Development of nonradioactive microtiter plate assays for nuclease activity.

We have developed two microtiter plate assays for the detection of DNA cleavage by nucleases, using 3'-biotinylated oligonucleotide substrates. In the covalently linked oligonucleotide nuclease assay (CLONA), the biotinylated substrates are phosphorylated at the 5' end to facilitate their covalent immobilization on CovaLink NH plates. The cleavage of the covalently immobilized substrate by nucleases results in biotin release.

A method for the detection and screening of catalytic anti-DNA antibodies.

We have developed a microtiter plate assay for the detection and screening of anti-DNA hydrolytic antibodies. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5'-end of the 3'-biotinylated DNA strands. The substrate binds specifically to the wells of streptavidin-coated microtiter plates where the reaction takes place.

Combining phage display and molecular modeling to map the epitope of a neutralizing antitoxin antibody.

Crotoxin is a potent presynaptic neurotoxin from the venom of the rattlesnake Crotalus durissus terrificus. It is composed of the noncovalent and synergistic association of a weakly toxic phospholipase A2, CB, and a nontoxic three-chain subunit, CA, which increases the lethal potency of CB. The A-56.

Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis.

Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR-2) mediates vascularization and tumor-induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF-KDR interaction. A phage epitope library was screened by affinity for membrane-expressed KDR or for an anti-VEGF neutralizing monoclonal antibody.

Phage-displayed mimotopes elicit monoclonal antibodies specific for a malaria vaccine candidate.

The phage-displayed peptide CGRVCLRC (C15) has been isolated from a random library by affinity screening with the D14-3 monoclonal antibody, which was raised to the 42 kDa C-terminal fragment of the major merozoite surface protein 1 of Plasmodium vivax (Pv42). In order to investigate the use of such mimotopes as possible vaccine components, we studied the antibody response in Biozzi mice immunized with C15. High titers of antibodies cross-reacting with Pv42 were generated and the IC50 of all immune sera were in the 5 x 10(-9) M range.