Isolation of a high-affinity Bet v 1-specific IgG-derived ScFv from a subject vaccinated with hypoallergenic Bet v 1 fragments.

BACKGROUND: Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies, and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild-type allergens. However, so far no treatment-induced IgG antibodies have been characterized. OBJECTIVE: To clone, express, and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a nonallergic subject. METHODS: A phage-displayed combinatorial single-chain fragment (ScFv) library was constructed from blood of the immunized subject and screened for Bet v 1-reactive antibody fragments. ScFvs were tested for specificity and cross-reactivity to native Bet v 1 and related pollen and food allergens, and epitope mapping was performed. Germline ancestor genes of the antibody were analyzed with the ImMunoGeneTics (IMGT) database. The affinity to Bet v 1 and cross-reactive allergens was determined by surface plasmon resonance measurements. The ability to inhibit patients' IgE binding to ELISA plate-bound allergens and allergen-induced basophil activation was assessed. RESULTS: A combinatorial ScFv library was obtained from the vaccinated donor after three injections with the Bet v 1 fragments. Despite being almost in germline configuration, ScFv (clone H3-1) reacted with high affinity to native Bet v 1 and homologous allergens, inhibited allergic patients' polyclonal IgE binding to Bet v 1, and partially suppressed allergen-induced basophil activation. CONCLUSION: Immunization with unfolded hypoallergenic allergen derivatives induces high-affinity antibodies even in nonallergic subjects which recognize the folded wild-type allergens and inhibit polyclonal IgE binding of allergic patients.

Biomolecular interaction analysis : strategies for antibody and antigen characterization.

One of the most important classes of reagents for clinical diagnosis is antibodies, either in polyclonal preparations, as monoclonal antibodies (MAbs), or as customized reagents prepared by genetic engineering. The enormous range of antibodies produced by hybridoma and recombinant technologies, together with the requirements of clinical diagnosis for sensitivity and selectivity, make heavy demands on the processes of selecting and characterizing suitable antibody reagents.

Molecular characterization of a cloned idiotypic cascade containing a network antigenic determinant specific for the human carcinoembryonic antigen.

The monoclonal anti-idiotypic antibody (maId) 6G6.C4, directed against the carcinoembryonic antigen (CEA)-specific T84.66 immunoglobulin, was recently shown to act as a surrogate antigen for CEA in experimental animals. In this report, we have extended our studies. 1) The kinetics of complex formation in this CEA-specific idiotypic cascade were investigated using Biosensor technology. 2) Bacterial expression studies show that the mimicked epitope can be delimited to the A3 domain of CEA. 3) We cloned and characterized the genes coding for maId 6G6.C4. 4) Comparison of this epitope-bearing domain with the hypervariable region sequences of 6G6.C4 yields substantial amino acid similarity. Sequence homology between maId 6G6.C4 and anti-CEA antibodies binding to the T84.66 epitope led us to investigate the interaction of maId 6G6.C4 and CEA. Surprisingly, 6G6.C4 specifically binds to CEA but not CEA-related antigens in Western blots. 6G6.C4 and T84.66 recognize different epitopes on CEA. Our results suggest that the T84.66 epitope functionally mimicked by maId 6G6.C4 may be involved in the homophilic binding between CEA molecules, and that heterophilic interactions in the CEA-family are mediated by different binding sites. A model for the intermolecular adhesion of CEA is presented.

[Possibilities of using ultrasonic tools in changing endoprostheses]. / Möglichkeiten der Anwendung von Ultraschallwerkzeug bei Endoprosthesenwechsel.

In joint replacement surgery an exchange of endoprostheses is technically most difficult, time consuming, and extremely unpleasant for the patient. Removal of the implant without damaging the bone entails many problems. Experience has shown that, in addition to the normal operative technique, the ultrasonic method may be very helpful. Ultrasonic implements that melt thermoplastic implants facilitate the removal of those implants (e.g., polymethylmethacrylate, polyethylene), protect the tissue, and save time. This method is not an alternative to the normal operative technique, but an additional help.

Flexible bandpass filtering using a coherent optical feedback technique.

A certain flexibility in an optical filter experiment can be achieved with coherent optical feedback because the transfer function of such systems depends on an adjustable phase. With a simple filter in the optical system, a spatial bandpass characteristic is attainable. The tuning frequency of the bandpass is a function of the phase of the feedback signal. The variable bandpass effect is demonstrated with different inputs.