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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.

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.

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.

By means of a sensitive radio-immunoassay an antibody-like activity against ampicillin was detected in both IgM and IgG immunoglobulin classes of patients with infectious mononucleosis, being fifteen times the level (for IgM) and six and a half times the level (for IgG) of control subjects. Such ampicillin antibody occurred without obvious relation to prior therapy with ampicillin. This antibody which may originate in a similar manner to Paul-Bunnell antibody was nevertheless immunologically unrelated. The possibility is discussed that the ampicillin rash in infectious mononucleosis results from a disseminated reaction of the small blood vessels to circulating ampicillin-antibody complexes.