Enzyme-linked immunosorbent assay for detection of antibodies to the unmodified beta-lactam ring.

Dose- and pH- dependent carbodiimide-mediated coupling of Penicillin-G to polystyrene microtiter-plates that leaves the beta-lactam ring unchanged is described. A new ELISA method was developed using Penicillin-G coated plates. The binding of 3 different monoclonal antibodies as well as human IgG antibodies of the IgG1 and IgG3 subclasses is demonstrated, whereas IgG2, IgG4 and IgE antibodies did not bind. Thus, covalently coupled Penicillin-G can be used to study the immune-response to the unchanged beta-lactam ring in patients receiving penicillin therapy. The new method is complementary to hitherto described techniques, which generally only allow detection of antibodies binding to penicilloyl-groups.

Mechanism of allergic cross-reactions--II. Cross-stimulation, by chemically unrelated ligands, of rat basophilic leukemia cells sensitized with an anti-DNP IgE antibody.

Serotonin release from rat basophilic leukemia (RBL) cells, sensitized with a DNP-binding monoclonal IgE, was stimulated with solid surface (polystyrene)-bound DNP-amino acids. The stimulatory potency of DNP-amino acids was dependent on the structure of amino acid attached to DNP. Generally, DNP-amino acids with high affinities to the sensitizing IgE (I(50) less than 10 microM) were stimulatory in polystyrene-bound form; DNP-amino acids with lower affinities (Pro, Cys, Trp), and aliphatic aromatic DNP-amino acid derivatives were inactive. In addition to structural analogues of DNP, lymecycline, that is chemically unrelated to DNP but was found to have high affinity to IgE(aDNP), was also stimulatory in this system. This drug, and various quinones (e.g. acenaphthene-quinone) in BSA-conjugated forms also stimulated serotonin release from RBL cells sensitized with IgE(aDNP). These studies suggest that (1) There is a threshold of intrinsic ligand binding affinities at approximately I(50) = 10-100 microM; ligands with lower affinities do not stimulate mediator release even if they are presented in multivalent forms; (2) The above affinity threshold for mediator cell stimulation is valid for various ligands, irrespective of their chemical similarity to the immunogen; (3) Multispecific stimulation of mediator release may contribute to the frequently observed allergic cross-reactions, false positive tests for allergies, and anaphylactic reactions to drugs upon first exposure.

Mechanism of allergic cross-reactions--I. Multispecific binding of ligands to a mouse monoclonal anti-DNP IgE antibody.

A recently developed solid-phase binding assay was used to investigate the specificity of ligand binding to a mouse monoclonal anti-dinitrophenyl IgE [IgE(aDNP)]. All DNP-amino acids, that were tested, inhibited the binding of radio-labeled IgE(aDNP) to DNP covalently attached to polystyrene microtiter plates; however, the concentration for 50% inhibition varied within four orders of magnitude, DNP-L-serine being the most, DNP-proline the least potent inhibitor. In addition to DNP analogues a large number (2074) of drugs and other compounds were tested for their ability to compete with DNP for the binding site of IgE(aDNP). At the concentrations used for screening 59% of the compounds had no significant inhibition; 19% inhibited the binding of IgE(aDNP) more than 50%. Several families of compounds (tetracyclines, polymyxines, phenotiazines, salicylates and quinones) of effective competitors were found. Within these families change in the functional groups attached to the "family stem" had major effects on the affinity of ligand binding. The occurrence frequencies of interactions of ligands with IgE(aDNP) is in good agreement with a semi-empirical model for multispecific antibody-ligand interactions.

Contamination of the medical air supply with oxygen: a clinical engineering incident investigation.

Two incidents of oxygen contamination in the hospital medical air supply occurred on consecutive weekends. This paper describes those incidents and the investigation that followed. The cause of the contamination was determined to be faulty check valves in some Bird air/oxygen blenders. The investigation of the incidents was hindered because the conventional blender test methods did not detect the problem. Only after monitoring the medical air and oxygen pressures and simulating the real conditions did the cause of the problem surface.

Binding of a mouse monoclonal IgE (anti-DNP) antibody to radio-derivatized polystyrene-DNP complexes.

Polystyrene (PS) labware products (microtiter plates, test tubes, etc.) show radiation (gamma) dose-dependent carbodiimide (EDC)-mediated uptake of carboxyl-containing molecules such as DNP-[3H]Gly. The presence of water during irradiation increases, and oxygen decreases the coupling capacity of irradiated PS. The EDC-mediated attachment of DNP-Gly to PS is stable because it resists prolonged exposure to acids at low or high salt concentrations; the bond is sensitive to bases and oxidizing agents. Commercially available radiation-sterilized PS labware products also show elevated EDC-mediated uptake of DNP-[3H]Gly. Nonirradiated PS products or non-PS materials in these studies were inactive. Commercially irradiated PS microtiter plates were coated with DNP-amino acids in an EDC-mediated reaction, and the binding of a radiolabeled mouse monoclonal IgE (aDNP) to DNP-coated plates was studied. 1) The minimal ligand (DNP-Gly) and reagent (EDC) concentrations for plate coating to reach saturating antibody binding were found to be 0.2 mM and 0.2 mg/ml, respectively. High coating densities were suboptimal for antibody binding; 2) five to 60 min of coupling times yielded optimal coating densities; 3) The binding of antibody to DNP-Gly-coated plates reached half-maximal levels in approximately 40 min; 4) Dissociation of antibody from DNP-Gly and DNP-Ser-coated plates was very slow. Intra- and interassay coefficients of variation of antibody binding to plates coated with DNP-Gly under optimal conditions were generally below 3%. We conclude that the PS-bound DNP produced by this method is recognizable by anti-DNP antibodies. The optical quality of PS is not affected by radio-derivatization, and the ligand-coated plates obtained by these methods are suitable for colorimetric assays. All brands of heavily irradiated PS examined were suitable carriers for EDC-mediated coupling of DNP-aminoacids.

Eczema-like, erythematous, infiltrated plaques: a common side effect of subcutaneous heparin therapy.

Erythematous, infiltrated plaques appear to be a common but neglected cutaneous reaction to heparin. Erythematous, infiltrated plaques are unrelated to heparin necrosis and sometimes closely mimic contact dermatitis. We report 15 patients (14 women and 1 man, the first to be reported in the literature) in whom erythematous, infiltrated plaques developed 3 to 21 days after commencement of subcutaneous heparin therapy. The clinical appearance, routine histopathologic and immunohistopathologic findings, and results of various skin tests provided circumstantial evidence for the presence of a delayed hypersensitivity reaction. Subcutaneous provocation tests proved superior to intracutaneous or epicutaneous tests for the diagnosis of erythematous, infiltrated plaques. Erythematous, infiltrated plaques were caused by heparin constituents in all female patients, whereas chlorocresol was implicated as the cause in the only man.

[Eosinophilic spongiosis and ICS antibodies in a child with strophulus-like dermatosis]. / Eosinophile Spongiose und ICS-Antikörper bei einem Kind mit strophulusartiger Dermatose.

Eosinophilic spongiosis associated with in vivo-bound antibodies to the epidermal intercellular space (ICS) were consistently observed in a recurrent strophulus-like eruption in an 11-year-old boy, thus suggesting pemphigus. The clinical course, however, ruled this diagnosis out since neither acantholysis nor the clinical picture of pemphigus developed in a period of 2.5 years. Since in vivo-bound ICS-antibodies have been described in several case reports of bullous impetigo we speculate that immune reactions to bacterial antigens may be involved in producing eruptions mimicking pemphigus vulgaris.

Junctional blisters in acquired bullous disorders of the dermal-epidermal junction zone: role of the lamina lucida as the mechanical locus minoris resistentiae.

The level of cleavage was determined in a variety of acquired bullous diseases of the dermal-epidermal junction zone (bullous pemphigoid, dermatitis herpetiformis, porphyria cutanea tarda and epidermolysis bullosa acquisita). We used an indirect immunofluorescence technique to examine the basal membrane zone with anti-type IV collagen and anti-laminin antisera and bullous pemphigoid sera. The majority of blisters examined proved to be junctional, including those from disorders hitherto considered to be dermolytic. Dermolytic cleavage was encountered only sporadically in microvesicles of dermatitis herpetiformis, in one small vesicle and in one out of five large blisters of porphyria cutanea tarda and in a large lesion of epidermolysis bullosa acquisita. We conclude that in acquired bullous disorders of the dermal-epidermal junction zone the preferential site of split formation is the lamina lucida which appears to act as a locus minoris resistentiae; dermolytic split formation of substantial extent occurs only when the sublaminal fibrillar apparatus is mechanically compromised.