Development and validation of an immunochromatographic assay for rapid multi-residues detection of cephems in milk.

A one-step immunochromatographic assay (ICA) was developed for the detection of seven kinds of cephems in milk. Polyclonal antibodies (PcAb) with group-specific to cephems were raised in rabbits after immunization with cephalexin-keyhole limpet hemocyanin (KLH) conjugate. The specificity of anti-sera was determined by indirect competitive enzyme-linked immunosorbent assay (icELISA), and the 50% inhibitions (IC(50)) of cephalexin and cefadroxil were obtained at 1.5 ngmL(-1); IC(50) of cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were 4, 3.7, 3.2, 4.5 and 5 ngmL(-1), respectively. The PcAb against cephems were conjugated to colloidal gold particles as the detection reagent for ICA strips to test for cephems. This method achieved semi-quantitative detection of cephems in <5 min, with high sensitivity to cephalexin and cefadroxil (both 0.5 ngmL(-1)). At the same time, cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were detected at <100 ngmL(-1) in spiked processed-milk samples. This method was compared with an enzyme-linked immunosorbent assay by testing 40 milk samples, and the positive samples were validated by a high-performance liquid chromatographic method, with an agreement rate of 100% for both comparisons. In conclusion, the method was rapid and accurate for the multi-residue detection of cephems in milk.

Equal allergenic potency of beta-lactam antibiotics produced by chemical or enzymatic manufacturing--mouse IgE test.

BACKGROUND: Cephalexin and amoxicillin are semisynthetic beta-lactam antibiotics with a broad spectrum of antibacterial activity against gram-positive and gram-negative microorganisms. Both antibiotics are produced by a new 'green' process in which enzyme technology is used to combine the intermediate structure and the side chain in an aqueous medium to yield cephalexin or amoxicillin, thus avoiding the use of several chemical reagents and volatile organic solvents. As a result of the enzyme technology a new residual protein impurity has been identified. To check for the sensitizing capacity of the residual protein, a mouse IgE test was used to detect differences in the production of specific IgE by chemical or enzymatic preparations of the antibiotics. METHODS: Balb/c female mice were immunized intraperitoneally with alum and conjugates of different amoxicillins or cephalexins with ovalbumin (OVA). After 16 days, the amoxicillin mice were injected with one half the original amount of antigen. After 19-23 days, the sera were tested for specific IgE by the passive cutaneous anaphylaxis assay in Sprague-Dawley rats. The greatest dilution of sera which resulted in a positive response was the titer of specific IgE. RESULTS: No significant differences were found between the titers of specific IgE caused by the chemically and enzymatically produced beta-lactam antibiotics, indicating that the antibiotics are equal in allergenicity. CONCLUSIONS: The data show that a residual level of 35 ppm protein did not affect the allergenic potency of these beta-lactam antibiotics as determined by the mouse allergenicity model.

The development of an enzyme-linked immunosorbent assay (ELISA) for cephalexin.

Cephalexin was structurally modified by the attachment of a spacer at the carboxylic acid through which it was subsequently covalently attached to BSA. This method permitted the molecule to be attached without cleavage of the beta-lactam ring giving a conjugate distinct from previously described immunogenic preparations of penicillins and cephalosporins. This approach required the development of a novel spacer molecule, and its synthesis and characterisation are reported. Rabbits were used to raise antisera and the antibodies produced were characterised with respect to their reactivity with cephalexin and various analogues, other cephalosporins, and a number of penicillins.

Immunological properties of cephalexin-induced delayed type hypersensitivity reaction in guinea pigs.

Immunological properties of delayed-type hypersensitivity (DTH) reaction induced by cephalexin (CEX) in guinea pigs were investigated. The animals were immunized with CEX using Freund's complete adjuvant. The time course of CEX-induced erythema showed some differences compared with that of classical DTH reaction. The erythema appeared at 6 h after intradermal administration of CEX, reached maximum size at 12 to 24 h and to be visible until 72 h. By enzyme-linked immunosorbent assay, anti-CEX antibody was detected in only one of 15 animals tested. Normal animals (recipients) which had received immune sera from CEX-sensitized animals (donors) showed no skin reaction to CEX. In contrast, reaction to CEX was observed in recipient animals which had received a local transfer of lymphocytes or T cells from CEX sensitized animals. In immunopharmacological study, cyclosporin A suppressed the skin reaction but cyclophosphamide did not. Administration of carrageenan, an inhibitor of macrophage function, had no effect on expression of the reaction. Post administration (1 or 15 h) of clemastine, an anti-histamine drug, did not affect the reaction. By histological examination, the infiltrating cell-types at the reaction site were mainly composed of mononuclear cells and neutrophils, but no basophils, indicating that CEX-induced DTH reaction is tuberculin-type DTH and not a cutaneous basophil hypersensitivity reaction.

Cross-antigenicity between penams and cephems by intradermal skin test and leucocyte migration test in guinea pigs.

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.

Anti-cephalexin monoclonal antibodies and their cross-reactivities to cephems and penams.

Three cell lines producing monoclonal antibodies (MAbs), Cep1-2, 2-2 and 6, against cephalexin were established and the immunoglobulin class of the MAbs was IgM. The cross-reactions of the MAbs with penams and cephems were examined by enzyme-linked immunosorbent assay (ELISA). The cross-reactivities of Cep1-2 and 2-2 were scarcely influenced by the structures of acyl side chains of cephems and penams. The cross-reactivities of Cep1-2 were affected by the presence or absence of dihydrothiazolidine ring of cephem nucleus in hapten-protein conjugates which were prepared by alkaline method, MBS method and activated ester method but the cross-reactivities of Cep2-2 were not. The findings suggest that Cep1-2 recognize the degradate product(s) of cephem nucleus and Cep2-2 recognize a new antigenic determinant (NAD), which is formed by the conjugation of beta-lactam and carrier protein. On the other hand, the cross-reactivities of Cep6 were influenced by the structure of amino acyl side chain. It seems that Cep6 recognize specifically the acyl side chain at the C-7 of cephem. In ELISA inhibition test, three MAbs showed different inhibition pattern. The reaction of Cep1-2 with cephalexin-HSA was inhibited by cephalexin lysate. Cep2-2 and Cep6 were weakly inhibited by the binding to cephalexin-HSA by cephalexin lysate. Furthermore, the reactions of all MAbs were remarkably inhibited by penicillamine. The above results indicate that the MAbs can recognize at least three epitopes of the degradate product(s) of cephem nucleus, NAD and acyl side chain in cephalexin-protein conjugate.

[Immunomodulating properties of interferon inducers]. / Immunomoduliruiushchie svoistva induktorov interferona.

Data on the immunomodulating activity of interferon inductors are presented. It was revealed that the inductors increased the animal vaccinal response. Schemes for combined use of the interferon inductors and immunomodulators were developed. The immunomodulators were shown to increase the host interferon response evident from synergistic increasing of the interferon titers or prolongation of interferon circulation in blood of the animals. The efficiency of the schemes for combined use of the interferon inductors and immunomodulators was obvious from stimulation of the antibody production. As a result the time of the antibody circulation in blood increased. The effect of the combined use of the immunomodulators and interferon inductors was studied. The combined use of the preparations significantly increased the average life-span of the animals and the rate of their survival.

[A study on dose-response relationship of occupational allergy in a pharmaceutical plant].

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.