Z-Orn-Arg(NO2)-Pro- Pro-Gly-Phe-Ser(Bzl)-Pro-Pro-OAdp (VIII) was synthesized by a 9 + 1 scheme. This peptide was also selectively deblocked. Comparing reaction abilities of Z-Pro-OAdp and Z-Pro-OBut, it was demonstrated that the Adp ester is cleaved by 3% TFA/DCM 230 times faster than the Bu(t) ester. For peptides IV and VIII the ratios between the rates of competitive elimination of the Adp and Boc groups by 3% TFA/DCM are 19 and 64, respectively.

Specific and non-specific phagocytosis of ligand-grafted PLGA microspheres by macrophages

We evaluated the influence of ligand grafting on the rate and intensity of uptake of poly( d, l-lactide-co-glycolide) microparticles by alveolar macrophages. Microspheres with a mean diameter of 2.5 μm were obtained by spray drying. Three ligands (WGA, an RGD containing peptide and mannose-PEG 3-NH 2) and a cationic molecule (PLL) were covalently grafted on the particle surface using the carbodiimide method. Their grafting efficiency was quantified, and WGA grafting was characterized by confocal laser scanning microscopy (CLSM) and by atomic force microscopy (AFM). The uptake by macrophages of surface-modified microspheres was quantified by CLSM. This work showed that the uptake of negatively charged ligand-grafted microspheres (−26 to −51 mV) was increased up to two to four times according to the ligand compared to ungrafted microspheres (−81 mV) and displayed saturation as opposed to the cationic PLL-grafted microspheres. Moreover, a specific receptor-mediated phagocytosis mechanism was suggested based on free ligand, cytochalasin D and +4 °C incubation that decreased the microparticle uptake. Furthermore, this work clearly showed that the relative contribution of specific and non-specific processes to the overall uptake varied greatly according to the ligands, and was dependent on the particle-to-cell ratio. In conclusion, this work showed that ligand grafting can enhance the uptake of microparticles, with a variable relative contribution of specific and non-specific uptake mechanism.

A comparative investigation of methods for protein immobilization on self-assembled monolayers using glutaraldehyde, carbodiimide, and anhydride reagents

Three different approaches to the immobilization of proteins at surfaces have been compared. All rely on the creation of surface groups that bind primary amines on lysine residues. Carboxylic acid terminated self-assembled monolayers (SAMs) have been activated using a water soluble carbodiimide to yield an active ester functionalized surface and with trifluoroacetic anhydride to yield a surface anhydride, and amine terminated SAMs have been activated using glutaraldehyde. Although the degree of surface derivatization by n-alkylamines was greater using the carbodiimide and anhydride methods under anhydrous conditions, the glutaraldehyde activation of amine terminated SAMs yielded significantly greater attachment of streptavidin than is achieved using either of the other methods. This is attributed to the susceptibility to hydrolysis of the active species formed by activation of the carboxylic acid terminated monolayers. Patterned protein structures may be formed by using both glutaraldehyde activation of amine terminated thiols and carbodiimide activation of carboxylic acid terminated thiols, in conjunction with selective photo-oxidation of oligo(ethylene glycol) terminated SAMs.

Preparation and characterization of PE38KDEL-loaded anti-HER2 nanoparticles for targeted cancer therapy

The clinical use of immunotoxins is severely limited by nonspecific toxicity. To overcome this limitation, PE38KDEL was used as a model protein toxin to prepare PE38KDEL-loaded poly(lactic-co-glycolic acid) (PLGA) antibody modified nanoparticles (NPs), which were covalently conjugated with Fab' fragments of a humanized anti-HER2 monoclonal antibody (rhuMAbHER2) by a two-step carbodiimide method. The characterization of the PE38KDEL-loaded nanoparticles-anti-Fab' bioconjugates (PE-NPs-HER), such as particle size, zeta potential and morphology, were evaluated by dynamic light-scattering detector and transmission electron microscope (TEM). Micro BCA assay was used to determine the drug encapsulation efficiency and the quantity of Fab' conjugated with NPs. The binding affinity and internalization efficiency of PE-NPs-HER were demonstrated by flow cytometry and laser-scanning confocal microscopy. In comparison with PE38KDEL-loaded nanoparticles (PE-NPs) that lack anti-HER2 Fab', PE-NPs-HER had superior in vitro cytotoxicity against HER2-overexpressing breast cancer cell lines. Progressively, PE-NPs-HER has superior protective antitumor activity in HER2-overexpressing tumor-bearing mice than the control immunotoxin PE-HER constructed by chemically coupling PE38KDEL to rhuMAbHER2. Most strikingly, in developed HER2-overexpressing tumor xenograft model, administration of PE-NPs-HER (0.9 mg/kg) showed a much better therapeutic efficacy in inhibiting tumor growth compared with PE-HER and other controls: final mean tumor load was 13 ± 6 mm 3 (mean ± SD; n = 8, significantly smaller than all other groups by ANOVA at 95% confidence interval). In addition, PE-NPs-HER were well tolerated in mice with a much higher MTD (maximally tolerated dose) than PE-HER (2.92 mg/kg vs. 0.92 mg/kg), indicating the systemic toxicity of PE38KDEL was dramatically reduced by PLGA encapsulation. Thus, the bioconjugates PE-NPs-HER may represent a potentially useful strategy for cancer therapy.

First synthesis of novel spin-labeled derivatives of camptothecin as potential antineoplastic agents

In an effort to improve the stability of labile lactone ring and water solubility of camptothecin, five novel spin-labeled camptothecin derivatives were synthesized in quantitative yield by a simple modification of the carbodiimide method using the combination of scandium triflate (Sc(OTf) 3) and 4-dimethylaminopyridine (DMAP), and the in vitro pharmacokinetic determination of the lactones of representative compound 13a showed that the biological life span of their lactone forms in human and mouse plasma significantly increased when compared with their mother compound camptothecin. Also, the in vitro cytotoxicity of compounds 13a– 13e against human bladder cancer T-24 showed either similar or better activity than that of the parent drug, camptothecin, and clinically available drug, irinotecan.

An Enzyme-linked Immunosorbent Assay for the Detection of Bifenthrin

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the quantitative detection of bifenthrin. Two haptens, LBc ((2-methyl[1,1′-biphenyl]-3-methoxy) carbonyl propionic acid) carbonyl propionic acid) and LBy ((2-methyl[1,1′-biphenyl]-3-yl)carboxylic acid), were synthesized from 2-methyl-3-phenylbenzyl alcohol, a metabolite of bifenthrin. The LBc was conjugated to bovine serum albumin (BSA) for the immunogen by the carbodiimide method, and the LBc and LBy were conjugated to ovalbumin (OVA), respectively, for the coating antigens by the mixed anhydride method. Polyclonal antibodies was raised against LBc-BSA conjugate in rabbits and hapten-OVA conjugates as coating antigens were screened and selected for the assay in the homologous and heterologous ELISA systems, and the titers of antiserum (5.12 × 10 4) were determined by non-competitive ELISA procedure. Under the optimum assay conditions of pH 7.5, PBS of 0.3 M Na + and 30% methanol, the heterologous assay system was more sensitive. The IC 50 value for bifenthrin was (2.16 ± 0.32) mg l −1, and the detection limit was (0.016 ± 0.002) mg l −1. Pyrethroids, such as cyhalothrin, deltamethrin, cypermethrin, fenvalerate, fenpropathrin, and pyrethroid metabolite, 3-phenoxybenzoic acid, did not cross-react significantly in this assay.

Raman spectroscopic study of amidated food proteins

Various amounts of tryptophan were attached to three food protein products: soy protein isolates, spray-dried egg white and gluten, using a water-soluble carbodiimide method. The extent of amidation was determined by a spectrophotometric method. Raman spectra (600–2000 cm −1) of the modified proteins were obtained and analyzed. The phenyl stretching vibration at 1552 cm −1, directly attributed to the attached tryptophan, was used as a marker band, and increases in band intensity were observed in the modified protein samples. Calibration curves were constructed by plotting the intensity ratio of the 1552 cm −1 band to the 1003 cm −1 phenylalanine stretching band (used as an internal standard) against the amount of tryptophan attached. High correlation coefficients, ( r) ⩾ 0.99, were obtained from these calibration curves. The Raman spectral data showed a transition from ordered conformation to random coil structures in the amidated food protein products.