Development of a Biosensor-based Immunoassay for Screening of Chloramphenicol Residues in Milk

Biosensor immunoassays were developed recently for antibiotics with an established maximum residue limit (MRL). In this study, according to the regulatory banning of chloramphenicol (CAP) use for food producing animals, the main objectives were: the specificity of the biosensor assay and the lowest detection limit possible. The assay was based on the inhibition of the binding of polyclonal antibodies against CAP to immobilized CAP on a sensor chip by CAP in solution. The response varied inversely with the antibiotic concentration in the sample. Two different antibodies and two immobilization protocols were tested. As in ELISA tests the antibody influenced the assay performances. Moreover, we showed that particular care should be concentrated on the immobilization step because it is a critical point in the assay development. Three different protocols were developed in milk. The best assay was obtained with antibody 1 in milk on the CAP base surface because of its very low detection limit (0.1 μg l^-1) and the decreased consumption of antibody (four times less than on the CAP surface). This assay is rapid (3 min/run), sensitive, and specific for CAP and CAP glucuronide. It could be integrated in a multi-residue screening test and applied to other matrices (bile, urine, meat).     

Direct Versus Competitive Biosensor Immunoassays for the Detection of (Dihydro)Streptomycin Residues in Milk

A monoclonal antibody (MAb) against dihydrostreptomycin (4G8) was developed and its performance compared with a previously developed MAb against streptomycin (4E2) in biosensor immunoassays (BIAs) using a surface plasmon resonance (SPR)-based biosensor (BIACORE 3000). Direct BIAs for the detection of dihydrostreptomycin (DHS; 583 Da) and streptomycin (STREP; 581 Da) were developed by immobilising the MAbs on the sensor chip (CM5). These direct BIAs were compared with competitive inhibition BIAs, using a STREP- protein conjugate immobilized on the chip. The sensitivities of the direct and competitive BIAs for both drugs in buffer were comparable (10-20 ng ml- 1 at 50% binding or inhibition). With milk, interferences, probably due to the nonspecific binding of proteins to the sensor chips, were observed in both BIAs. These interferences could be largely reduced using ultra filtration (UF) as sample pre-treatment. Another option was the use of a reference flow channel to correct for nonspecific binding. Using this option with five times diluted milk, MAb 4G8 was found to be suited for the direct BIA of both drugs with a limit of detection (LOD) of 20 ng ml- 1 and both MAbs could be applied in the competitive BIA format with similar LODs.     

Quantification of plasma-derived blood coagulation factor VIII by real-time biosensor measurements

Plasma-derived blood coagulation factor VIII was analyzed in real time using biosensor technology. Monoclonal antibodies directed against the heavy and against the light chain of factor VIII were immobilized on different carboxymethyl dextran surfaces. Different factor VIII concentrations were injected over the antibody surfaces in parallel and response levels were determined from the dissociation phase at a fixed time after sample injection. Serial dilutions of plasma-derived factor VIII with known concentrations determined by a commercial FVIIIC:Ag ELISA were used as standards. A quantification limit of 0.9 I.U./ml with antibody 530p and 1.5 I.U./ml with antibody 531p was calculated. Intra-assay precision expressed as percent coefficient of variation was below 10% for concentrations above 0.6 I.U./ml. Inter-assay precision for antibody 530p was below 20% for concentrations higher than 0.6 I.U./ml. For 531p, inter-assay precision was below 10% for concentrations higher than 2 I.U./ml. A sensor chip lifetime in respect to regeneration of at least 100 cycles for both antibodies was found. The small sample requirement of 35 microl allows fast analysis of different FVIII products and the use of two monoclonal antibodies directed against two different FVIII domains provides additional information about the integrity of the FVIII molecule.     

Induction of early gene expression in murine macrophages by synthetic lipid A analogs with differing endotoxic potentials.

Numerous lipid A analogs have been synthesized in an attempt to dissociate endotoxic activities from beneficial immunomodulatory activities. In the present study, we have evaluated select lipid A analogs in macrophages for their ability to induce a panel of lipopolysaccharide (LPS)-inducible genes to gain insights into the molecular mechanisms which underlie endotoxicity. We evaluated three monosaccharide lipid A analogs: SDZ MRL 953, an agonist with an improved therapeutic margin over endotoxin; SDZ 281.288, a more toxic analog; and SDZ 880.431, an analog with proven LPS-inhibitory activity. In addition, three disaccharide lipid A analogs (i.e., lipid IVA, SDZ 880.611, and SDZ 880.924) that differ in acylation and phosphorylation patterns were also examined and compared with synthetic lipid A. With the exception of SDZ 880.431, each of these structurally diverse analogs was able to induce the complete panel of LPS-inducible genes, specifically genes which encode tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, 75-kDa type 2 TNF receptor (D7), IP-10, D3, and D8. These results underscore that macrophage stimulation by lipid A analogs is permissive to considerable structural diversity. Structures with favorable therapeutic indices (SDZ MRL 953, SDZ 880.611, and SDZ 880.924) were not different from structures with poor therapeutic indices (lipid A, lipid IVA, and SDZ 281.288) with regard to gene induction. Nonetheless, the nontoxic SDZ MRL 953 was approximately 1,000-fold less potent than synthetic lipid A at inducing TNF-alpha secretion, and perhaps this contributes to the lack of toxicity exhibited by this compound. The ability of compound SDZ 880.431 to inhibit TNF-alpha secretion induced by both SDZ MRL 953 and smooth LPS suggests that the monosaccharide and smooth LPS share a receptor or a portion thereof. A pattern of protein tyrosine phosphorylation similar to that induced by LPS was stimulated by the monosaccharide SDZ MRL 953 and SDZ 281.288 and disaccharides lipid IVA, SDZ 880.924, and SDZ 880.611, providing evidence for a common signalling pathway.     

Development of an immunobiosensor assay for the beta-adrenergic compound zilpaterol

A surface plasmon resonance biosensor method was developed to measure zilpaterol residues in sheep urine. A CM-5 sensor chip previously reacted with ethylenediamine to produce an aminoethyl group was coupled with 4-carboxybutyl zilpaterol activated using EDC/NHS. Five polyclonal and four monoclonal antibodies were screened for their suitability to detect low levels of zilpaterol using the biosensor technology. Total binding was greater for polyclonal than monoclonal antibodies, but a less diluted antibody solution was required for polyclonal antibodies. A fixed antibody concentration and various concentrations of zilpaterol were injected to obtain a standard curve for each antibody to allow for B₀ and IC₅₀ determination. The stability of the assay was assessed by the consistency of B₀ in repeated experiments extending at least six hours. A measure of non-specific binding allowed the assessment of the specificity of the antibody-immobilized ligand interaction. The effect of varying concentrations of urine on B₀ and IC₅₀ was evaluated to assess the degree of “matrix” effect that would be present in an assay. Based on these criteria the most promising antibody (2E10, a monoclonal antibody) was selected for further evaluation. This antibody had good sensitivity with IC₅₀=4.47±0.41 ng/ml (n=11) in buffer). Both intra- and inter-assay variation studies showed excellent recovery and reproducibility for concentrations between 2 ng/ml and 8 ng/ml. A comparison of the biosensor method with a previously developed ELISA demonstrated that both methods give equivalent results (slope of the correlation plot = 1.02) with a high correlation (r²=0.91) between them.     

Penicillin-specific antibodies: Monoclonals versus polyclonals in ELISA and in an optical biosensor

Two penicillin-specific monoclonal antibodies mAb 19C9 and mAb 9H3 and the penicillin-specific polyclonal antibodies pAb K2 were evaluated for their use in a competitive ELISA and in the BIAcore™ optical biosensor. In the ELISA, an ampicillin-protein conjugate was used as a coating molecule. For the biosensor assay, ampicillin was immobilized on a CM5 chip. With both monoclonal antibodies and in both test systems, ampicillin, amoxicillin and benzylpenicillin were better recognized than oxacillin, cloxacillin and dicloxacillin. Because the reproducibility was better in the biosensor (CV = 1.6%) than in the ELISA (CV = 8.9%), the limit of detection for ampicillin in buffer solution using mAb 19C9 was lower in the biosensor (46 ng ml^-1) as compared to the ELISA (356 ng ml^-1). Ampicillin can thus be detected below the MRL (50 ng ml^-1) in the biosensor assay but not in the ELISA. Both the ELISA and biosensor assay using the polyclonal antibodies pAb K2 were more sensitive as compared to the assays with the monoclonals. The ELISA using pAb K2 allowed the detection of all tested penicillins below the MRL. In the biosensor assay, ampicillin was also detected below the MRL (IC50 = 10 ng ml^-1). In contrast to the binding of the monoclonals, no spontaneous dissociation was observed after injection of the polyclonal antibodies in the biosensor. Whereas the monoclonals were completely removed from the sensor surface using ampicillin in the buffer solution as regeneration solution, stronger conditions were necessary for the pAb binding.     

Analysis of β-Lactam Antibiotics Using a Microbial Receptor Protein-based Biosensor Assay

An optical biosensor assay for detection of β-lactam antibiotics in milk based on a microbial receptor protein was developed. The assay uses a general sensor surface previously described with a small organic molecule (H1) immobilized. A conjugate between a β-lactam (cephalosporin C) and a monoclonal H1 antibody is injected across the sensor surface before injection of the sample mixed with receptor protein. Receptor inhibited by β-lactam residues in the milk sample will not bind to the sensor surface and the reduction in response is inversely related to the β-lactam concentration of the sample. The detection limit for a number of commonly used β-lactams was below or near the respective maximum residue limit and the relative standard deviation (CV) for penicillin G in milk was 6-12% in the interval 2.0-12.5 μ g kg^-1. For application in the field further optimization is needed to solve problems related to non-specific binding to the sensor surface.     

Effects of pretreatment with SDZ MRL 953, a novel immunostimulatory lipid A analog, on endotoxin-induced acute lung injury in guinea pigs.

SDZ MRL 953 (SDZ), a novel immunostimulatory lipid A analog, has been reported to have immunopharmacological activities similar to those of lipopolysaccharide (LPS) but to have little of the toxicity of LPS. We investigated the effects of pretreatment with SDZ on Escherichia coli endotoxin-induced acute lung injury in guinea pigs. Four experimental groups consisted of saline control (n = 16), SDZ (-12 h) plus LPS (2 mg/kg of SDZ per kg of body weight injected intravenously 12 h before intravenous injection of 2 mg of LPS per kg; n = 15), SDZ (-10 min) plus LPS (SDZ injected 10 min before LPS injection; n = 10), and LPS alone (n = 16). The animals were sacrificed, and lung tissue was sampled 4 h after LPS or saline infusion. Lung injury was assessed by measuring the wet weight-to-dry weight ratio and the level of 125I-labeled albumin accumulation in bronchoalveolar lavage fluid relative to that in plasma. In the SDZ (-12 h) plus LPS group, these two parameters of acute lung injury were decreased compared with those in the LPS alone group. However, they were not decreased in the SDZ (-10 min) plus LPS group. We conclude that SDZ attenuates endotoxin-induced acute lung injury when it is administered 12 h before LPS injection. The attenuating effects of SDZ are speculated to be due to down regulation of the response to endotoxin rather than to receptor blocking.     

Detection of the mycovirus OMSV in the edible mushroom, Pleurotus ostreatus, using an SPR biosensor chip

A surface plasmon resonance (SPR) biosensor chip was developed for the rapid detection of the oyster mushroom spherical virus (OMSV), which causes a mushroom die-back disease, the symptoms of which include malformed fruiting bodies and retarded mycelial growth in the cultivated edible mushroom, Pleurotus ostreatus. An anti-OMSV monoclonal antibody (mAb) was generated initially using purified OMSV viral particles. For the fabrication of the biosensor chip, the anti-OMSV mAb was layered onto an activated carboxymethyl-dextran (CM-Dex) gold thin film. Analysis on the SPR angle shift showed that the bound mAb was 6.7 ng/mm2 of the chip surface. Subsequently, the biosensor chip was applied to the detection of OMSV in the mushroom mycelial extract. It detected specifically OMSV in the extract in a concentration-dependent manner. Finally, the biosensor chip was employed for the detection of OMSV in the mushroom fruiting bodies collected from 10 commercial farms. Among the tested samples, OMSV was found to infect fruiting bodies from a farmland, and this was confirmed further via immunoblot analysis and a TAS-ELISA assay. In conclusion, the SPR biosensor chip combined with an anti-OMSV mAb evidenced superior performance, particularly with regard to the prompt detection of OMSV infection.     

Novel assay format permitting the prolonged use of regeneration-based sensor chip technology

A polyclonal antibody raised against morphine-3-glucuronide (M3G, the main metabolite of heroin and morphine) was used in the development of a novel assay format using a surface plasmon resonance (SPR)-based biosensor. Previously developed assays have generated calibration curves based on differences in the quantity of response units binding to the surface of a chip coated with the analyte. The novel assay described here was based on the development of a standard curve using the slope of a series of consecutive binding interactions. Using this format, regeneration between each assay cycle was no longer required. This increased the useable life span of the chip surface and, as a result, decreased the cost associated with the assay. Thus, at least 15 binding interactions could be carried out before the saturation of antibody on the surface of the chip caused the response to deviate significantly from linearity. After 15 nonregenerated binding interactions, the slope still remained within 1.5% of the slope after a single binding event. Analysis time, and the sample volumes required were also markedly decreased while sensitivity was enhanced. The inhibition assay developed had a detection range of 270 to 17,500 pg ml(-1).     

A Biosensor-based Immunoassay for Rapid Screening of Deoxynivalenol Contamination in Wheat

A surface plasmon resonance (SPR) based indirect inhibitive immunoassay was developed for the rapid quantification of concentrations of the trichothecene mycotoxin deoxynivalenol (DON). A DON-biotin conjugate was synthesized and immobilized to a streptavidin coated SPR sensor surface to measure free anti-DON antibody added to a sample. For analysis, ground wheat was extracted with 10% (v/v) methanol in water with 6% (w/v) polyvinyl-pyrrolidone, filtered and cleaned up with MycoSep™ columns. Extracts were mixed with a polyclonal anti-DON antibody and pre-incubated prior to injection into a BIAcore® device. The sensor surface was regenerated with a rinse of 6 M-guanidinechloride in 10 mm-glycine (pH 2.9). The assay had a working range between 0.13 and 10.0 μg ml -1 of DON, a 50% inhibition concentration (IC 50 ) of 0.72 μg ml -1 , and a detection limit of 2.5 pg μl -1 . Recovery of DON in spiked wheat was 104 ±15%. The system enables analysis of a sample to be completed in 15 min including sample preparation (10 min) and quantification (5 min). The assay was applied to the analysis of wheat samples with different levels of DON contamination. Statistical analysis revealed a positive, linear correlation between concentrations of DON measured with the new biosensor and GC/MS or HPLC as reference methods. Coefficients of correlation of R 2 = 0.9464 (GC/MS data) and R 2 = 0.9066 (HPLC data) were found.     

A Direct (Non-Competitive) Immunoassay for Gentamicin Residues with an Optical Biosensor

Using sensor chip immobilized anti-gentamicin monoclonal antibodies in an optical biosensor (BIACORE 3000) resulted in the first single antibody based and label free noncompetitive immunoassay for the direct detection of residues of a low molecular weight compound in a food product. Calibration curves for gentamicin (Mw 466 Da) in buffer and milk showed 50% binding at 20 and 35 ng ml^-1, respectively, which is well below the maximum residue limit of 100 ng ml^-1.     

Development and validation of a biosensor-based immunoassay for progesterone in bovine milk

We have developed a rapid automated immunoassay, using the BIACORE surface plasmon resonance (SPR) biosensor, to measure progesterone in bovine milk. The assay was designed as an inhibition assay with progesterone covalently immobilised to the carboxymethyl dextran matrix of a CM5 sensor chip. A fixed amount of monoclonal anti-progesterone antibody 39C5H7 was mixed 9:1 with the sample and the amount of free antibody was then determined using biomolecular interaction analysis (BIA) by injection of the mixture over the immobilised progesterone sensor surface. The assay was designed to cover the concentration range 0.5 to 50 ng/ml. The limit of detection (LOD) was 3.56 ng/ml. Reproducibility of the assay was very good with both intra-assay and inter-assay coefficients of variation <5%. As results become available within minutes of injection and the procedure involves fully automated instrumentation, we believe that this BIA assay for progesterone in milk could be used in-line in the milking parlour and, thus, provide an important tool for reproductive management of dairy cattle to detect heat and predict pregnancy.     

Appearance of IgG and IgA antibodies in human bile after tetanus toxoid immunization

Humans immunized intramuscularly with one dose of tetanus toxoid exhibited IgG, and in some cases IgA antibody, in their bile as well as serum. Both isotypes appeared in bile transiently with titres declining after about day 10 for both classes. These kinetics resembled those of the serum IgA response but were markedly different to those for IgG antibody in serum. Measured IgG titres in bile were between 0.07 and 4.2% of those in paired sera, and IgA titres were between 6.8 and 124% of sera. Peak responses in bile, while generally of smaller size, exceeded those of paired sera when expressed as antibody/mg of IgG or IgA present. This calculation showed that during the peak response bile was up to nineteen-fold more abundant in IgG antibody than was serum taken at the same time, and up to forty-five-fold more for IgA. Enrichment of antibody in bile is not consistent with the Ig of bile being solely conferred by plasma, and may mean the involvement of local synthesis too. This study indicates that tetanus toxoid immunization of humans results in biliary antibody and raises the possibility of intra-hepatic antibody production for export to the intestinal tract in man.     

Aberrant expression of GM1 on lymph node cells of MRL/Mp-lpr/lpr mice: influences on the autoreactivities of anti-asialo GM1 antibodies.

Although changes in surface carbohydrate expression of abnormally expanded MRL/Mp-lpr/lpr (MRL/lpr) lymph node (LN) cells have previously been described, the composition and function of glycolipids present on these cells as well as the spectrum of specificity of anti-carbohydrate antibodies reactive with these cells remains obscure. Analysis of antibodies to a panel of 22 carbohydrate structures using a liposome immune lysis assay (LILA) showed that, except for anti-asialo GM2 (GA2) antibodies, marked reduction of antiglycolipid antibody levels was observed in sera from 4-mo-old MRL/lpr mice compared with these from MRL/Mp(-)+/+ (MRL/+) mice. Absorption experiments revealed that both anti-asialo GM1 (GA1) and globoside antibodies had binding capacity to MRL/lpr LN cells. To elucidate the glycolipid profiles of MRL/lpr LN cells, glycolipids were extracted from LN cells of both MRL/lpr and MRL/+ mice and analysed. A 30-fold elevation of GM1 was found in MRL/lpr LN cells compared with MRL/+ LN cells. From the results of LILA using GA1/GM1 mixed liposomes, aberrantly expressed GM1 inhibited the classical complement pathway but did not interfere with the binding of anti-GA1 antibodies to liposomal GA1. These findings suggest that a drastic GM1 increase on MRL/lpr LN cells would inhibit the action of anti-GA1 antibodies and complement on the cell surface. This may explain the escape of these cells from an activated self directed immune response.     

Fine Specificity and Isotype Expression of Anti-PC Antibodies in BALB/c, MRL/Mp-1pr/1pr, and −+/+ Mice at Different Ages

The MRL/Mp congenic mouse strains develop autoimmune disease with age. We have investigated age- and autoimmune-related changes in fine specificity, isotype spectra and T15 idiotype expression of the anti-phosphorylcholine (PC) response in BALB/c, MRL/Mp- + and -lpr congenic mice and in (BALB/c x MRL/Mp-lpr) F1 hybrids. Two groups of anti-PC antibodies with distinct fine specificity are elicited in the memory response. Group I antibodies recognize the PC moiety and express the T15 idiotype. Antibodies of group II are specific for phenyl-phosphorylcholine and are found predominantly in the memory response. In the MRL/Mp-lpr and - + strains only a minor population of antibodies expresses the T15 idiotype at all ages. However, a third group of antibodies was observed which binds to PC-coated proteins and to Diplococcus pneumoniae R-36A. This binding was not inhibited by PC-chloride and appeared mainly in the memory response at old age. The isotype distribution among anti-PC antibodies was similar in all strains analysed. In the initial response primarily mu, gamma 3 and gamma 1 isotypes were produced, while in the memory response gamma 1 was dominant. Thus autoimmune defects and ageing result in altered anti-PC antibody and idiotype profile, probably related to altered states in both the T- and B-cell compartments.     

The effect of hormones and of an osmotic gradient on the structure and properties of mammalian foetal urinary bladder in vitro.

1. Water and isotope fluxes were measured by incubating urinary bladders of foetal pigs and sheep in vitro in the presence and absence of a concentration and osmotic gradient. The structure of the urinary bladder of foetal pigs under various conditions was studied by electron microscopy. Its ultrastructure was found to be closely similar to that of foetal sheep. 2. Antidiuretic hormone (ADH) (0-2 U. ml-1) enhanced the enlargement of intercellular spaces caused by dilute mucosal medium in pig bladders; prolactin (1 u. ml-1) prevented osmotic dilatation of the intercellular spaces. 3. The hydraulic conductivity, Lp, was estimated to be 0-5 X 10 (-7) cm.s-1atm-1 in sheep and pigs at about 100 days gestation; the ratio of osomotic to diffusional permeability, (LpRT/VW)/PD, in the presence and absence of ADH, was 2-1 and 1-6 respectively. These are similar to the values found in fish gills. 4. Prolactin reduced bulk flow of water to zero in seven out of eight bladders investigated. Incubation with ADH or vasotocin (55 mu. ml-1) in the presence of prolactin restored water flux to 22% and 45% of control values respectively. 5. There was no significant net flux of sodium from mucosa to serosa in pig bladder except in the presence of prolactin. No net flux of sodium occurred from mucosal to serosal side of pig or sheep bladders in the presence of an adverse electrochemical gradient, although in sheep the permeability ratio was significantly greater than one. 6. The diffusional flux ratio for water remained unity under all conditions; vasotocin increased unidirectional fluxes and prolactin reduced them. The flux ratios were unaffected by the direction of bulk fluid flow, probably because diffusion was rapid compared to flow: the ratio of diffusional flux to volume flow was between 11 and 18.     

Design and Use of a General Capturing Surface in Optical Biosensor Analysis of Sulphamethazine in Milk

A new optical biosensor assay, based on a general capturing surface, for detection of the antimicrobial agent sulphamethazine (SMZ) was evaluated and compared with a previously described biosensor assay. At the general surface, the immobilisation is thought to be independent of type of analyte. Monoclonal antibodies against a small molecule (hapten H1) were immobilised and used to capture a conjugate between H1 and SMZ. Polyclonal SMZ antibodies were added to the milk sample and the amount of antibodies bound to the surface was in inverse proportion to the SMZ concentration in the milk sample. The detection limit of the new assay was 0.5 microg kg -1 and within-assay repeatability was 2.4%. This is in agreement with previous results obtained when SMZ was directly immobilised on the surface. Incurred samples from SMZ-treated cows were analysed, and non-specific binding was studied by analyses of individual cow's milk. The advantages of the new assay format include analyte-independent immobilisation and regeneration. Furthermore, the assay enables measurements with covalent interactions between analyte and detecting molecule. The main disadvantage is the requirement of a conjugate between analyte and the hapten H1. Moreover, it is likely that the antibody surface will have a shorter life span than a surface with the antimicrobial immobilised.     

Detection of Yersinia pestis fraction 1 antigen with a fiber optic biosensor.

A fiber optic biosensor was used to detect the fraction 1 (F1) antigen from Yersinia pestis, the etiologic agent of plague. The instrument employs an argon ion laser (514 nm) to launch light into a long-clad fiber and measures the fluorescence produced by an immunofluorescent complex formed in the evanescent wave region. This sensing area is a short section (12.5 cm) at the end of the optical fiber from which the cladding has been removed and in which the silica core has been tapered. Capture antibodies, which bind to F1 antigen, were immobilized on the core surface to form the basis of the sandwich fluoroimmunoassay. The ability to detect bound F1 antigen was provided by adding tetramethylrhodamine-labeled anti-plaque antibody to form fluorescent complexes. The evanescent wave has a limited penetration depth (< 1 lambda), which restricts detection of the fluorescent complexes bound to the fiber's surface. The direct correlation between the F1 antigen concentration and the signal provided an effective method for sample quantitation. This method achieved a high level of accuracy for determining F1 antigen concentrations from 50 to 400 ng/ml in phosphate-buffered saline, serum, plasma, and whole blood, with a 5-ng/ml limit of detection. Subsequent blind studies, which included serum samples from patients, yielded results in good agreement with measurements by enzyme-linked immunosorbent assay. A major advantage of the fiber optic biosensor is that results can be generated within minutes while isolating the user from hazardous samples. These factors favor development of this biosensor into a facile and rapid diagnostic device.     

SDZ ASM 981

black triangle SDZ ASM 981 is an anti-inflammatory macrolactam which binds with high affinity to macrophilin-12. The resulting complex inhibits calcineurin, thus blocking the synthesis of inflammatory cytokines. black triangle Twice daily application of topical SDZ ASM 981 1% cream was effective in the treatment of atopic dermatitis in adults and children in clinical trials. black triangle Summarised results from 260 patients with atopic dermatitis indicate that the efficacy of SDZ ASM 981 is dose dependent. The highest concentration evaluated (1% cream) was not as effective as betamethasone valerate 1% cream in this 3-week trial. black triangle The efficacy of SDZ ASM 981 and clobetasol ointments, used under occlusion, did not differ significantly in 10 patients with chronic psoriasis. Likewise, SDZ ASM 981 0.6% and betamethasone valerate 1% creams were similarly effective in 66 patients with allergic contact dermatitis. black triangle Concentrations of SDZ ASM 981 in the blood during topical treatment were invariably below 2.1 microg/L. black triangle Oral SDZ ASM 981 20mg or 30mg twice daily were effective in a dose dependent manner in the reduction of psoriasis in adults with no evidence of adverse effects. black triangle SDZ ASM 981 was well tolerated in the available trials, exhibiting no potential for systemic adverse reactions and no atrophogenic potential, a problem commonly associated with corticosteroid treatment.