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. Uncleaved substrate retains the digoxigenin label, which is then detected with an enzyme-labeled anti-digoxigenin antibody. We first assessed the efficiency of this assay by measuring S1 nuclease and DNase I activities and the inhibitory effect of EDTA on the reaction. The ALONA procedure was then successfully applied to the screening of a high number of hybridoma clones derived from nonimmunized (NZB x NZW)F1 mice with spontaneous lupus erythematosus. We detected three potential catalytic antibodies and investigated their substrate specificity. Overall, our findings demonstrate the value of the ALONA method for high throughput screening of potential nucleases and catalytic antibodies. Although this assay was designed for the selection of catalysts active in DNA hydrolysis, it can be adapted to detect most types of substrate cleavage reaction.

Application of a modified version of Habeeb's trinitrophenylation method for the characterization of hapten-protein conjugates in a reversed micellar medium.

We describe here a protocol for determining the epitope density of hapten-carrier conjugates at the nanomolar level. Conjugates of bovine serum albumin (BSA) and hen egg albumin (OVA) were prepared with two model haptens: 4-acetyl benzoic acid (ABA) as a chromophoric carboxylic hapten and cholesterol hemisuccinate (Chol HS) as a carboxylic derivative of a nonchromophoric hydroxylated hapten. Small-scale but valuable haptenization of carriers was achieved (approximately 4.3 nmol with an input molar ratio of hapten to carrier within the range from 50:1 to 100:1) that proved yet compatible with immunogenicity and antibody detection. We used a modified version of the amide bond-generating mixed anhydride method of Erlanger performed in a reversed micellar medium. Microsample aliquots of the coupling reaction (carriers plus activated haptens) or control experiment (carriers plus nonactivated haptens) mixtures were directly subjected to trinitrophenylation in the reversed micellar medium. The results for carrier substitution were strongly correlated (r2=0.94; n=12) with those obtained by other methods such as UV analysis (for ABA) and chromatographic determination (for Chol HS). This method was found directly applicable to other hapten-carrier conjugates coupled by the same procedure, provided that the haptens do not absorb at about 335 and 420 nm (absorption peaks of trinitrophenyl groups). With this simple, rapid, reproducible and low-cost analysis method, the separation of uncoupled hapten and conjugate is unnecessary. This facilitates the optimization of reaction conditions. Finally, using this procedure, kinetic studies of conjugate formation can be carried out in a very simple manner.

Variation in rRNA operon number as revealed by ribotyping of Bacillus anthracis strains.

Ribotyping of various Bacillus strains with one restriction enzyme (AccI) revealed significant similarity between Bacillus anthracis strains, Bacillus thuringiensis and Bacillus cereus strains, which are all members of the Bacillus cereus group. A further ribotyping study of 10 virulent and 8 attenuated B. anthracis strains, using 4 endonucleases and both 23S and 16S probes independently, was performed. The discrimination index D of Hunter and Gaston showed that the best combination for future large-scale ribotyping studies would be either the combination of AccI and 23S, or that of EcoRI and 16S. Depending on the B. anthracis strain analyzed 10 or 11 rRNA operons were found. In all cases, many strains were grouped into 2 to 3 patterns. Attenuated strains, including a laboratory-cured strain, yielded aberrant patterns.

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. The uncleaved substrate molecules are detected with an enzyme-avidin conjugate. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5' end of the 3'-biotinylated DNA strand. The substrate binds specifically to the wells of streptavidin-coated microtiter plates, in which the nuclease reaction takes place. Uncleaved substrate retains the digoxigenin label, which is detected with an enzyme-labeled anti-digoxigenin antibody. We assessed the efficiency of these two assays by measuring S1 nuclease and DNase I activities, and the inhibitory effect of EDTA and aurintricarboxylic acid on the reaction. Both methods are more convenient than the standard radioactive nuclease assay and are suitable for high-throughput screening of potential nuclease inhibitors, nucleases, and catalytic antibodies. The ALONA assay was found to be more sensitive than the CLONA assay, with a performance similar to that of the standard nuclease assay.