Expanding the conformational diversity by random insertions to CDRH2 results in improved anti-estradiol antibodies.

The length of the heavy chain complementarity-determining region 2 (CDRH2) was extended beyond what is found in germline genes to improve the binding properties of an anti-estradiol antibody. The previous immunochemical characterization and the molecular modeling of the high affinity (Ka=3.9x10(8)) murine anti-estradiol antibody 57-2 suggested that a part of the antigen was loosely recognized by the antibody. The CDRH2, because of its close location but scarce contacts with the hapten, was considered as a conceivable target for mutagenesis. Libraries with either two, three or four random amino acid insertions in the tip of the CDRH2 loop were constructed and displayed on the M13 filamentous phage as Fab fragments. Mutations were introduced also into the rest of the VHdomain by error-prone polymerase chain reaction to allow the surrounding structures to adapt to the extended CDRH2. After the panning of the libraries with an antigen off-rate-based selection, a number of active clones, most of which showed significantly improved affinity and specificity, were isolated, characterized and sequenced. The results indicate that the structure of the antibody can tolerate a number of different insertions in the CDRH2 region. They also suggest that the repertoire of antibody libraries can be expanded by extending the length of the CDR loops beyond that naturally provided by the given set of germline genes. This kind of mutagenesis can be generally useful for the engineering of hapten-binding antibodies.

A recombinant Escherichia coli sensor strain for the detection of tetracyclines.

A bioluminescent Escherichia coli K-12 strain for the specific detection of the tetracycline group of antibiotics is described. A sensor plasmid, containing five genes from bacterial luciferase operon of Photorhabdus luminescens inserted under the control of tetracycline-responsive elements of the transposon Tn10, was constructed. Usage of the full-length luciferase operon in the sensor resulted in tetracycline-dependent light production without additions, i.e., self-luminescent phenotype, since all the substrates were intrinsically produced by the recombinant organism. The time needed for optimal induction of light emission was 90 min. Maximal induction of approximately 100-fold over uninduced levels by using 20 ng of tetracycline, and picomole sensitivities for the seven different tetracyclines tested, were obtained without added Mg2+ ions. The higher the pH and the magnesium ion concentration in the assay medium the higher was the amount of membrane-impermeable tetracycline-Mg2+ chelate complex. In consequence, by adjusting the pH and the Mg2+ ion concentration, the sensitivity of the assay can be modified for different analytical purposes. Different non-tetracycline antibiotics did not cause induction of light emission.

Intraembryonic haemopoietic cells and early T cell development.

T cell precursors in the chick embryo have been localized into the intraembryonic mesenchyme (IEM) and into the para-aortic region before the first wave of the thymic colonization on embryonic day (ED) 6,5-8. The cell surface markers of avian prethymic stem cells are not known. It is also not known whether these precursor cells are already committed to the T cell lineage before their thymic colonization. In 7-day-old chick embryos Ov+ cells were found in the para-aortic region. Also the endothelial cells of the embryonic dorsal aorta were positively stained. Ov antigen might represent a very primitive marker for precursor cells having the potentiality to differentiate both to haemopoietic and endothelial cells. Scattered CD45+ cells were observed in the same para-aortic area as in many haemopoietic areas in the loose embryonic mesenchymal tissues. CD8 alpha (MoAb 3-298) expressing haemopoietic cells were detected before thymic colonization on ED6. In flow cytometric analysis of IEM precursors Ov, CD45 and CD8 alpha expressing cells seemed to form distinct subsets suggesting heterogeneity of these haemopoietic cells.