Model systems for the study of drug hypersensitivity

I. It was not possible to produce anti-tetracycline antibody in laboratory animals by any of the methods tried. Tetracycline protein conjugates were prepared and characterized. It was shown that previous reports of the detection of anti-tetracycline antibody by in vitro-methods were in error. Tetracycline precipitates non-specifically with serum proteins. The anaphylactic reaction reported was the result of misinterpretation, since the observations were inconsistent with the known mechanism of anaphylaxis and the supposed antibody would not sensitize guinea pig skin. The hemagglutination reaction was not reproducible and was extremely sensitive to minute amounts of microbial contamination. Both free tetracyclines and the conjugates were found to be poor antigens.

II. Anti-aspiryl antibodies were produced in rabbits using 3 protein carriers. The method of inhibition of precipitation was used to determine the specificity of the antibody produced. ε-Aminocaproate was found to be the most effective inhibitor of the haptens tested, indicating that the combining hapten of the protein is ε-aspiryl-lysyl. Free aspirin and salicylates were poor inhibitors and did not combine with the antibody to a significant extent. The ortho group was found to participate in the binding to antibody. The average binding constants were measured.

Normal rabbit serum was acetylated by aspirin under in vitro conditions, which are similar to physiological conditions. The extent of acetylation was determined by immunochemical tests. The acetylated serum proteins were shown to be potent antigens in rabbits. It was also shown that aspiryl proteins were partially acetylated. The relation of these results to human aspirin intolerance is discussed.

III. Aspirin did not induce contact sensitivity in guinea pigs when they were immunized by techniques that induce sensitivity with other reactive compounds. The acetylation mechanism is not relevant to this type of hypersensitivity, since sensitivity is not produced by potent acetylating agents like acetyl chloride and acetic anhydride. Aspiryl chloride, a totally artificial system, is a good sensitizer. Its specificity was examined.

IV. Protein conjugates were prepared with p-aminosalicylic acid and various carriers using azo, carbodiimide and mixed anhydride coupling. These antigens were injected into rabbits and guinea pigs and no anti-hapten IgG or IgM response was obtained. Delayed hypersensitivity was produced in guinea pigs by immunization with the conjugates, and its specificity was determined. Guinea pigs were not sensitized by either injections or topical application of p-amino-salicylic acid or p-aminosalicylate.

Immobilization of cell-binding peptides on poly-ε-caprolactone film surface to biomimic the peripheral nervous system.

Cell-material interactions are crucial for cell adhesion and proliferation on biomaterial surfaces. Immobilization of biomolecules leads to the formation of biomimetic substrates, improving cell response. We introduced RGD (Arg-Gly-Asp) sequences on poly-ε-caprolactone (PCL) film surfaces using thiol chemistry to enhance Schwann cell (SC) response. XPS elemental analysis indicated an estimate of 2-3% peptide functionalization on the PCL surface, comparable with carbodiimide chemistry. Contact angle was not remarkably reduced; hence, cell response was only affected by chemical cues on the film surface. Adhesion and proliferation of Schwann cells were enhanced after PCL modification. Particularly, RGD immobilization increased cell attachment up to 40% after 6 h of culture. It was demonstrated that SC morphology changed from round to very elongated shape when surface modification was carried out, with an increase in the length of cellular processes up to 50% after 5 days of culture. Finally RGD immobilization triggered the formation of focal adhesion related to higher cell spreading. In summary, this study provides a method for immobilization of biomolecules on PCL films to be used in peripheral nerve repair, as demonstrated by the enhanced response of Schwann cells.

Composite electrospun gelatin fiber-alginate gel scaffolds for mechanically robust tissue engineered cornea.

A severe shortage of good quality donor cornea is now an international crisis in public health. Alternatives for donor tissue need to be urgently developed to meet the increasing demand for corneal transplantation. Hydrogels have been widely used as scaffolds for corneal tissue regeneration due to their large water content, similar to that of native tissue. However, these hydrogel scaffolds lack the fibrous structure that functions as a load-bearing component in the native tissue, resulting in poor mechanical performance. This work shows that mechanical properties of compliant hydrogels can be substantially enhanced with electrospun nanofiber reinforcement. Electrospun gelatin nanofibers were infiltrated with alginate hydrogels, yielding transparent fiber-reinforced hydrogels. Without prior crosslinking, electrospun gelatin nanofibers improved the tensile elastic modulus of the hydrogels from 78±19 kPa to 450±100 kPa. Stiffer hydrogels, with elastic modulus of 820±210 kPa, were obtained by crosslinking the gelatin fibers with carbodiimide hydrochloride in ethanol before the infiltration process, but at the expense of transparency. The developed fiber-reinforced hydrogels show great promise as mechanically robust scaffolds for corneal tissue engineering applications.

Composite electrospun gelatin fiber-alginate gel scaffolds for mechanically robust tissue engineered cornea

A severe shortage of good quality donor cornea is now an international crisis in public health. Alternatives for donor tissue need to be urgently developed to meet the increasing demand for corneal transplantation. Hydrogels have been widely used as scaffolds for corneal tissue regeneration due to their large water content, similar to that of native tissue. However, these hydrogel scaffolds lack the fibrous structure that functions as a load-bearing component in the native tissue, resulting in poor mechanical performance. This work shows that mechanical properties of compliant hydrogels can be substantially enhanced with electrospun nanofiber reinforcement. Electrospun gelatin nanofibers were infiltrated with alginate hydrogels, yielding transparent fiber-reinforced hydrogels. Without prior crosslinking, electrospun gelatin nanofibers improved the tensile elastic modulus of the hydrogels from 78±19. kPa to 450±100. kPa. Stiffer hydrogels, with elastic modulus of 820±210. kPa, were obtained by crosslinking the gelatin fibers with carbodiimide hydrochloride in ethanol before the infiltration process, but at the expense of transparency. The developed fiber-reinforced hydrogels show great promise as mechanically robust scaffolds for corneal tissue engineering applications. © 2013 Elsevier Ltd.

Interaction Between Tripeptide Gly-Gly-His and Cu2+ Probed by Microcantilevers

Gly-Gly-His tripeptide modified microcantilever was developed by carbodiimide attachment of the Gly-Gly-His tripeptide onto a 3-mercaptopropionic acid(MPA) modified gold surface. The interaction of peptide with Cu2+ ion was studied. At a relative high concentration of Cu2+, the cantilever bent toward the gold side initially as the N atom of imidazole ring and carboxyl group in different peptide coordinate with Cu2+, which results in a tensile surface stress. And then the reversed deflection of microcantilever was observed, which implies that the peptide-Cu2+ complex are formed with conformation transition. In another case, i.e., at a relative low concentration Of Cu2+, only the process of conformation transition was observed due to the coordination mode can not be formed initially. The influences of pH and salt concentration of the test solution on the performance of the sensor were studied. The results show that the maximum deflection was obtained at pH 7 and the bonding Of Cu2+ to the Gly-Gly-His tripeptide was inhibited due to the formation Of CuClx2-x.

Annealing of nylon-1010 under high pressure

Full Paper: A study has been made on the annealing of nylon-1010 under high pressures. Heat treatment of melt-crystallized nylon-1010 was performed at 250degreesC for 30 min in the pressure range 0.7 similar to 2.5 GPa. It was found that the triclinic crystals of virgin nylon-1010 were retained at pressures less than 1.0 GPa or larger than 1.2 GPa. The X-ray diffraction intensity of (100) planes decreased with increasing pressure. The diffraction peaks shifted slightly to higher angles (2theta) relative to the virgin nylon-1010, indicating dense packing of polymer chains at high pressures. The highest melting temperature was 208degreesC for the sample annealed at 1.5 GPa. No extended-chain crystals were formed under the experimental conditions. Crosslinking occurred in the pressure range 1.0 similar to 1.2 GPa. The structure of the crosslinked samples was characterized by means of infrared spectroscopy and X-ray photoelectron spectroscopy. It is concluded that a mechanism of crosslinking via carbodiimide can explain the nature of crosslinking of nylon-1010 annealed at high pressures. The remarkable changes of the structure of annealed nylon-1010 are also discussed in this article.

Grafting of diaminoalkane on glassy carbon surface and its functionalization

Diaminoalkanes (NH2(CH2)(n)NH2, n = 7,10,12) were grafted onto a glassy carbon electrode (GCE) surface by amino cation radical formed during electrooxidation of amino group. The presence of diamine grafted layer at the GCE is demonstrated by X-ray photoelectron spectroscopy. The effect of the grafted layer at the GCE surface on the redox responses of Ru(NH3)(6)(3+) and Fe(CN)(6)(3-) redox probes has been investigated. Electrochemical impedance experiments indicate that the kinetics of electron transfer are slowed down when the scan rate taken to modify the GCE is low, and that diaminoalkane with longer alkyl-chain used has higher blocking characteristics. The amine-functionalized GCE is versatile not only to further covalently immobilize ferrocene acetic acid via carbodiimide coupling, but also as a charge-rich substrate to successfully adsorb heteropolyanion P2W18 in acidic solution by electrostatic interaction. (C) 2000 Elsevier Science S.A. All rights reserved.

Analysis of primary aromatic amines using precolumn derivatization by HPLC fluorescence detection and online MS identification

2-(2-Phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and 2-(9-acridone)-acetic acid (AAA), two novel precolumn fluorescent derivatization reagents, have been developed and compared for analysis of primary aromatic amines by high performance liquid chromatographic fluorescence detection coupled with online mass spectrometric identification. PPIA and AAA react rapidly and smoothly with the aromatic amines on the basis of a condensation reaction using 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (EDC) as dehydrating catalyst to form stable derivatives with emission wavelengths at 380 and 440 nm, respectively. Taking six primary aromatic amines (aniline, 2-methylaniline, 2-methoxyaniline, 4-methylaniline, 4-chloroaniline, and 4-bromoaniline) as testing compounds, derivatization conditions such as coupling reagent, basic catalyst, reaction temperature and time, reaction solvent, and fluorescent labeling reagent concentration have also been investigated. With the better PPIA method, chromatographic separation of derivatized aromatic amines exhibited a good baseline resolution on an RP column. At the same time, by online mass spectrometric identification with atmospheric pressure chemical ionization (APCI) source in positive ion mode, the PPIA-labeled derivatives were characterized by easy-to-interpret mass spectra due to the prominent protonated molecular ion m/z [M + H](+) and specific fragment ions (MS/MS) m/z 335 and 295. The linear range is 24.41 fmol-200.0 pmol with correlation coefficients in the range of 0.9996-0.9999, and detection limits of PPIA-labeled aromatic amines are 0.12-0.21 nmol/L (S/N = 3). Method repeatability, precision, and recovery were evaluated and the results were excellent for the efficient HPLC analysis. The most important argument, however, was the high sensitivity and ease-of-handling of the PPIA method. Preliminary experiments with wastewater samples collected from the waterspout of a paper mill and its nearby soil where pollution with aromatic amines may be expected show that the method is highly validated with little interference in the chromatogram.

2-(2-Phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and its application for determination of amines by high performance liquid chromatography with fluorescence detection and identification with mass spectroscopy/atmospheric pressure chemical ionization

A simple, sensitive, and mild method for the determination of amino compounds based on a condensation reaction with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCI) as the dehydrant with fluorescence detection has been developed. Amines were derivatized to their acidamides with labeling reagent 2-(2-phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA). Studies on derivatization conditions indicated that the coupling reaction proceeded rapidly and smoothly in the presence of a base catalyst in acetonitrile to give the corresponding sensitively fluorescent derivatives with an excitation maximum at lambda(ex) 260nm and an emission maximum at lambda(em) 380nm. The labeled derivatives exhibited high stability and were enough to be efficiently analyzed by high-performance liquid chromatography. Identification of derivatives was carried out by online post-column mass spectrometry (LC/APCI-MS/MS) and showed an intense protonated molecular ion corresponding m/z [MH](+) under APCI in positive-ion mode. At the same time, the fluorescence properties of derivatives in various solvents or at different temperature were investigated. The method, in conjunction with a gradient elution, offered a baseline resolution of the common amine derivatives on a reversed-phase Eclipse XDB-C-8 column. LC separation for the derivatized amines showed good reproducibility with acetonitrile-water as mobile phase. Detection limits calculated from 0.78 pmol injection, at a signal-to-noise ratio of 3, were 3.1-18.2 fmol. The mean intra- and inter-assay precision for all amine levels were < 3.85% and 2.11%, respectively. Excellent linear responses were observed with coefficients of > 0.9996. The established method for the determination of aliphatic amines from real wastewater and biological samples was satisfactory. (c) 2006 Elsevier B.V. All rights reserved.

Simple derivatization method for sensitive determination of fatty acids with fluorescence detection by high-performance liquid chromatography using 9-(2-hydroxyethyl)-carbazole as derivatization reagent

A simple and sensitive method for the determination of short and long-chain fatty acids using high-performance liquid chromatography with fluorimetric detection has been developed. The fatty acids were derivatized to their corresponding esters with 9-(2-hydroxyethyl)-carbazole (HEC) in acetonitrile at 60 degreesC with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as a coupling agent in the presence of 4-dimethylaminopyridine (DMAP). A mixture of esters of C-1-C-20 fatty acids was completely separated within 38 min in conjunction with a gradient elution on a reversed-phase C-18 column. The maximum fluorescence emission for the derivatized fatty acids is at 365 nm (lambda (ex) 335 nm). Studies on derivatization conditions indicate that fatty acids react proceeded rapidly and smoothly with HEC in the presence of EDC and DMAP in acetonitrile to give the corresponding sensitively fluorescent derivatives. The application of this method to the analysis of long chain fatty acids in plasma is also investigated. The LC separation shows good selectivity and reproducibility for fatty acids derivatives. The R.S.D. (n = 6) for each fatty acid derivative are <4%. The detection limits are at 45-68 fmol levels for C-14-C-20 fatty acids and even lower levels for

Determination of alcohols by high-performance liquid chromatography with fluorimetric detection after pre-column derivatisation with carbazole-9-N-acetic acid and chromatographic behaviour of alcoholic derivatives

Alcohols were derivatised to their carbazole-9-N-acetic acid (CRA) esters with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC . HCl) as the dehydrating agent. Studies on derivatisation conditions indicated that the coupling reaction proceeded rapidly and smoothly in the presence of a base catalyst in acetonitrile to give the corresponding sensitively fluorescent derivatives. The retention behaviour of alcohol derivatives was investigated by varying mobile phase compositions (ACN-water and MeOH-water). The parameters from the equation log k'=A-BX were evaluated by retention data of derivatives using an isocratic elution with different mobile phases. The results indicated that the parameters derived allowed computation of retention factors in good agreement with experiments. At the same time, a general equation was derived that makes possible predictions of partition coefficient in binary mobile phases with different proportions of organic solvent to water based on some simple regression analysis. The LC separation for the derivatised alcohols containing higher carbon alcohols showed good reproducibility on a reversed-phase C-18 column with gradient elution. The detection limits (excitation at 335 nm, emission at 360 nm) for derivatised alcohols (signal-to-noise ratio=3:1) were in the range of 0.1-0.4 pg per injection. (C) 2001 Elsevier Science B.V. All rights reserved.

Enhanced surface attachment of protein-type targeting ligands to poly(lactide-co-glycolide) nanoparticles using variable expression of polymeric acid functionality

The density of reactive carboxyl groups on the surface of poly(lactide-co-glycolide) (PLGA) nanoparticles (NP) was modulated using a combination of high-molecular weight (MW) encapped and low MW non-encapped PLGA. Carboxyl groups were activated using carbodiimide chemistry and conjugated to bovine serum albumin and a model polyclonal antibody. Activation of carboxyl,groups in solution-phase PLGA prior to NP formation was compared with a postformation activation of peripheral carboxyl groups on intact NP. Activation before or after NP formation did not influence conjugation efficiency to NP prepared using 100% of the low-MW PLGA. The effect of steric stabilization using poly(vinyl alcohol) reduced conjugation of a polyclonal antibody from 62 mu g/(mg NP) to 32 mu g/(mg NP), but enhanced particulate stability. Increasing the amount of a high-MW PLGA also reduced Conjugation, with the activation post-formation still superior to the preformation approach. Drug release studies showed that high proportions of high-MW PLGA in the NP produced a longer sustained release profile of a model drug (celecoxib). It can be concluded that activating intact PLGA NP is superior to activating component parts prior to NP formation. Also, high MW PLGA could be used to prolong drug release, but at the expense of conjugation efficiency on to the NP surface. (C) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 87A: 873-884, 2008

Immunocolloidal Targeting of the Endocytotic Siglec-7 Receptor Using Peripheral Attachment of Siglec-7 Antibodies to Poly(Lactide-co-Glycolide) Nanoparticles

Purpose: To prepare a nanoparticulate formulation expressing variable peripheral carboxyl density using non-endcapped and endcapped poly(lactide-co-glycolide), conjugated to antibodies recognising the siglec-7 receptor, which is expressed on most acute myeloid leukaemias. The aim is to exploit this receptor as a therapeutic target by constructing an internalising drug-loaded nanoparticle able to
translocate into cytoplasm by siglec receptor-mediated internalisation.

Materials and Methods: Antibodies to the siglec-7 (CD33-like) receptor were conjugated to dye-loaded nanoparticles using carbodiimide chemistry, giving 32.6 mg protein per mg of nanoparticles using 100% of the non-endcapped PLGA. Binding studies using cognate antigen were used to verify preservation of antibody function following conjugation.

Results: Mouse embryonic fibroblasts expressing recombinant siglec-7 receptor and exposed to NileRed-loaded nanoparticles conjugated to antibody accumulated intracellular fluorescence, which was not observed if either antibody or siglec-7 receptor was absent. Confocal microscopy revealed internalised perinuclear cytoplasmic staining, with an Acridine Orange-based analysis showing red staining in localised foci, indicating localisation within acidic endocytic compartments.

Conclusions: Results show antibody-NP constructs are internalised via siglec-7 receptor-mediated internalisation. If loaded with a therapeutic agent, antibody-NP constructs can cross into cytoplasmic
space and delivery drugs intracellularly to cells expressing CD33-like receptors, such as natural killer cells and monocytes.

Production and characterization of specific antibodies for evaluation of glycated insulin in plasma and biological tissues

Previous studies have shown that glycation of insulin occurs in pancreatic beta -cells under conditions of hyperglycaemia and that the site of glycation is the N-terminal Phe(1) of the insulin B-chain. To enable evaluation of glycated insulin in diabetes, specific antibodies were raised in rabbits and guinea-pigs by using two synthetic peptides (A: Phe-Val-Asn-Gln-His-Leu-Cys-Tyr, and B: Phe-Val-Asn-Gln-His-Leu-Tyr-Lys) modified by N-terminal glycation and corresponding closely to the N-terminal sequence of the glycated human insulin B-chain. For immunization, the glycated peptides were conjugated either to keyhole limper haemocyanin or ovalbumin using glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester or 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride. Antibody titration curves, obtained using I-125-tyrosylated tracer prepared from glycated peptide A, revealed high-titre antisera in five groups of animals immunized for 8-28 weeks. The highest titres were observed in rabbits and guinea-pigs immunized with peptide B coupled to ovalbumin using glutaraldehyde. Under radioimmunoassay conditions, these antisera exhibited effective dose (median) (ED50) values for glycated insulin of 0.3-15 ng/ml and 0.9-2.5 ng/ml respectively, with negligible cross-reactivity against insulin or other islet peptides. The degree of cross-reaction with glycated proinsulin was approximately 50%. Glycated insulin in plasma of control and hydrocortisone-treated diabetic rats measured using rabbit 3 antiserum (1:10 000 dilution; sensitivity

Specific label-free and real-time detection of oxidized low density lipoprotein (oxLDL) using an immunosensor with three monoclonal antibodies

Increased levels of plasma oxLDL, which is the oxidized fraction of Low Density Lipoprotein (LDL), are associated with atherosclerosis, an inflammatory disease, and the subsequent development of severe cardiovascular diseases that are today a major cause of death in modern countries. It is therefore important to find a reliable and fast assay to determine oxLDL in serum. A new immunosensor employing three monoclonal antibodies (mAbs) against oxLDL is proposed in this work as a quick and effective way to monitor oxLDL. The oxLDL was first employed to produce anti-oxLDL monoclonal antibodies by hybridoma cells that were previously obtained. The immunosensor was set-up by selfassembling cysteamine (Cyst) on a gold (Au) layer (4 mm diameter) of a disposable screen-printed electrode. Three mAbs were allowed to react with N-hydroxysuccinimide (NHS) and ethyl(dimethylaminopropyl)carbodiimide (EDAC), and subsequently incubated in the Au/Cys. Albumin from bovine serum (BSA) was immobilized further to ensure that other molecules apart from oxLDL could not bind to the electrode surface. All steps were followed by various characterization techniques such as electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV). The analytical operation of the immunosensor was obtained by incubating the sensing layer of the device in oxLDL for 15 minutes, prior to EIS and SWV. This was done by using standard oxLDL solutions prepared in foetal calf serum, in order to simulate patient's plasma with circulating oxLDL. A sensitive response was observed from 0.5 to 18.0 mg mL 1 . The device was successfully applied to determine the oxLDL fraction in real serum, without prior dilution or necessary chemical treatment. The use of multiple monoclonal antibodies on a biosensing platform seemed to be a successful approach to produce a specific response towards a complex multi-analyte target, correlating well with the level of oxLDL within atherosclerosis disease, in a simple, fast and cheap way.