Bivalency and epitope specificity of a high-affinity IgG3 monoclonal antibody to the Streptococcus group A carbohydrate antigen. Molecular modeling of a Fv fragment.

The binding of Strep 9, a mouse monoclonal antibody (mAb) of the IgG3 subclass directed against the cell-wall polysaccharide of Group A Streptococcus (GAS), has been characterized. The intact antibody and proteolytic fragments of Strep 9 bind differently to GAS: the intact mAb and F(ab)2' have greater affinity for the carbohydrate epitope than the monomeric Fab or F(ab)'. A mode of binding in which Strep 9 binds bivalently to portions of the polysaccharide on adjacent chains on GAS is proposed. A competitive ELISA protocol using a panel of carbohydrate inhibitors shows that the branched trisaccharide, beta-D-GlcpNAc-(1-->3)-[alpha-L-Rhap-(1-->2)]-alpha-L-Rhap, and an extended surface are key components of the epitope recognized by Strep 9. Microcalorimetry measurements with the mAb and two synthetic haptens, a tetrasaccharide and a hexasaccharide, show enthalpy-entropy compensation as seen in other oligosaccharide-protein interactions. Molecular modeling of the antibody variable region by homology modeling techniques indicates a groove-shaped combining site that can readily accommodate extended surfaces. Visual docking of an oligosaccharide corresponding to the cell-wall polysaccharide into the site provides a putative model for the complex, in which a heptasaccharide unit occupies the site and the GlcpNAc residues of two adjacent branched trisaccharide units occupy binding pockets within the groove-shaped binding site.

Real-time, sequence-specific detection of nucleic acids during strand displacement amplification.

Strand displacement amplification (SDA) is an isothermal nucleic acid amplification method based on the primer-directed nicking activity of a restriction enzyme and the strand displacement activity of an exonuclease-deficient polymerase. Here we describe fluorogenic reporter probes that permit real-time, sequence-specific detection of targets amplified during SDA. The new probes possess the single-strand half of a BsoBI recognition sequence flanked on opposite sides by a fluorophore and a quencher. The probes also contain target-binding sequences located 3' to the BsoBI site. Fluorophore and quencher are maintained in sufficiently close proximity that fluorescence is quenched in the intact single-stranded probe. If target is present during SDA, the probe is converted into a fully double-stranded form and is cleaved by the restriction enzyme BsoBI, which also serves as the nicking agent for SDA. Fluorophore and quencher diffuse apart upon probe cleavage, causing increased fluorescence. Target replication may thus be followed in real time during the SDA reaction. Probe performance may be enhanced by embedding the fluorogenic BsoBI site within the loop of a folded hairpin structure. The new probe designs permit detection of as few as 10 target copies within 30 min in a closed-tube, real-time format, eliminating the possibility of carry-over contamination. The probes may be used to detect RNA targets in SDA mixtures containing reverse transcriptase. Furthermore, a two-color competitive SDA format permits accurate quantification of target levels from the real-time fluorescence data.

Quantitative reverse transcription strand displacement amplification: quantitation of nucleic acids using an isothermal amplification technique.

Recent advances in nucleic acid amplification techniques have allowed for quantitation of viral nucleic acid levels in clinical specimens. The most prevalent testing is carried out for HIV viral load. Strand displacement amplification (SDA) is an isothermal DNA amplification system utilizing a restriction enzyme and a DNA polymerase with strand displacement properties. SDA was adapted for quantitative RNA amplification (QRT-SDA) of an HIV gag sequence by including AMV reverse transcriptase, a quantitative control sequence, and 32P-labeled detector oligonucleotides for the HIV and the control sequences. We have also improved the amplification efficiency by including the single-strand binding protein from gene 32 of T4 bacteriophage (T4gp32) to enhance strand displacement replication. In a preliminary analytical demonstration of the technique, RT-SDA was quantitative to within twofold over a range of 500-500,000 transcripts that were generated from a plasmid bearing an HIV gag sequence. QRT-SDA potentially represents a convenient alternative for viral load testing in a clinical setting.

Confirmation of the presence of Mycobacterium tuberculosis and other mycobacteria in mycobacterial growth indicator tubes (MGIT) by multiplex strand displacement amplification.

Multiplex strand displacement amplification (mSDA) is capable of amplifying three distinct DNA sequences simultaneously. These include sequences present in most genera of mycobacteria, a sequence specific for Mycobacterium tuberculosis, and an internal control. mSDA was used to detect the presence of these target sequences in 154 (72 positive, 76 negative, and 6 failed) clinical specimens cultured in the mycobacterial growth indicator tube (MGIT) system. A wide variety of specimen types were processed and cultured. Once these cultures were deemed positive by MGIT fluorescence or were deemed negative after 8 weeks of incubation, MGIT culture aliquots were processed for mSDA analyses. A chemiluminescent microwell assay was used to detect the amplified products. The procedure was relatively simple and took less than 6 h to complete. The sensitivity of mSDA for detecting acid-fast bacilli was 96.4% compared to that of MGIT culture. Sensitivity and specificity were 97.2 and 96.1%, respectively, when all clinical criteria were considered. mSDA was shown to be a rapid and effective method for confirming the presence of M. tuberculosis and other mycobacteria in positive MGIT cultures.

Detection of M. tuberculosis DNA using thermophilic strand displacement amplification.

Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying DNA that is based upon the combined action of a DNA polymerase and restriction enzyme. Previously, a form of SDA was developed which utilizes the exonuclease deficient Klenow fragment of E. coli polymerase I (exo Klenow) and the restriction enzyme HincII to achieve 10(8)-fold amplification in 2 h at 37 degrees C (Walker, G.T., 1993, PCR Methods and Applications 3; 1-6). A new thermophilic form of SDA is reported here which uses a restriction endonuclease from Bacillus stearothermophilus (BsoBI) and a 5'-->3' exonuclease deficient polymerase from Bacillus caldotenax (exo Bca). SDA was used to amplify DNA from Mycobacterium tuberculosis. An amplification factor of 10(10)-fold was achieved after 15 min of SDA at 60 degrees C. The new thermophilic system is much more specific than the previous mesophilic system as evidenced by a dramatic decrease in background amplification products. Thermophilic SDA was also optimized with dUTP substituted for TTP to enable amplicon decontamination using uracil-DNA glycosylase.

Multiplex strand displacement amplification (SDA) and detection of DNA sequences from Mycobacterium tuberculosis and other mycobacteria.

Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying a DNA target sequence prior to detection [Walker et al (1992) Nucleic Acids Res., 20, 1691-1693]. Here we describe a multiplex form of SDA that allows two target sequences and an internal amplification control to be co-amplified by a single pair of primers after common priming sequences are spontaneously appended to the ends of target fragments. Multiplex SDA operates at a single temperature, under the same simple protocol previously developed for single-target SDA. We applied multiplex SDA to co-amplification of a target sequence (IS6110) that is specific to members of the Mycobacterium tuberculosis-complex and a target (16S ribosomal gene) that is common to most clinically relevant species of mycobacteria. Both targets are amplified 10(8)-fold during a 2 hour, single temperature incubation. The relative sensitivity of the system was evaluated across a number of clinically relevant mycobacteria and checked for crossreactivity against organisms that are closely related to mycobacteria.