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.

Characterization of B-cell epitopes on IpaB, an invasion-associated antigen of Shigella flexneri: identification of an immunodominant domain recognized during natural infection.

The invasion plasmid antigen B (IpaB), a 62-kDa plasmid-encoded protein associated with the ability of shigellae to invade epithelial cells, is the bacterial antigen most strongly and consistently recognized by the host during infection. The strong systemic and mucosal immune responses observed against this invasin prompted us to map its B-cell epitopes. For this purpose, IpaB was first overexpressed in Shigella flexneri and used to raise rabbit polyclonal antiserum and murine monoclonal antibodies, which were subsequently used to screen a lambda gt11 ipaB library.

Identification and characterization of B-cell epitopes of IpaC, an invasion-associated protein of Shigella flexneri.

Invasion plasmid antigen C (IpaC) is a 43-kDa plasmid-encoded protein associated with the ability of shigellae to invade epithelial cells. This protein is consistently strongly recognized by sera from convalescent patients and monkeys experimentally infected with shigellae. The strong immunogenicity of IpaC in the course of natural infection makes it a good candidate as a potentially protective antigen.

Identification of allergenic epitopes on Der f I, a major allergen of Dermatophagoides farinae, using monoclonal antibodies.

The antigenic and allergenic structure of Der f I, a major allergen of the house dust mite Dermatophagoides farinae (Df) was investigated by means of a panel of 11 selected monoclonal antibodies (mAb) obtained from BALB/c mice immunized with purified Der f I. The species specificity of these mAb, tested with Der f I and Der p I--the homologous allergen from Dermatophagoides pteronyssinus--was generally restricted to Der f I since 10 out of 11 mAb reacted only with this allergen. Epitope specificity of the mAb was determined by both competitive inhibition and sandwich ELISA experiments.

Production and characterization of neutralizing and nonneutralizing monoclonal antibodies against listeriolysin O.

Listeriolysin O (LLO) is a thiol-activated toxin secreted by the facultative intracellular pathogen Listeria monocytogenes. LLO is essential for the survival of the bacterium in the infected cell because it promotes lysis of the phagosome membrane and escape of the bacterium into the cytosol. LLO was used as an antigen for the production of nine monoclonal antibodies (MAbs) in mice.

Calibration of target amounts of DNA in hybridization experiments using monoclonal anti-nucleoside antibodies.

Two anti-nucleoside monoclonal antibodies (A-16 and G-K21) were raised after immunizing mice with adenosine or guanosine coupled to bovine serum albumin by periodate oxidation. They were selected for their ability to detect these immunogens and single-stranded DNA in an enzyme-linked immunosorbent assay test. The antibodies were purified from ascitic fluids, their isotypes were determined and their ability to detect DNAs and RNAs on nitrocellulose membranes was tested.

Transhybridomas from fusion gene transgenic lymphocytes can be used to produce foreign protein of biological interest.

A method is described for achieving the efficient production of proteins of biological interest by the establishment of hybridomas from lymphocytes of transgenic animals carrying a fusion gene having promoter-regulatory sequences functional in lymphocytes fused to the coding sequences of the protein to be produced. While possible improvements in the method are discussed, it is anticipated that the method will ultimately be applicable to the production of any protein of interest.

Newborn Xid mice carry the genetic information for the production of natural autoantibodies against DNA, cytoskeletal proteins, and TNP.

Spleen cells from 6-day-old unimmunized male and female (CBA/N X BALB/c)F1 mice, fused with the nonsecreting hybrid SP2/0, gave 184 hybrids secreting immunoglobulins (183 IgM class; 115 from males and 69 from females) which were screened for antibody (Ab) activity against actin, tubulin, myosin, TNP-BSA, dsDNA, and denatured DNA. Eleven hybrids from the male series (9.65%) and eight hybrids from the female series (11.

Characterization of two ectocellularly oriented 67 K presynaptic proteins. Lack of effect of their removal on acetylcholine release.

A presynaptic plasma membrane fraction was purified after subfractionation of pure cholinergic synaptosomes prepared from Torpedo electric organ. Two 67 kdalton proteins were highly enriched in the synaptosomal plasma membrane (SPM): the hydrophobic form of AChE and a protein against which we raised a monoclonal antibody (C1-8). These two proteins exhibit similar biochemical properties: both exist as disulphide linked dimers with the same molecular weight; they are glycoproteins binding Concanavalin A; they are exposed on the external surface of the SPM and detached as almost entire molecules by Pronase.

Murine hybridomas secreting natural monoclonal antibodies reacting with self antigens.

Spleen cells from nonimmunized BALB/c mice were fused with two nonsecreting myeloma lines. The hybrids were selected in HAT medium and screened for Ig production and for antibody activity against actin, tubulin, myosin, thyroglobulin, myoglobin, spectrin, dsDNA, fetuin, and transferrin. Among 161 hybrids secreting Ig, three were found to react with DNA, one with thyroglobulin, and one mainly with myosin.