Search

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 β-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 β-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 β-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.

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.

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., (© 2017 S. Karger AG, Basel.)

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

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.

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.

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.

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).

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.

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.

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.

Single-chain Fv (scFv) antibody libraries were constructed from mice immunized with an ampicillin-bovine serum albumin conjugate. Several antibodies with specificity for intact ampicillin were selected by phage display and characterized. The antibody scFv fragment aL2 binds to intact ampicillin and shows no detectable cross-reactivity with hydrolyzed ampicillin. We determined the X-ray structures of two crystal forms of w.t. aL2, which differ mainly in the side-chain conformation of Trp H109 (according to a new consensus nomenclature Kabat residue number H95) in the extremely short (three residues) CDR H3 and the presence or absence of a well-resolved molecule of 2-methyl-pentane-2,4-diol in the bottom of the binding pocket. Attempts to co-crystallize aL2 with its antigen or to diffuse ampicillin into the wild-type aL2 crystals were unsuccessful, since crystal contacts obstruct the binding pocket. However, a mutant with two point mutations near the N terminus (Gln H6 replaced by Glu and Ala H10 (Kabat H9) replaced by Gly) crystallized in a form compatible with antigen-binding. Although the mutations affect the conformation of framework I, the conformations of the binding pocket of the uncomplexed wild-type aL2 and of the mutant complex were almost identical. The structure explains the specificity of the antibody for intact ampicillin and the degree of cross-reactivity of aL2 with a wide variety of ampicillin analogs. This antibody system will be very useful as a diagnostic reagent for antibiotics use and abuse, as a model for the effect of expression of antibiotic binding molecules in Escherichia coli, and for directed evolution towards high antibiotic resistance., (Copyright 2001 Academic Press.)

The N-terminal segment (FR-H1) of the heavy chain (V(H)) of antibodies shows significant conformational variability correlating with the nature of the amino acids H6, H7 and H10 (Kabat H9). In this study, we have established a causal relationship between the local sequence and the structure of this framework region and linked this relationship to important biophysical properties such as affinity, folding yield and stability. We have generated six mutants of the scFv fragment aL2, covering some of the most abundant amino acid combinations in positions H6, H7 and H10 (according to a new consensus nomenclature, Kabat H9). For the aL2 wild-type (w.t.) with the sequence 6(Q)7(P)10(A) and for two of the mutants, the X-ray structures have been determined. The structure of the triple mutant aL2-6(E)7(S)10(G) shows the FR-H1 backbone conformations predicted for this amino acid combination, which is distinctly different from the structure of the w.t, thus supporting our hypothesis that these residues determine the conformation of this segment. The mutant aL2-6(E)7(P)10(G) represents a residue combination not occurring in natural antibody sequences. It shows a completely different, unique structure in the first beta-strand of V(H), not observed in natural Fv fragments and forms a novel type of diabody. Two V(H) domains of the mutant associate by swapping the first beta-strand. Concentration-dependent changes in Trp fluorescence indicate that this dimerization also occurs in solution. The mutations in amino acids H6, H7 and H10 (Kabat H9) influence the dimerization behavior of the scFv and its thermodynamic stability. All the observations reported here have practical implications for the cloning of Fv fragments with degenerate primers, as well as for the design of new antibodies by CDR grafting or synthetic libraries., (Copyright 2001 Academic Press.)

The effects of antibiotics on the antigen-specific humoral immune response are not known. Macrolides, tetracyclines, and beta-lactams are commonly prescribed antibiotics. The first two are known to have immunomodulatory activities. The effects of clarithromycin, doxycycline, and ampicillin on the primary and secondary antibody responses to tetanus toxoid, a pneumococcal polysaccharide vaccine, a hepatitis B virus surface antigen (HBsAg) vaccine, and live attenuated Salmonella typhi (Ty21a) were investigated using a mouse model. For the mice receiving the tetanus toxoid, the immunoglobulin M (IgM) level of the clarithromycin group at day 7 was significantly lower than the corresponding antibody level of the normal saline (NS) group. For the mice receiving the pneumococcal polysaccharide vaccine, the total antibody and IgM levels of the clarithromycin group and the IgM level of the doxycycline group at day 7 were significantly lower than the corresponding antibody levels of the ampicillin and NS groups. For the mice receiving the HBsAg vaccine, the IgM level of the doxycycline group at day 7 was significantly lower than the corresponding antibody levels of the clarithromycin and NS groups, while the IgM level of the clarithromycin group at day 28 was significantly lower than the corresponding antibody levels of the doxycycline, ampicillin, and NS groups. For the mice receiving all three vaccines, there were no statistically significant differences between any of the antibody levels of the ampicillin group and the corresponding antibody levels of the NS group. For the mice receiving Ty21a, the total antibody levels of the ampicillin group at days 7 and 21 were significantly higher than the corresponding antibody levels of the NS group. Moreover, the IgM levels of the clarithromycin, doxycycline, and ampicillin groups at days 7 and 21 were significantly higher than the corresponding antibody levels of the NS group. Furthermore, the total antibody level of the ampicillin group at day 21 was significantly higher than the corresponding antibody level of the doxycycline group. For all four vaccines, there were no statistically significant differences among the serum levels of interleukin-10 and gamma interferon for the mice treated with the various antibiotics. We conclude that clarithromycin and doxycycline, but not ampicillin, suppress the antibody responses of mice to T-cell-dependent and T-cell-independent antigens, whereas all three antibiotics enhance the antibody response to live attenuated mucosal bacterial vaccines.

Background: Subjects with IgE-mediated allergic reactions to penicillins can develop urticaria or anaphylactic shock. Urticaria is mainly associated with positivity to the major determinant of benzylpenicillin (BPO), and anaphylactic shock with minor determinants (MDM). The presence of IgG antibodies to BPO is thought to be mainly associated with urticaria, possibly protecting from anaphylactic shock. We aimed to study the skin test response to BPO and MDM, amoxicillin (AX), and ampicillin (AMP) in a group of subjects allergic to penicillins, and to evaluate the role of specific IgG., Methods: We studied a group of patients with immediate allergic reactions to penicillins, comparing urticaria and anaphylactic shock. Skin tests were done with BPO, MDM, AX, and AMP. Specific IgE and IgG antibodies to benzylpenicilloyl-poly-L-lysine (BPO-PLL) and amoxicilloyl-poly-L-lysine (AXO-PLL) were determined by RAST and ELISA, respectively., Results: Fifty-nine patients were studied (30 with anaphylactic shock and 29 with urticaria). Skin test positivity to BPO was associated with urticaria (P<0.001), and positivity to MDM, AX, and AMP with anaphylactic shock (P=0.006, P<0.001, and P=0.002, respectively). Specific anti-BPO-PLL and AXO-PLL IgG values were higher in patients than controls (P<0.001), but no differences were observed between urticaria and anaphylactic shock., Conclusions: Positivity to minor determinants of penicillins is associated more with anaphylactic shock than urticaria, but the role of IgG antibodies in helping to prevent the development of anaphylactic shock could not be confirmed.

Background: Literature reports dealing with cell-mediated allergy to betalactams have appeared with increasing frequency in the last years., Objective: To evaluate patients with such reactions and to identify cross-reactivities among betalactams in order to provide safe guidelines for their further clinical management., Methods: Thirty consecutive subjects with cell-mediated allergy to betalactams (history of adverse reactions to these antibiotics; serum total IgE within the normal range; absence of serum specific IgE antibodies to penicillin G and V, amoxicillin, and ampicillin; negative skin tests with a wide pattern of betalactam preparations; and positive patch-test to at least one betalactam antigenic determinant) were investigated. The subjects admitted to the study were patch tested with a wide variety of betalactam preparations in order to identify alternative molecules tolerated by the patient. To better evaluate the cross-reactivity pattern, tolerance challenges with patch-negative betalactams were also performed in each subject., Results: Both specific IgE and skin tests were negative in all patients. The skin biopsies performed on the positive patch-tested area in four patients showed a clear T-lymphocyte, CD4+-type infiltrate, thus definitely proving the occurrence of a cell-mediated response. A total of 44 adverse reactions (mean: 1.47 episodes for each patient) were reported in history, with a mean interval of 15 hours after betalactam administration. The reported symptoms were mainly cutaneous (maculo-papular rash and urticaria) and the responsible drugs were chiefly aminopenicillins (86.4% of cases) and penicillin G (9.1%). We were able to identify three separate groups of patients on the basis of clinical history, patch-test, and tolerance challenge pattern: allergy to the side chain of aminopenicillins in 16 patients (53.3%); allergy to the thiazolidine ring in 3 patients (10.0%); undetermined specificity in the remainder 11 patients (36.7%). Cross-reactivity among different betalactam molecules (revealed by positive tolerance tests performed with patch-negative betalactams) was found in 4.8% of cases only (23.3% of all investigated patients). This fact demonstrates a very high (95.2%) predictive value of a negative patch-test in excluding the occurrence of a cross-reactivity. The mis-match between patch and tolerance tests was observed in 3 out of 178 cases only (1.7% of cases, 10.5% of patients) in groups A and B, and in as much as 12.2% of cases (45.5% of subjects) in group C (P < .05)., Conclusions: Delayed allergy to betalactams (mainly to aminopenicillins) may be exerted by a cell-mediated response. Patch tests and tolerance challenges are extremely useful and safe for diagnosis and further clinical treatment of these patients, helping to identify safe alternative betalactam molecules that could be successfully tolerated by the allergic subjects.

Monoclonal antibodies (Mab) against ampicillin were prepared by immunization of mice with an ampicillin-keyhole limpet hemocyanin conjugate coupled by a glutaraldehyde method. Sensitivity and specificity of these antibodies were tested in a direct competitive enzyme immunoassay, in which an ampicillin-horseradish peroxidase conjugate prepared by a carbodiimide method served as the labelled antigen. According to their cross-reactivities with the other beta-lactam antibiotics, the Mabs could be divided into two groups, which are represented by the clones designated 1D1 and 3B5. While Mab 3B5 (IgG1) showed no major cross-reactions with the other penicillins frequently used in veterinary medicine except for amoxicillin (108%), Mab 1D1 (IgG2a) had marked cross-reactivities with most of the 17 tested beta-lactam antibiotics (e.g., amoxicillin 187%, penicillin G 31%, cloxacillin 30%, dicloxacillin 44%, and oxacillin 14%). The detection limits for ampicillin, calculated from the antibiotic concentration giving 30% binding inhibition, were 11.7 (Mab 3B5) and 16.6 ng ml-1 (Mab 1D1). To prepare multi-immunoaffinity chromatography columns, Mab 1D1 and a previously described antibody against cloxacillin (Mab 1F7) were each coupled to CNBr activated sepharose. The capacity of the resulting immunosorbents was approximately 6.6 and 5.4 micrograms ml-1 gel for ampicillin and cloxacillin, respectively. Recoveries of amoxicillin, ampicillin, cloxacillin, dicloxacillin, penicillin G and oxacillin (in buffer solutions) from the produced immunoaffinity columns were in the range from 67 to 100%.

Aim: To study whether or not the freshly prepared benzylpenicillin could induce different non-IgE antibody response from aged benzylpenicillin., Methods: Antibody response was determined by enzyme-linked immunosorbent assay (ELISA). Antigen molecules recognized by antibodies and antigenic cross reactions were tested by hapten inhibition assay., Results: Isotypes of specific non-IgE antibodies induced by freshly prepared benzylpenicillin were mainly IgM, and then IgG and IgA. Some parts of specific antibodies recognized benzylpenicillin molecule and major parts combined with degraded or transforming products. Isotypes of antibodies responsible for cross reaction were mainly IgG between benzylpenicillin and ampicillin and IgM between benzylpenicillin and piperacillin., Conclusion: Freshly prepared and aged benzylpenicillin induced different non-IgE antibody response.

Background: Ampicillin (AMP) is a drug that has been prescribed extensively. Reactions that have been reported include exanthema, desquamative contact eczema, urticaria and anaphylaxis. Experimental evidence indicates that the side chain of AMP is a structure that may induce a selective immune response either at the humoral or lymphocyte T-cell level. With regard to IgE reactions, the selectivity and specificity of the response needs to be studied in humans., Objectives: To study the specificity of the IgE response in a group of subjects who had an immediate allergic reaction after the administration of AMP., Methods: Subjects developing an immediate response (anaphylaxis or urticaria) after the administration of AMP or an aminopenicillin derivative with the same side chain as AMP were studied. Skin tests were made to determinants generated from benzyl penicillin (BP): benzyl penicilloyl (BPO) and minor determinant mixture (MDM), as well as amoxicillin (AX) and AMP. Specific IgE antibodies were determined to benzyl penicilloyl polylisine (BPO-PLL), amoxicilloyl-polylisine (AX-PLL) and ampicilloyl-polylisine (AMP-PLL). The specificity of the IgE antibody response was studied by RAST and RAST inhibition. Subjects were classified in three categories: group A: those who were skin test and/or RAST positive to determinants derived from benzylpenicillin, group B: those who were negative to determinants derived from benzylpenicillin but were skin test and/or RAST positive to determinants derived from AX and AMP and group C: those who were exclusively positive to determinants derived from AMP., Results: A total of 48 subjects was included in the study. In group A there were 35 cases, in group B 10 cases, and in group C three cases. RAST inhibition studies showed that in some instances the side chain of AMP could induce specific responses with a variable degree of crossreactivity between BP and AX., Conclusions: Although AMP can induce an immediate IgE response in subjects allergic to betalactams and the structure of the side chain may contribute to the specificity of the response, our results indicate that in most instances crossreactivity with the other penicillins exists and that in the groups studied selective reactions to just AMP derived determinants were uncommon.

The purpose of this study was to determine if the sustained release of ampicillin from a biodegradable drug-delivery system (microencapsulated ampicillin anhydrate (MEAA)) will increase or decrease the intensity of a hypersensitivity reaction compared with that observed with free drug. Ovalbumin, which is known to elicit a marked hypersensitivity reaction in guinea pigs, and microencapsulated ovalbumin (MOVA) were tested in parallel with ampicillin and MEAA. Guinea pigs were sensitized biweekly by subcutaneous and intramuscular injections of ampicillin, MEAA, ovalbumin, MOVA or placebo microspheres (test articles), each mixed with Freund's adjuvant, and challenged 2 weeks later, intradermally, with the free compounds. In a separate set of experiments, guinea pigs were sensitized by implantation of the same agents in the caudal thigh of anaesthetized animals. Skin allergic reactions were tested at 1 and 3 weeks following local implantation of the test articles. Sera of sensitized guinea pigs were tested for specific IgG antibodies by enzyme-linked immunosorbent assay, and skin samples from the site of the inflammatory reaction were fixed, stained and evaluated histologically. Guinea pigs sensitized systemically with MEAA or MOVA showed smaller, but not statistically different skin allergic response than animals given corresponding free compounds. However, guinea pigs sensitized by local implantation of MEAA showed a significantly lower inflammatory response (P < 0.0001) than those given an equivalent dose of the free drug. Guinea pigs sensitized with placebo microspheres showed a low inflammatory skin reaction which was similar to those sensitized with all doses of MEAA. There was no significant difference in specific IgG antibody response in the sera of guinea pigs sensitized locally with either free or microencapsulated ampicillin or ovalbumin. Histology of skin revealed a milder inflammatory reaction with MEAA or MOVA than with ampicillin or ovalbumin, respectively. We conclude that the encapsulated ampicillin or ovalbumin and subsequent release of each agent will elicit a reduced hypersensitivity reaction in guinea pigs than will the free agent.

Background: The cross-reactivity between penicillins and cephalosporins can be influenced by different factors, which are not all well known. The chemical structure of the side chain may contribute to the cross-reactivity., Objective: The study was carried out in allergic subjects who are selectively responsive to amoxicillin to determine allergenic cross-reactivity with a cephalosporin containing a side chain identical to that of amoxicillin, cefadroxil, and one containing a different side chain, cefamandole., Methods: Allergic subjects with a selective response to amoxicillin were chosen according to the following criteria: history of an immediate allergic reaction to amoxicillin, negative skin test responses to benzylpenicilloyl and minor determinant mixture of benzylpenicillin, negative RAST response to benzylpenicilloyl, and good tolerance to benzylpenicillin and phenoxymethyl penicillin challenges. In addition, subjects had to have a positive skin test response to amoxicillin and/or positive RAST response to amoxicilloyl or, if these test results were negative, a positive challenge test response to amoxicillin. In vivo cross-reactivity to cefadroxil was assessed by giving oral cefadroxil at increasing doses from 5 to 500 mg. In vitro cross-reactivity was determined by RAST inhibition studies with amoxicilloyl RAST disks and the following monomeric conjugates in the fluid phase: amoxicillin-butylamine, cefadroxil-butylamine, and the side chain para-hydroxy-phenylglycine. Tolerance to cefamandole was determined by giving 100 mg and then 500 mg parenterally., Results: Twenty-one patients with a selective response to amoxicillin were included in the study. Eight subjects (38%) had a positive response to cefadroxil, and none reacted to cefamandole. In vitro RAST inhibition studies indicated that cefadroxil-butylamine monomers cross-reacted with amoxicillin butylamine and the side chain contributed relevantly to the inhibition., Conclusions: These results indicate that the percentage of cross-reactivity between penicillins and cephalosporins with an identical side chain is high and that this critical part of the molecule seems to be an important contributor to these results. The value is higher than previously reported data from similar studies of non-side-chain-related cephalosporins.

Background: There are instances where individuals may come into contact with penicillins without being aware of it. This non-therapeutic exposure from different sources may cause sensitization and even clinical manifestations in subjects allergic to penicillins., Objective: To determine the capacity that inadvertent contact with penicillins may have in inducing resensitization and symptoms in patients diagnosed as allergic to penicillins who were followed over a long period of time after their initial evaluation., Methods: A follow-up study of penicillin-allergic subjects who agreed to be regularly tested for in vitro and in vivo control of their sensitivity. Skin tests were carried out with major and minor determinants of benzylpenicillin (BPO and MDM), amoxicillin (AX), and ampicillin (AMP), and specific IgE antibodies were determined by radioallergosorbent test (RAST). A questionnaire was sent to and answered by the subjects in order to see if they experienced symptoms at any time during the follow-up period. In addition, if any unexplained symptoms occurred, a bleeper system was used to contact the allergy centre., Results: Seven subjects experienced anaphylactic reactions with no obvious cause. At the time of their initial allergic reaction, which was caused by exposure to prescribed penicillin, the subjects had one or more positive skin tests and/or RAST results to penicillin related reagents. However, over the following 2-4 years all their tests became negative. After reporting their unexplained reaction all seven had one or more positive skin tests and/or RAST results again and when retested 1 week later RAST measurements showed that levels of penicillin-specific IgE were maintained or increased. None of the subjects had knowingly received penicillin but the questionnaire showed that six had been exposed to it and in the seventh case exposure was likely. In two cases contact was by sexual intercourse with a partner who was receiving penicillin, three subjects had handled penicillin formulations and one had drunk from a glass previously used for giving penicillin. In the seventh case exposure could have occurred whilst in hospital for surgery, although this was not proven., Conclusions: these results show that non-therapeutic exposure to penicillin can cause severe symptoms and that in vitro and in vivo testing can help in the diagnosis of such cases.

Pharmacological or immunological activation of human basophil induces the expression of membrane markers such as CD63 and the release of histamine and leukotriene C4 (LTC4). LTC4 release test has the advantages of a cellular test which is obviously closer to the physio-pathological reaction than the direct determination of circulating antibodies. Moreover, the determination of a newly formed mediator eliminates the spontaneous release of mediator by simple cellular damage. We present here, after having controlled the reliability of the method for aero-allergens, preliminary results of LTC4 release obtained for haptens (ampicillin, methylprednisolone and acetylsalicylic acid) which are in favour of the use of this method in the complex field of drug allergy diagnosis.

Ampicillin and benzylpenicillin conjugated to human serum albumin were used as immunogens in order to obtain antihaptenic IgG responses in outbred guinea pigs according to different schedules, all involving complete Freund's adjuvant. The individual responses were characterized by ELISA and by ELISA inhibition using ampicillin, benzylpenicillin, and carbenicillin peptidic conjugates for coating and for inhibition. In several instances, drastically reduced cross-reactivity and even its absence were observed, although the penicillin antigens differ only in the side-chain. The notion that the invariantly present thiazolidine ring will always provide significant binding to antibodies against all penicillins differing only in the side-chain has to be dropped. The experiments were performed in relation to newer findings of clinical penicillin-allergy skin testing which suggest that benzylpenicillin-based reagents alone are not able to detect or predict all reactions against semisynthetic penicillins. The experimental evidence here obtained corroborates this conclusion.

The diagnosis of drug-induced hepatitis is usually based on clinical criteria, with emphasis on both the temporal relationship between drug intake and liver injury and the exclusion of alternative causes. In vitro tests of lymphocyte sensitization to drugs are considered to have a low sensitivity. We investigated the possibility of detecting lymphocyte reactivity to drugs in drug-induced hepatitis by analyzing the lymphocyte proliferative responses to ex-vivo drug or metabolite antigens to improve the sensitivity of the in vitro test. Lymphocyte proliferative responses to five different concentrations of the drug and to ex-vivo drug antigens (serum collected from normal subjects after the ingestion of the drugs) were analyzed in 25 patients with a clinical diagnosis of drug-induced hepatitis, 27 healthy subjects and 10 individuals with a recent exposure to the same drugs without development of adverse drug reactions. In seven of the 25 patients, lymphocyte reactivity to drugs was detected (28%). The use of sera collected from healthy volunteers after drug intake (ex-vivo drug antigens) and the addition of a prostaglandin inhibitor to the cultures allowed the detection of lymphocyte sensitization in seven additional cases, increasing the detection ability from 28% to 56%. We suggest that the use of ex-vivo drug antigens may represent a significant contribution to the identification of the drug involved in cases of drug-induced hepatitis.

Two cell lines producing monoclonal antibodies, Abp4 (IgM) and Abp7 (IgG1) against ampicillin were established. The epitopes and the cross-reactions of the antibodies with various beta-lactams were examined by enzyme-linked immunosorbent assay (ELISA) and ELISA inhibition test. Abp4 showed broad cross-reaction to human serum albumin (HSA) conjugates of several beta-lactams, 6-aminopenicillanic acid and 7-aminocephalosporanic acid. Abp7 reacted only with ampicillin and cephalexin, which have the same acyl side chain. In ELISA inhibition tests, Abp4 inhibited binding to ampicillin-HSA by benzylpenicilloyl-epsilon-amino-n-caproic acid, and strongly inhibited the binding by penicillamine. Abp7 strongly inhibited the reaction by aminobenzylpenicilloyl-epsilon-amino-n-caproic acid, but benzylpenicilloyl-epsilon-amino-n-caproic acid was less effected. These data suggest that Abp4 recognizes the thiazolidine ring and Abp7 recognizes the acyl side chain. Therefore, the thiazolidine ring-epitope acts in broad cross-reaction among beta-lactams, and the acyl side chain acts only in the cross-reactivity between penam and cephem, which have a similar acyl side chain.

The majority of the 78 enteropathogenic (EPEC) and the 151 non-EPEC Escherichia coli strains isolated from preterm neonates during an outbreak of gastroenteritis in a hospital in Nairobi, Kenya, were resistant to trimethoprim-sulfamethoxaxole, chloramphenicol, oxytetracycline and ampicillin, but only a few strains were resistant to cefazolin, cefamandole, cefotaxime, amikacin and nalidixic acid. Fourteen different antimicrobial resistance patterns were observed in the 229 strains of E. coli analysed. Eighty-two percent of the EPEC strains belonged to two resistance pattern compared with 79% of non-EPEC strains which exhibited three resistance patterns. There was no consistent relationship between plasmid profile group and antimicrobial resistance pattern, although one resistance pattern was more frequently observed in EAF-positive strains belonging to the dominant plasmid profile group. Nine percent of the EPEC strains were resistant to gentamicin compared to 37% in the non-EPEC group. No correlation was observed between administration of gentamicin and percentage of resistant strains isolated. None of the nine neonates receiving gentamicin died during the outbreak. Gentamicin resistance was observed in E. coli strains from six out of these nine neonates. Five out of fourteen neonates who received other antimicrobials, or no antibiotic treatment at all, died.

A patient with hypocomplementemic urticarial vasculitic syndrome (HUV) is presented. This is an immunological pathology, limited to skin or multisystemic, that requires a differential diagnosis with erythematosus systemic lupus on the same occasions. The ever-present symptom is skin participation, such as urticaria-angioedema or fixed exanthema; biopsy shows necrotizing venulitis with polymorphonuclear infiltration and leukocytoclastic powder. Typical laboratory data are: diminished C3, C4 and C1q; C1 inhibition can be low or normal; the more characteristic finding is the presence of C1q associated immunocomplexes. Leukocytoclastic necrotizing vasculitis was found in the skin biopsy. During the course of illness (three years) the patient presented moderate cutaneous symptoms and asthma, without other systemic participation. During this period, antihistamines and, occasionally, corticoids were administered with improvement. Moreover, the patient presented urticaria related to ampicillin ingestion, and furthermore, the presence of anaphylaxis to beta-lactam was diagnosed in vivo and specific IgE was found in the laboratory study. This feature was previously observed by other authors; however, we cannot determine why the IgE-mediated allergy to beta-lactam and a complement pathology like HUV are related.

The delayed-type hypersensitivity (DTH) reactions for penams or cephems of beta-lactam antibiotics were investigated by intradermal skin test and leucocyte migration test (LMT) in guinea pigs. The animals were immunized with ampicillin (ABPC) or cephalexin (CEX) using Freund's complete adjuvant. The cross-reactivities among ABPC, penicillin G (PCG) and cloxacillin as penam and CEX, cephalothin (CET) and cephalosporin C (CEPC) as cephem and phenylglycine (PhGly), which is the amino acyl side chain of ABPC and CEX, were examined. By intradermal reaction, ABPC-sensitized animals showed a cross-reaction with CEX, PCG and CET, but CEX-sensitized animals did not cause cross-reaction with ABPC. The CEX-sensitized group exhibited slight cross-reactions to CET and PhGly. PhGly exhibited low immunogenicity only in maximization test of guinea pig. The above results indicate that there is the difference in cross-reactivity between penams and cephems in skin test. In LMT, all the ABPC-sensitized animals reacted with ABPC and showed cross-reactions with all drugs tested. The CEX-sensitized group reacted with 4 out of 7 animals with CEX and exhibited cross-reactivities to ABPC, PCG, CET, CEPC and PhGly. The cross-reactivity between intradermal skin reaction and LMT elicited some different results.

A patient with ampicillin-induced toxic epidermal necrolysis (TEN) showed pronounced delayed hypersensitivity to ampicillin sodium by both intradermal and patch tests. In addition, a positive lymphocyte transformation test to ampicillin was demonstrated by an in vitro study. On the basis of these findings and the data found in the literature, we believe that delayed hypersensitivity plays a crucial role in the development of drug-induced TEN. We recommend patch testing with suspected medication in such cases.

The immunogenicity of four penicillin preparations was comparatively investigated by titration of penicilloyl (PO) specific hemagglutinating antibodies found in sera of rabbits inoculated with equimolecular amounts of these penicillins incorporated in Freund incomplete adjuvant. Benzylpenicillin (BP) was shown to be endowed with the highest immunogenicity, while oxacillin (OX), ampicillin (AM) and Amoxil (AX) were found significantly less immunogenic. By examining comparatively the titers of hemagglutination reactions given by the sera of each animal with erythrocytes coated with the penicillin used for its immunization as well as with the other penicillins investigated, important cross immunological reactions were found in all of the cases. Interestingly, the cross reactions produced by AM- and AX-specific antisera with heterologous penicillins were apparently higher than the reactions obtained with the homologous penicillin. No significant differences were found concerning the ability of the four penicillins to form PO conjugates with BGG. Thus, the differences in immunogenicity exhibited by the four penicillin preparations may not be explained in this way.

Antibody responses to the penicilloyl moiety were recorded in patients treated intramuscularly with ampicillin for respiratory infections. For practical reasons this was performed at two clinics. At each clinic, two commercially available ampicillin preparations were used in a single-blind, randomised study. One of the preparations at each clinic was of high-grade purity and the other was slightly contaminated, according to a radioimmunoassay. There was the same heterogeneity in the patient material between the two clinics. The results showed a slight but significant (p less than 0.001) increase in IgM penicilloyl antibodies in the group of patients treated with the pure penicillin but not in those treated with the contaminated penicillin. When the patients in the two studies were analysed jointly, the increase in IgM antibodies was significantly greater (p less than 0.05) in the group treated with pure penicillin than in that treated with impure penicillin. The difference in antibody formation between the patient groups treated with pure or contamined ampicillin at each clinic was not significant, however. An immunological activity of the impurities suppressing the IgM antibody response is suggested.

The dose-response relationship between the frequencies or concentration of exposure to powdered drug allergens and drug induced allergic onsets was studied in a pharmaceutical plant for 15 years from 1974 to 1984. The subjects were 41 male workers and the target allergens were two kinds of anti-inflammatory enzymes (Bromelain and Trypsin) and three kinds of antibiotics (Ampicillin, Amoxycillin and Cephalexin). The allergic onsets were confirmed by periodic allergological examinations and occasional clinical findings. Statistical analysis was made by the person-year method. The results showed that in workers who had any allergic history, the incidence rates of allergic onsets increased with elevation in the frequencies or concentration of exposure to these allergens, while in the cases without such history, the incidence rates increased only in those with high frequencies of exposure to the allergens. The findings suggest that the incidence rates of occupational drug allergy were dependent on the frequencies and concentration of exposure to allergens.

A case presentation describes a young woman with a history of two reactions to ampicillin therapy and a reproducible skin test reaction of intermediate timing that disappeared within 48 hours. The skin test response was to ampicillin only and not to other penicillin-related skin test reagents. Tests for serum IgE and IgG antibody to ampicillin were negative. The histology was that of a mononuclear and neutrophilic cellular infiltrate with neutrophil margination in the vessels. There was no immunoglobulin, complement, or fibrin deposition. The skin test reaction began and ended earlier than would be expected for a classic delayed hypersensitivity reaction. It is considered to be an isolated late cutaneous response but cannot yet be designated a late cutaneous allergic response. Reactants characteristic of an Arthus reaction were not present, and no alternative immunologic basis was confirmed.

A new method for the preparation of ampicillin-BSA conjugate by a three step procedure was developed. The first step is the introduction of a maleimide residue to ampicillin by a cross-linking reagent, MBS. The second step is reductive cleavage of disulfide bonds in BSA. The third step is thioether formation between the introduced maleimide residues and the reduced thiol groups. The obtained ampicillin-BSA conjugated raised an anti-ampicillin serum in rabbits. A new reagent, MPGS, was used for enzyme labelling of ampicillin to avoid immunological cross reaction. Using the enzyme labelled ampicillin and anti-ampicillin serum, enzyme immunoassay of ampicillin was successful in detecting 4 ng to 1 mug. Cross reactivities of anti-ampicillin to ampicillin analogs were studied by the enzyme immunoassay to find that the antiserum is specific to penicillin especially to ampicillin but hardly reacts with cephalosporins or the penicilloic acid derivative of ampicillin.

In 90 patients with clinical symptoms of penicillin allergy, the onset of the skin reaction was related to the type of penicillin administered (small- or broad-spectrum) and to the type of immune reactivity. Early onset reactions (urticaria, Quincke's edema, Arthus-reactions) were frequently seen after the use of benzylpenicillin, while late onset reactions, mostly macular papular rashes, were mainly seen after the use of aminopenicillins. The clinical symptoms of penicillin hypersensitivity were not related to one single type of immunological (cellular or humoral) reactivity.

BALB/C mice were immunized with conjugate of benzylpenicillin or ampicillin with Ascaris suum extract. The mice developed IgE antibodies to penicillin, which were found to react with commercially available penicillin preparations in the PCA system. Elimination of polymerized penicillin by Sephadex chromatography from tested preparations could not diminish their activities to elicit PCA. High pressure liquid chromatographic (HPLC) separation of PcG preparation yielded fractions unable to elicit PCA. On the other hand, all fractions from ABPC retained the activity even after the HPLC purification. A comparative estimation of cross-reactivity of IgE and IgE antipenicillin antibodies showed that IgE antibodies cross-reacted with a variety of beta-lactam antibiotics in a high degree.

35 individuals showing reactions to penicillin of anaphylactic shock, angioedema or urticaria were investigated. Their skin sensitivity was analysed using 16 different penicillin derivatives. In addition, the content of circulating reagins against the penicilloyl structure in the patient's sera were analysed using RAST. 17 of the patients had negative skin reactions and RAST results to all substances tested. The other 18 were skin test-positive to at least one derivative but showed markedly heterogeneous patterns of skin reactivity. 14 had positive reactions against penicilloyl structures accompanied by anti-penicilloyl reagins. Four patients showed doubtful reactions only to penicillin or penicilloate and/or penilloate. These patients also had very low levels of reagins against penicilloyl in their sera. Positive skin test results using monovalent penicillin derivatives such as penicillin, penicilloate, penilloate, penicilloyl amide, penicilloyl-formyl-lysine, penicillamine, which cannot form a multivalent antigen with penicillyol specificity, indicated formation of other derivatives of importance in penicillin allergy, e.g., penicillamine protein conjugates. Three patients showed skin reactions to ampicillin polymer and two to benzyl-penicillin polymer. The skin tests performed with the penicillin derivatives used do not seem to give more information on the sensitivity of the patients than does the RAST using penicilloyl structures.

A 47-year-old group A, Rh1Rh1 woman treated with intravenous ampicillin for chronic pyelonephritis received two units of blood and also received oral cephalexin. Three months after the transfusions she was noted to have allo-anti-E and anti-c, and a 2+ positive direct antiglobulin test. Anti-E and anti-c could be eluted from her cells, yet neither antigen could be demonstrated on the patient's circulating red blood cells. Also present in the serum and in the eluate was anti-ampicillin antibody. Studies of the patient's red blood cell eluates using ampicillin-treated R1R1 and untreated R2R2 cells demonstrated anti-E complexed with anti-ampicillin in a drug-related example of the Matuhasi-Ogata phenomenon. Artificially created mixtures of anti-E and drug antibody could reproduce the effect in vitro. No effect of cephalexin could be demonstrated. The variability of the Matuhasi-Ogata phenomenon is discussed with regard to the sequence of antibody attachment, and the possible relationship to cephalexin is discussed. Drug antibodies may be involved in the Matuhasi-Ogata phenomenon in cases where another red blood cell antibody cannot be shown to be present.