A fiber optic nanoplasmonic biosensor for the sensitive detection of ampicillin and its analogs.

A novel optical immunosensor for the screening of ampicillin (Amp) residues has been developed. The biosensor is based on fiber optic particle plasmon resonance detection and uses an enhancement method called as fiber optic nanogold-linked immunosorbent assay (FONLISA) for the sensitive detection of antibiotics. A commercial antibody which had a higher affinity for ampicillin than for other ß-lactam antibiotics was chosen. A surface competitive binding assay was used in which a fixed concentration of antibiotic-conjugated gold nanoparticles (AuNPs) competes with free unlabeled antibiotic molecules to measure the amount of binding with antibody molecules immobilized on an optical fiber. The synthesis of the 11-mercaptoundecanoic acid (MUA)-ampicillin conjugate facilitates the attachment of the Amp molecules to AuNPs via MUA which acts as a linker between them. This AuNP-Amp conjugate was then used for the detection of ß-lactam antibiotics. The practical limit of detection obtained for Amp was 0.74 ppb (7.4 × 10-10 g/mL) which is lower than the recommended maximum residue limit (MRL) for ß-lactams. The method also shows a wide linear range of 4 orders. Its applicability to the determination of ampicillin in spiked milk samples has been demonstrated with good recovery and reproducibility. Graphical abstract.

Small molecule detection with aptamer based lateral flow assays: Applying aptamer-C-reactive protein cross-recognition for ampicillin detection.

Aptamer-based lateral flow assays (LFAs) are an emerging field of aptamer applications due to numerous potential applications. When compared to antibodies, potential advantages like cost effectiveness or lower batch to batch variations are evident. The development of LFAs for small molecules, however, is still challenging due to several reasons, primarily linked to target size and accessible interaction sites. In small molecule analysis, however, aptamers in many cases are preferable since immunogenicity is not required and they may exhibit even higher target selectivity. We report the first cross-recognition of a small molecule (ampicillin) and a protein (C-reactive protein), predicted by in-silico analysis, then experimentally confirmed - using two different aptamers. These features can be exploited for developing an aptamer-based LFA for label-free ampicillin detection, functioning also for analysis in milk extract. Most importantly, the principal setup denotes a novel, transferable and versatile general approach for detection of small molecules using competitive LFAs, unlikely to be generally realized by aptamer-DNA-binding otherwise.

Life-Threatening Atypical Case of Acute Generalized Exanthematous Pustulosis.

Antibiotics are known to cause severe cutaneous adverse reactions, such as the rare acute generalized exanthematous pustulosis (AGEP). Unlike Stevens-Johnson syndrome or toxic epidermal necrolysis, AGEP is rarely life-threatening. Systemic involvement is not typical, and if present usually coincides with a mild elevation of the hepatic enzymes and a decrease in renal function. Hence, AGEP is known to have a good prognosis and to be life-threatening only in elderly patients or patients with chronic diseases. Herein, we report a case of AGEP in a young healthy male leading to systemic inflammatory response syndrome and to treatment in an intensive care unit after being treated with 5 different antibiotics. Initial symptoms were not indicative for AGEP and the patient's course of disease led promptly to critical cardiorespiratory symptoms and systemic inflammatory response syndrome. We assume that the administration of the 5 different antibiotics resulted in type IV allergy as well as secondary infection with Enterococcus faecium and Staphylococcus aureus, while the underlying periodontitis also contributed to the severity of this case.

Use of antibody gene library for the isolation of specific single chain antibodies by ampicillin-antigen conjugates.

Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cell-surface display formats. We describe a new method which allows the selection of Escherichia coli cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (anti-fluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single chain antibody binding biotin by growing E. coli BL21 Star (DE3) containing the library in presence of a biotin-ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects, e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps.

A computerized protein-protein interaction modeling study of ampicillin antibody specificity in relation to biosensor development.

Nonspecific interactions between immobilized biomolecules and interfering proteins significantly impede biosensor development and commercialization. Advances in bioinformatics and computer technology have facilitated a greater understanding of biological interactions. We employed two different protein-protein docking programs to simulate the nonspecific interaction between ampicillin antibody and potential interfering proteins (human serum albumin and ovalbumin). To evaluate the contact and probability of association with the active site of the antibody, different amino acid chains from human serum albumin (HSA) and ovalbumin (OVA) were modeled in the simulation. In addition, a well-known specific immune complex, lysozyme and lysozyme antibody, was simulated for comparison. The results demonstrated that the cluster density of nonspecific interactions was smaller than the specific interaction between lysozyme and antibody, and that the dock scores were scattered. However, the active site of ampicillin antibody was prone to nonspecific protein interactions. The strength of interaction was different for specific binding and nonspecific binding. These results provide a platform for detecting the probability of nonspecific interactions and for improving methods of biosensor detection construction with reduced nonspecific adsorption.

The specificity of tests for anti-beta-lactam IgE antibodies declines progressively with increase of total serum IgE.

BACKGROUND: Immediate allergic reactions to beta-lactam antibiotics are mediated by specific IgE antibodies. The Phadia CAP System FEIA is a commercial method for quantification of specific IgE. We wished to determine anti-beta-lactam IgE antibodies in patients without penicillin allergy but with high levels of total IgE. METHODS: Sera from 41 patients (31 with high total IgE, 10 with low total IgE) were analyzed for IgE antibodies specific to penicilloyl G, penicilloyl V, amoxicilloyl and ampicilloyl using the CAP FEIA((R)) method that was available up to 2006. Seven sera that tested positive were rechecked in a new improved system available after 2006. RESULTS: In patients without a history of penicillin allergy, the specificities of commercial tests for anti-beta-lactam IgE antibodies were 100%, 60%, 27% and 20% at total IgE levels of 8-263 kU/l, 500-664 kU/l, 1000-2000 kU/l and > 2000 kU/l, respectively. In seven retested sera, only 2 (28%) were still positive for penicillin-specific IgE antibody. CONCLUSION: Before 2006, tests for anti-beta-lactam IgE antibody in patients with total IgE > 500 kU/l were probably often false positive. Patients who were diagnosed as penicillin allergic before 2006 solely on the basis of a positive CAP FEIA test for specific IgE should be considered for diagnostic reevaluation.

Effect of UV irradiation and magnetic field on immunometabolic effects of antibiotics immobilized in cell carriers.

The effects of UV and magnetic radiation on the immunometabolic activity of ampicillin and cephazolin immobilized in erythrocytic and leukocytic carriers were studied in intact Wistar rats and animals infected with staphylococci. Erythrocytic and leukocytic carriers with antibiotics were obtained. Injection of free antibiotics stimulated the immunosuppressive, pro-oxidant, and hepatotoxic effects, associated with staphylococcal infection. Treatment with antibiotics in erythrocytic and leukocytic carriers stimulated (to different degrees) the activity of the immune system and stabilized the parameters of LPO, antioxidant defense, cytolysis, and cholestasis. Ultraviolet irradiation and magnetic field modified (to different measures) the immunometabolic effects of ampicillin and cephazolin, immobilized in erythrocytic and leukocytic carriers, in animals with staphylococcal infection.

Skin testing and oral penicillin challenge in patients with a history of remote penicillin allergy.

BACKGROUND: Penicillin administration is usually contraindicated in penicillin-allergic patients with positive skin test results. OBJECTIVE: To examine whether penicillin oral challenge for patients with a history of remote non-life-threatening allergic reaction to penicillin can be well tolerated irrespective of skin test results. METHODS: In a prospective open-label trial, 8,702 individuals were screened between November 1998 and January 2000. Of 687 patients with a non-life-threatening allergic reaction to penicillin, occurring longer than 3 years earlier, 169 were enrolled. Regardless of the response to penicillin skin testing, patients received the usual 1-day dosage of penicillin and amoxicillin, on 2 separate occasions. Two to 6 years later, a follow-up was conducted to assess the outcomes of further penicillin administration. RESULTS: A total of 272 combined skin tests and oral challenges were performed on 169 patients. Among 137 challenges with a positive skin test result and 135 patients with a negative skin test result, 9 (6.6%) and 5 (3.7%) (P = .29), respectively, developed a mild rash to oral challenge. At follow-up, 2 to 6 years afterward, 3 of 55 patients (5.5%) who were given a full treatment course of penicillin developed a mild skin eruption. CONCLUSIONS: Positive penicillin skin test results for patients with a remote history of non-life-threatening allergic reaction to penicillin were not associated with a greater prevalence of adverse reactions to oral challenge with penicillin than negative results. Because skin testing is considered the gold standard and the safest method for predicting tolerance to penicillin administration, oral penicillin challenge may be used as a diagnostic method only in these specific patients when skin testing is not feasible.

[Enzyme immunoassay of ampicillin in milk].

An indirect immunoassay for quantitative determination of ampicillin (range, 10-1000 ng/ml) in buffer or milk has been developed. Polyclonal antibodies were obtained against ampicillin conjugated with bovine serum albumin; the conjugate was synthesized by direct condensation using carbodiimide. The antibodies were specific for ampicillin and exhibited low cross-reactivity to other penicillins (azlocillin, 17%; penicillin G, 10%; piperacillin, 5%; and carbenicillin, 4%). Matrix effects were minimized by combining the use of a casein-supplemented buffer (content of casein, 1%) with sample dilution. The threshold of ampicillin detection in milk (diluted tenfold) was equal to 5.0 ng/ml (which corresponded to 50 ng/ml of the original sample).

[Studies of the specificity of immediate allergic reactions to ampicillin]. / Badania nad swoistoscia natychmiastowych reakcji alergicznych wywolywanych przez ampicyline.

Ampicillin belongs to the drugs causing most frequently IgE-dependent allergic reactions, but the current specificity of these reactions is poorly known. Some experimental data suggest that the side chain of ampicillin may induce synthesis of drug-specific IgE antibodies. In our study we have decided to explain the specificity of ampicillin-induced allergic reactions type I appearing in patients after drug administration. Thirty-eight subjects developing an immediate response after administration of ampicillin entered the study. In all the patients skin tests with penicillin, ampicillin and amoxicillin determinants were performed. We have observed positive skin tests results with penicillin determinants in 23 subjects, in 32 subjects with ampicillin and in 17 cases with amoxicillin. Only in three persons the results were positive exclusively to ampicillin. The results of our study indicate that in the Polish population ampicillin induces an immediate IgE response with variable degree of cross-reactivity to other tested drugs, and selective response to ampicillin is uncommon.

The novel synthetic immune response modifier R-848 (Resiquimod) shifts human allergen-specific CD4+ TH2 lymphocytes into IFN-gamma-producing cells.

BACKGROUND: In experimental models, imidazoquinolines exhibit several immunomodulatory activities via Toll-like receptor signaling on cells of the innate immunity. OBJECTIVE: The aim of our study was to investigate whether R-848 (Resiquimod), a small-molecular-weight synthetic compound belonging to the imidazoquinoline family and known for its ability to substantially delay the onset of recurrent genital herpes lesions in both animals and human beings, could influence, at least in vitro, the cytokine production profile of human hapten- or allergen-specific T cells. METHODS: Ampicillin- and Der p 1-specific T-cell lines were derived from peripheral blood of allergic donors in the absence or presence of R-848 and assessed by flow cytometry at the single-cell level for their ability to produce IL-4 and/or IFN-gamma. RESULTS: R-848 induced both hapten- and allergen-specific circulating T cells, including T(H)2 effectors, to produce IFN-gamma and even to lose the ability to produce IL-4, thus shifting their phenotype of cytokine production to a type 0 (T(H)0) or even T(H)1 profile. This effect was associated with an increase in the production of IL-12, IFN-alpha, IL-18, TNF-alpha, IL-10, and IL-15 by CD14(+) cells, as well as an increase in the proportions of IFN-gamma-producing CD3(-)CD16(+) (natural killer) cells. CONCLUSIONS: These results suggest that R-848, and probably other imidazoquinolines, might be used as adjuvants in view of novel allergen-specific immunotherapeutic regimens for the treatment of allergic disorders.

[Studies on cross reactivity to penicillins in patients with immediate allergic reactions caused by amoxicillin]. / Badania nad krzyzowa nadwrazliwoscia na antybiotyki penicylinowe u chorych z natychmiastowymi reakcjami alergicznymi wywolywanymi przez amoksycyline.

In this study we have explored the presence of cross reactivity to penicillin and ampicillin in patients with immediate allergic reactions caused by amoxicillin. Skin test results with amoxicillin were positive in 66.66% and in the remaining 33.34% were negative. In 81.48% patients we observed positive results of skin tests with penicillin and/or ampicillin. Only in 14.48% of the patients we observed selective response to amoxicillin. On the basis of obtained results we conclude that sensitivity of skin tests with amoxicillin is rather moderate and confirmation of amoxicillin hypersensitivity can be obtained using skin tests with other penicillins. In our material selective amoxicillin hypersensitivity can be defined as a relatively rare phenomenon. These data should have the important implications in antibiotics' selection in patients with amoxicillin allergy.