Competitive immunoassay of phenobarbital by microchip electrophoresis with laser induced fluorescence detection.

A microchip electrophoresis method with laser induced fluorescence detection was developed for the immunoassay of phenobarbital. The detection was based on the competitive immunoreaction between analyte phenobarbital and fluorescein isothiocyanate (FITC) labeled phenobarbital with a limited amount of antibody. The assay was developed by varying the borate concentration, buffer pH, separation voltage, and incubation time. A running buffer system containing 35 mM borate and 15 mM sodium dodecyl sulfate (pH 9.5), and 2800 V separation voltage provided analysis conditions for a high-resolution, sensitive, and repeatable assay of phenobarbital. Free FITC-labeled phenobarbital and immunocomplex were separated within 30s. The calibration curve for phenobarbital had a detection limit of 3.4 nM and a range of 8.6-860.0 nM. The assay could be used to determine the phenobarbital plasma concentration in clinical plasma sample.

Leaky gut and diabetes mellitus: what is the link?

Diabetes mellitus is a chronic disease requiring lifelong medical attention. With hundreds of millions suffering worldwide, and a rapidly rising incidence, diabetes mellitus poses a great burden on healthcare systems. Recent studies investigating the underlying mechanisms involved in disease development in diabetes point to the role of the dys-regulation of the intestinal barrier. Via alterations in the intestinal permeability, intestinal barrier function becomes compromised whereby access of infectious agents and dietary antigens to mucosal immune elements is facilitated, which may eventually lead to immune reactions with damage to pancreatic beta cells and can lead to increased cytokine production with consequent insulin resistance. Understanding the factors regulating the intestinal barrier function will provide important insight into the interactions between luminal antigens and immune response elements. This review analyses recent advances in the mechanistic understanding of the role of the intestinal epithelial barrier function in the development of type 1 and type 2 diabetes. Given our current knowledge, we may assume that reinforcing the intestinal barrier can offer and open new therapeutic horizons in the treatment of type 1 and type 2 diabetes.

Phenobarbital-induced DiHS and ceftriaxone hypersensitivity reaction: a case of multiple drug allergy.

Patients with DiHS show an increased risk of sensitization to multiple drugs. We report a case of a young woman who developed cutaneous rash, lymphoadenopathy, malaise and fever after the introduction of phenobarbitale. Because of these symptoms, she was treated with ceftriaxone and she experienced a severe flare-up of the cutaneous and general reaction. Allergological work-up, by cutaneous and lymphocyte transformation test, confirmed a double sensitization to phenobarbital and ceftriaxone. In conclusion, the high risk of DiHS during anticonvulsive therapy should suggest caution in using additional drugs, because of an increased risk of multiple reactions.

Pentobarbital desensitization in a 3-month-old child.

Barbiturates, a class of medications commonly used as antiepileptics and sedatives, are known to cause adverse reaction, with the most commonly reported immune-mediated reactions being anticonvulsant hypersensitivity syndrome. Other types of allergic reactions such as immediate hypersensitivity reactions also can occur. We present a 3-month-old child with refractory generalized convulsive status epilepticus who required pentobarbital therapy in the context of phenobarbital sensitivity because of progressive generalized seizures unresponsive to other aggressive therapies. Skin tests to pentobarbital and phenobarbital were negative. In the intensive care unit setting, intravenous pentobarbital desensitization was performed without reaction. To our knowledge, this is the first reported protocol for pentobarbital desensitization.

Effects of homeopathic preparations on the liver in rats with acute and chronic toxic hepatitis.

Ultralow doses of antibodies to phenobarbital and their mixture (1:1) with ultralow doses of antibodies to cholecystokinin reduced the severity of structural and metabolic disturbances in the liver of rats with acute CCl4-induced hepatitis. The mixture of antibodies had no effect on the course of CCl4-induced hepatitis.

Tritiated thymidine incorporation does not enhance sensitivity of the popliteal lymph node assay.

The popliteal lymph node (PLN) assay has been proposed as a tool to predict drugs and chemicals with the potential to induce systemic autoimmune reactions in man. In this assay, weight and cellularity indices typically are the measured endpoints. The present study was conducted to test whether incorporation of tritiated thymidine could improve sensitivity of the PLN assay. Male and female Balb/c mice were injected with 20 microCi of [3H]-methyl-thymidine intravenously 7 days after receiving 0.5, 1 or 2 mg of diphenylhydantoin, streptozotocin, sulfamethoxazole, ofloxacin, phenobarbital, or metformin intradermally. Results obtained with incorporation of tritiated thymidine were compared to weight indices. No consistent or marked differences in these endpoints were noted whatever the compound used. This study shows that incorporation of tritiated thymidine does not improve sensitivity of the PLN assay.

Characterization of drug-specific T cells in phenobarbital-induced eruption.

Phenobarbital has a high potential to elicit adverse reactions including severe skin eruptions and systemic involvements among the worldwide-prescribed drugs. Although phenobarbital hypersensitivity is thought to be mediated by T cells specific to the drug, its precise mechanism remains not fully elucidated. To characterize T cells reactive with phenobarbital, we generated drug-specific T cell clones and lines from PBMCs of patients with phenobarbital hypersensitivity showing various degrees of cutaneous and extracutaneous involvements. Although the TCR Vbeta repertoire and phenotype in the T cell clones/T cell lines were heterogeneous among the patients, Vbeta13.1(+) and Vbeta5.1(+) clones or lines were raised from the individuals examined who possessed different HLA haplotypes. Histopathological examination suggested that Vbeta5.1(+)CD8(+) T cells and Vbeta13.1(+) T cells played a role in cutaneous and extracutaneous involvements, respectively. A Vbeta13.1(+)CD4(+) clone was found to proliferate in response to the Ag with processing-impaired, fixed APCs. Most of the clones and lines belonged to the Th2 phenotype, producing IL-4 and IL-5 but not IFN-gamma upon phenobarbital stimulation. Clones/lines with Th1 or Th0 phenotypes also constituted minor populations. These observations clearly indicate the heterogeneity and a marked individual deviation of reactive T cell subsets among the patients in terms of CD4/8 phenotype, Vbeta repertoire, Ag recognition pattern, and cytokine production; and thus provide evidence whereby each pathogenic T cell subset contributes to special elements of clinical presentation.

Comparison of the conformations of two intact monoclonal antibodies with hinges.

Structures of two intact monoclonal antibodies were solved by X-ray diffraction analysis revealing, in both cases, the dispositions of all segments, as well as the structures of the hinge polypeptides. An IgG1, whose antigen is the drug phenobarbital, assumed a completely different conformation when compared with an IgG2a specific for canine lymphoma cells. Though neither IgG displays global two-fold symmetry, both maintain two pseudo dyad axes, one relating Fab segments, and the other the halves of the Fc. In both IgGs, the Fc segment is obliquely disposed with respect to the plane of the Fabs, making an angle of 128 degrees in the IgG2a, and 107 degrees in the IgG1. Hinge angles of the IgG1 are notably different at 78 degrees and 123 degrees, and unique as well from IgG2a values of 66 degrees and 113 degrees. Elbow angles within the IgG1 Fabs are the same at 155 degrees, but non-identical in IgG2a where they took on values of 143 degrees and 159 degrees. The IgG2a has an angle of 172 degrees between Fabs so that it exhibits a "distorted T" shape, whereas that angle in the IgG1 is a much more acute 115 degrees producing a "distorted Y". CH2 domains appear, in both antibodies, to be the most independently mobile of the paired IgG domains. This perhaps reflects their importance in modulating effector functions through exposure of binding loci on the CH2, at the CH2-CH3 interface, and on lower hinge polypeptides. Hinges in both antibodies contain disulfide-linked cores bounded by fluid regions above and below, which provide mobility to the Fabs and Fc respectively. The conformations seen in these two structures are undoubtedly only two among many but they illustrate the modes of flexibility inherent to the IgG architecture.

[Successful induction of tolerance in an epilepsy patient with phenobarbital allergy]. / Erfolgreiche Toleranzinduktion bei einer Epilepsiepatientin mit Phenobarbitalallergie.

We report on a 32-year-old female patient who had had a history of complex partial seizures since the age of 14. Phenobarbital was the most effective anticonvulsant drug in this patient. However, the drug treatment was complicated by a phenobarbital-induced exanthematous eruption. Reintroduction of the phenobarbital some years later resulted in a skin rash again; therefore, treatment with this substance had to be discontinued a second time. Because of the satisfactory antiepileptic efficacy, phenobarbital was introduced a third time using a desensitization procedure with increased oral doses, starting with a dose of 1 mg. After a daily dose of 90 mg phenobarbital, on day 6 an exanthematous eruption appeared. The exanthem disappeared parallel to a dose reduction of phenobarbital and with a gradually increasing dosage up to a maintenance dose of 200 mg. Tolerance to the allergic effect of phenobarbital was preserved and the seizure frequency was significantly reduced by phenobarbital monotherapy with a daily dose of 200-175 mg.

Crystallographic structure of an intact IgG1 monoclonal antibody.

The structure of an intact monoclonal antibody for phenobarbital, subclass IgG1, has been determined to 3.2 A resolution by X-ray crystallography. The molecule was visualized in a monoclinic unit cell having an entire immunoglobulin as the asymmetric unit. The two Fab segments, both with elbow angles of 155 degrees , were related by a rotation of 179.7 degrees plus a translation along the approximate dyad of 9 A. This is the first observation of such an Fab translation in a structurally defined antibody. The approximate 2-fold of the Fc was independent of that relating Fabs, making an angle of 107 degrees with the Fab dyad. The angle between long axes of the Fabs was 115 degrees, the most acute angle yet observed, yielding a distorted Y shaped molecule. This is in contrast to the distorted T shape of the only other intact IgG (2a) whose complete structure is known. Primary lattice interactions arise through formation of VH antiparallel beta ribbons whose strands are contributed by pseudo dyad related H2, and by L3 hypervariable loops from neighboring molecules. While one CH2 domain was mobile, Fabs and three domains of the Fc were well defined, as were hinge polypeptides connecting Fabs to the Fc, and the covalently attached oligosaccharides. Direct interactions are observed between hinge polypeptides, the glycosylated loop of one CH2 domain, and the oligosaccharide. Lattice interactions clearly influence, perhaps even determine the overall conformation of the antibody observed in this crystal. Comparison of this IgG1 with previously determined intact antibody structures extends the conformational range arising from segmental flexibility.

Analysis of antibody selection by phage display utilizing anti-phenobarbital antibodies.

The generation of new antibodies for diagnostic applications using phage display could greatly decrease the time and expense of new assay development but, to be effective, the process must yield antibodies with desired specificity and affinity comparable to those obtained by monoclonal antibody technology. In order to evaluate the ability of the phage selection process to yield antibodies with the desired specificity and affinity, a family of anti-phenobarbital antibodies were cloned as scFvs and Fabs and displayed on M13 gene III fusion proteins. All of the antibodies are derived from similar germline VL and VH genes and exhibit extensive sequence homology except in VH CDR3. Despite these similarities, the range of panning efficiencies was observed to vary by two orders of magnitude for expressed scFvs. Unexpectedly, the scFv with the highest panning efficiency has the lowest affinity. In competitive panning experiments this scFv is preferentially isolated over higher affinity antibodies. This scFv expresses high levels of soluble binding protein while higher affinity scFvs express lower levels of protein or nonfunctional protein. These results suggest that the efficiency of functional expression of scFv proteins can greatly influence the type of antibody selected by phage display. The range of panning efficiency for functional Fabs was significantly less (four-fold) than that observed for scFvs and did not correlate to the expression level of the secreted proteins. Based on the results of the Fabs examined, it is concluded that the expression properties of Fabs may not exhibit the extent of variability observed for scFvs when displayed and use of an Fab architecture may provide an advantage over scFv architecture in the selection process. The feasibility of selecting against undesirable cross-reactivities has also been demonstrated by simple modification of phage panning conditions. These combined results support the concept of obtaining antibodies with desirable specificity and affinity for diagnostic applications through the use of phage display.

Crystallization of intact monoclonal antibodies.

Attempts were made to crystallize four monoclonal antibodies, one IgG2a kappa and three IgG1 kappa. Using a PEG 3350 screen combined with detergents, and developed from our experiments with an IgG2a kappa antibody specific for canine lymphoma cells, crystals have now been obtained of two of these four immunoglobulins, an antiphenytoin and an antiphenobarbital antibody. A complex between the antiphenobarbital antibody and its drug antigen crystallized as well. The antibody for phenytoin has, to this point, produced only clustered microcrystals, marginally suitable for X-ray analysis. Single crystals of the IgG1 kappa antibody against phenobarbital, however, were characterized by X-ray diffraction to be primitive monoclinic, with unit cell dimensions a = 67 A, b = 193 A, c = 74 A, and beta = 110 degrees. These crystals have an entire IgG1 kappa molecule as the asymmetric unit and they diffract to at least 3.2 A resolution.

[Determination of phenobarbital by solid-phase immunoenzyme analysis. Choice of optimal strategy]. / Opredelenie fenobarbitala metodami tverdofaznogo immunofermentnogo analiza. Vybor optimal'noi strategii.

Two variants (direct and indirect) of enzyme linked immunosorbent assay (ELISA) of phenobarbital are compared. Both techniques were developed on the basis of the same monoclonal antibodies, and horse radish peroxidase was used as the label in both cases. When microtitration plates are used as the solid phase, indirect ELISA, in which phenobarbital of the sample competes with phenobarbital sorbed on plates in the form of a conjugate with protein for the binding with peroxidase-labeled antiphenobarbital antibodies, is preferable. In indirect ELISA, the sample volume was 5 microliters, the time of assay was 40 min, the variability coefficient was < 8%.

ELISA screening of monoclonal antibodies to haptens: influence of the chemical structure of hapten-protein conjugates.

An analysis has been made of the influence of the chemical structure of hapten-protein conjugates on ELISA based screening monoclonal antibodies to haptens. Evidence has been obtained that the size of haptens should be taken into account. For haptens with a relatively large molecular weight, one can use a conjugate with a carrier protein differing from the immunogen. For small haptens, the screening should be conducted with a conjugate differing from the immunogen with respect to both the carrier protein and the chemical linkage between the hapten and the protein. In addition, the hapten-protein coupling should involves different amino acid residues.

Some biochemical characteristics of the early phase of immunomodulation.

The study estimates some biochemical changes of chosen immunological and biochemical parameters after intraperitoneal application of a) immunomodulators of microbial origin--Propionibacterium acnes and Geotrichum candidum, b) chemical substances--indomethacin and phenobarbital. The estimation was concentrated above all to the changes of chemiluminiscence, phagocytic activities, the amount of cAMP in peritoneal macrophages, the amount of liver cytochrome P-450 and cAMP in the liver, spleen and thymus. The experiments also included histological examination of the spleen, thymus, lungs and diaphragm. The effect of Propionibacterium acnes was evident as soon as 24 hrs after application. In the frame of studied parameters, there was manifested the nonspecific activation of the immune system by the mechanisms independent to oxygen. The early changes were accompanied by the increase of cAMP in macrophages. In contrary, the intraperitoneal application of Geotrichum candidum activated the release of oxygen radicals from peritoneal macrophages. The amount of the cytochrome P-450 correlated to the intensity of immunomodulation. On the other hand, the induction of cytochrome P-450 by phenobarbital decreased the value of several immunity indices.

[Preparation of monoclonal antibodies to phenobarbital]. / Poluchenie monoklonal'nykh antitel k fenobarbitalu.

Monoclonal antibodies to phenobarbital have been developed. Spleen cells of BALB/c mouse immunized with conjugate keyhole limpet haemocyanin and phenobarbital were fused with P3-X63-Ag8.653 mouse myeloma cells. Three monoclonal antibodies, selected by indirect ELISA, were produced in mouse ascite fluids, purified and analyzed. Antibody 1A9 was selected for use in immunoassay, its association constant being 1.6 x 10(9) M-1.

Evaluation of the cloned enzyme donor immunoassay for measurement of phenytoin and phenobarbital in serum.

We report the evaluation of the cloned enzyme donor immunoassay (CEDIA) for the estimation of phenytoin and phenobarbital in serum. The assays were performed with a Hitachi 911 analyzer. Intra-assay coefficients of variation were from 1.5 to 4.4% for phenytoin and 1.6 to 5.5% for phenobarbital. Interassay coefficients of variation ranged from 1.8 to 5.3% for phenytoin and 2.9 to 4.8% for phenobarbital. Linearity was satisfactory, with a recovery of 103% at 11 mg/L and 110% at 22 mg/L phenytoin and 93% at 14 mg/L and 101% at 40 mg/L for phenobarbital. The detection limit was 1.2 mg/L for phenytoin and 0.6 mg/L for phenobarbital. Results of this assay correlated well with those of a conventional high-performance liquid chromatography (HPLC) method; r = 0.99 for phenytoin (n = 47) and r = 0.98 for phenobarbital (n = 48). The CEDIA was easy to handle and especially suitable for short turn-around time application.

[Determination of phenobarbital by polarized fluoroimmunoanalysis. The effect of immunogen structure and tracer on specificity and detection limit]. / Opredelenie fenobarbitala metodom poliarizatsionnogo fluoroimmunoanaliza. Vliianie struktury immunogena i treisera na spetsifichnost' i predel detektsii.

The influence of the immunogen's tracer's structure on properties of antibodies to phenobarbital was studied by the polarisation fluoroimmunoassay method. It was shown that in homogeneous method of polarisation fluoroimmunoassay the titer of antibodies to phenobarbital (1/1000-1/10,000) is affected by the position of linker binding the antigen with carrier-protein and by the structure of the linker. The affinity constant ((1-4) x 10(9) M-1), cross-reactivity, limit of the detection (0.6-2.6 micrograms/ml) phenobarbital with different combinations of antibodies and tracers were calculated. The antisera grew the more heterogeneous in affinity and specificity, the longer became the linker. The more heterogeneous antibodies are preferable in the class specific assay of barbiturates.

[An antiserum directed against phenobarbital with an organometallic tracer: production and characterization]. / Etude d'un antisérum dirigé contre le phénobarbital vis-à-vis d'un traceur organométallique: production et caractérisation.

In order to develop a new immunoassay procedure based on the use of organometallic labels, antisera are raising against sedative-hypnotic drug: phenobarbital. Immunization was performed in rabbits with parasuccinylamidophenobarbital coupled to bovine-serum-albumin. The evaluation of the specificity and the affinity constants of antisera are studied against different hypnotic or antiepileptic drugs and the metallotracer (phenobarbital labelled with an organometallic fragment derived from cymantrene). The most important result of this study indicates that the organometallic moiety of the metallotracer did not disturb neither the inhibition (% inhibition = 110) nor the affinity constant (Ka = 2.10(8) L/M) of the phenobarbital.

Single-reagent polarization fluoroimmunoassay for barbiturates in urine.

We describe a rapid polarization fluoroimmunoassay for screening for the presence of barbiturates in urine. The single reagent is prepared by pre-mixing antiserum with a fluorescein-labeled barbiturate derivative as tracer. Use of a mixture of two sheep antisera, raised against different barbiturate immunogens, results in an assay with a broad cross-reactivity spectrum for most common barbiturates. One adds urine to the pre-mixed reagent, incubates the solution for a few minutes at room temperature, and measures the fluorescence polarization. The tracer/antiserum reagent is stable for at least six months at 4 degrees C. Results compare favorably with two other commonly used screening techniques for barbiturates, thin-layer chromatography and the EMIT-stTM (Syva) system. Although designed as a qualitative screen, some drugs can be quantified by means of a standard curve of the relevant barbiturate.