Biomedical subjects

D Kenett

Publications and source records attributed to D Kenett.

5 recordsLinked to original sources

PubMed1990

Use of monoclonal antibodies in the detection of structural alterations occurring in lysozyme on heating.

Seven murine anti-hen egg-white lysozyme (HEL) monoclonal antibodies (MAbs), which recognize distinct epitopes of the native enzyme, were used as macromolecular probes to detect structural or conformational alterations occurring in HEL on heating at 95 degrees C, pH 5. As the interactions of the heat-treated HEL with its corresponding MAbs were carried out at room temperature, only irreversible structural and/or conformational alterations could be detected. The transformation of the native enzyme into its denatured form was followed electrophoretically and chromatographically. The denatured enzyme was more negatively charged at pH 8.4 and exhibited a longer retention time on reverse-phase HPLC than native HEL. Its specific catalytic activity was considerably lower than that of the native enzyme. Of the seven MAbs tested in competitive ELISA assays with native and heat-treated HEL only one, MAb D74.3, failed to recognize the heat-treated enzyme. This antibody, which is directed toward the active site region of the enzyme, was ineffective in inhibiting the catalytic activity of the heat-treated HEL using M. lysodeikticus as substrate. In contrast, the monoclonal antibody D1.3, which recognizes an epitope remote from the active site of HEL, inhibited the catalytic activity of the native as well as the heat-treated enzyme. The results indicate that the active site of HEL undergoes an irreversible structural alteration on heating for 2 hr at 95 degrees C, pH 5. No irreversible structural changes could be detected in the other regions of HEL recognized by the corresponding MAbs.

Animals↗

PubMed1989

Localization of a highly immunogenic region of carboxypeptidase A recognized by three different monoclonal antibodies and their use in the detection of subtle conformational alterations in this enzyme region.

Three murine monoclonal antibodies (mAb 100, 104, and 121) elicited against carboxypeptidase A (CPA) were prepared and characterized. All three mAbs recognize the same or partially overlapping sites of CPA. This is corroborated by the lack of antibody additivity in the ELISA assay carried out in the presence of pairs of mAbs, the similarity in molecular weight of the immunocomplex formed between CPA and one of the mAbs in the presence of another, and also a competition experiment in which one of the mAbs was labeled enzymatically. The three mAbs do not affect the enzymatic activity of CPA. Even at high concentrations, they do not recognize carboxypeptidase B (CPB) in spite of the similar tertiary structure and the 50% homology in amino acid sequence with CPA. This antigenic determinant is located on one of the four cyanogen bromide fragments of CPA. On the basis of the known sequences of the two enzymes, criteria which predict high antigenicity, and experimental data using synthetic peptides, such a determinant was found to be located within the amino acid sequence from residues 209 to 218 of the CPA molecule. The mAbs prepared detect conformational alterations in the above enzyme epitope when the enzyme is exposed to various conditions. The binding of the mAbs to CPA adsorbed onto a polystyrene plate is characterized by apparent binding constants higher by 1 or 2 orders of magnitude than those characterizing the interaction of the mAbs with CPA in solution. The mAbs also readily detect both conformational alterations of CPA on treatment with urea and subtle, reversible conformational alterations on removal of zinc from the active site of the enzyme.

Amino Acid Sequence↗

PubMed1988

A simple ELISA for the classification of monoclonal antibodies according to their recognition of native epitopes.

A sandwich ELISA for testing whether pairs of monoclonal antibodies recognize the same native antigenic site was developed. The assay was performed with murine anti-lysozyme monoclonal antibodies. A monoclonal antibody, adsorbed onto a microtiter plate, was used as a capture antibody for native lysozyme. After the reaction with the antigen a second monoclonal antibody, the test antibody, was added. The amount of bound antibody was quantitatively measured using rabbit anti-mouse immunoglobulin serum conjugated to alkaline phosphatase. The simultaneous binding of pairs of monoclonal antibodies to lysozyme was further substantiated by structural considerations.

Animals↗

PubMed1988

Quantitative ELISA for human lactate dehydrogenase isoenzyme 5.

Four monoclonal antibodies (Mab) derived from mice immunized with lactate dehydrogenase 5 (LDH5) react strongly with LDH5, but weakly with LDH2 which contains a single subunit of type M. Experimental evidence suggest that these antibodies are directed to an antigenic determinant in the interface between two subunits of type M. A sandwich ELISA procedure was devised, using these Mabs to identify and quantify LDH5. The procedure involves immobilization of one of these Mabs by its adsorption onto polyclonal anti-mouse IgG coated polystyrene plates, adsorption of LDH5, its identification by the same Mab as that used in the immobilization step, and finally color development by an enzyme labeled rabbit anti-mouse IgG antiserum. The method enables LDH5 to be assayed at a concentration range of 0-5 micrograms/ml.

Antibodies, Monoclonal↗

PubMed1987

Lysozyme bound to the D1.3 monoclonal antibody retains enzymatic activity in assays using N-acetylglucosamine oligomers as substrate.

An enzymatic assay using Micrococcus lysodeikticus as substrate had shown that hen egg-white lysozyme (HEL), complexed to the specific monoclonal antibody D1.3, was enzymatically inactive. However, a crystallographic study of the HEL-Fab D1.3 complex at 2.8 A resolution showed that Fab D1.3 is bound to the enzyme at a region remote from the catalytic site, and that no significant conformational change had occurred in the bound lysozyme. To resolve this apparent discrepancy, oligomers of N-acetylglucosamine, containing an average of six residues and which should not be sterically hindered by the monoclonal antibody, were used as substrate in enzyme assays. In these assays lysozyme complexed to antibody D1.3 retained enzymatic activity, thus supporting the results of the crystallographic study and indicating no major conformational change or rigidification of its structure.

Acetylglucosamine↗