Biomedical subjects
Publications and source records attributed to P Lafaye.
Two fixation sites for penicilloyl groups on human albumin were demonstrated. Using CNBr cleavage the first site was located between methionine 123 and methionine 297 and the second one between methionine 297 and the C-terminal residue. In both cases, penicilloyl groups were unmasked by pronase degradation or disulfide bond reduction.
Analysis of the antigenic structure of human serum albumin was undertaken using monoclonal antibodies. Nineteen antibodies were prepared and their specificities were studied using fragments which encompass the whole sequence of the albumin molecule. These antibodies recognized 13 different epitopes which are different from the one previously identified with two other monoclonal antibodies [Doyen et al., Immun. Lett. 3, 365-370 (1981)]. Among those 13 different epitopes, six were overlapping. Four epitopes were located on the N-terminal half of the albumin molecule. One of these required integrity of methionine 87 and the other three were overlapping and located around methionine 123. Eight epitopes were located on the C-terminal half of the albumin. Two of them were within the sequence, 330-422 and 299-496 respectively; the other six appeared to be topographic determinants which were altered or lost in the albumin fragments. A last epitope could not be located on any region of albumin. Four monoclonal antibodies directed against a given portion of the albumin molecule reacted slightly with another part of albumin, thus confirming the existence of an intramolecular cross-reactivity between the different domains of human albumin.
A high-affinity anti-tenanus toxoid (TT) human monoclonal antibody showing neutralizing activity was isolated from a fusion between mouse myeloma and human splenic cells. Fab fragments from this antibody were obtained using a recombinant phage surface-display expression system. The parental antibody and the corresponding Fab had identical immunological activities, including specificity and affinity. These results confirm the feasibility of developing Escherichia coli expression of monoclonal human Fab from hybridoma cells.
An enzyme-linked immunosorbent assay for measuring penicillin groups is described. Alkaline phosphatase conjugated to penicilloyl residue was reacted with purified anti-penicilloyl antibodies coated upon polystyrene plates. The inhibition of this reaction allowed a specific determination of penicilloyl residues at the picomole level. This assay was applied to the study of the penicilloyl groups linked to human albumin and to the measurement of some of these groups after enzymatic degradation or physical modifications of the albumin molecule.