Mimotope of Leptospira from phage-displayed random peptide library is reactive with both monoclonal antibodies and patients' sera.

The study aim was to use random heptapeptide library displayed by bacteriophage T7 for identifying mimotopes from 15 monoclonal antibodies (MAbs) specific to Leptospira spp., and from four leptospirosis patient sera, respectively. The bound phages, selected from fourth round of bio-panning with each antibody, were cloned by plaque isolation and the binding specificity of individual clones were confirmed by enzyme-linked immunosorbent assay, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. All together 150 phages were selected, mimotope from 86 phages (56.6%) were found to match with protein sequences of Leptospira from GenBank database. The predominant mimotopes were mimotope with sequence LTPCD that found in 27.3%, followed by TPCSK (16%), KSKKSS (4%), KTKRXAS (4%), SSKSYR (3.3%), DPNXNSF (3.3%), KSGRC (2.6%), TLINIF (2%), TPCI (2%), 1.33% each with mimotopes PKKS, PCNTKXTA, and CTKKK, and one phage each (0.66%) with mimotopes PTFGS, TNSKRK, SKSSRC, RSKRIR, VTNNTP, and CSNXSKR. Interestingly, mimotopes LTPCD, TPCSK, and TPCI were found to react with both MAb and patient's sera. The matched proteins from GenBank namely, leptospiral putative outer membrane protein (matched with mimotope PTFGS), thermolysin precursor protein (matched with mimotope TPCIXXGSAS), and hypothetical protein LIC12228 (matched with mimotope CSNXSKR), were found to locate at outer membrane of Leptospira. These phage mimotopes and matched proteins may have potential for further use as diagnostic reagent and immunogen against leptospirosis in the future. The results demonstrate that phage display technique has potential for rapidly identifying phage mimotopes that interact with leptospiral MAbs and patient's sera.

Epitope mapping of monoclonal antibodies specific to serovar of Leptospira, using phage display technique.

Random heptapeptide library displayed by bacteriophage T7 was used to characterize epitopes of five monoclonal antibodies that were specific to L. australis, L. bangkok, and L. bratislava. Phages selected by biopanning were cloned by plaque isolation, and the binding specificity of individual clones was confirmed by enzyme-linked immunosorbent assay, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. Almost all of the peptide epitopes were continuous or linear. Interestingly, in phages reacting with the monoclonal antibody (MAb) clones F11, F20, 2C3D4, and 8C6C4A12, the deduced amino acid sequence of the displayed peptides corresponded to a segment of hypothetical protein of the Leptospira genome (L. interrogans serovar Lai and Copenhageni). Considering the deduced amino acid sequences of phages reacting with the MAb clones F11, F20, 2C3D4, and 8C6C4A12, the consensus motif -SKSSRC-, -TLINIF-, -SSKSYR- and -CTPKKSGRC- appeared respectively. No similarity was observed among phage reacting with the MAb clone F21. The results demonstrate that T7 phage display technique has potential for epitope mapping of leptospiral MAbs, and for rapid analysis of the interactions between phage display peptides with the MAb. The finding of a phage peptide that binds to MAb with protective activity can be further tested as a candidate for leptospirosis vaccine in the future.

[Microbial diagnosis with PCR will become clinically beneficial with a faster analysis]. / Mikrobiell diagnostik med PCR blir kliniskt värdefull när analystiden kortas.

PCR was introduced in 1985 by Mullis and was immediately recognized as a valuable tool in biomedical research and was awarded the Nobel Prize in 1993. Two culture-negative meningitis cases are described where Haemophilus influenzae and Neisseria meningitidis were found by 16SRNA-PCR. The modern real time PCR technology using fluorescent probes (hybridization probes, lightup probes, molecular beacons etc) for detection of the PCR-product or on DNA microarray chips, is under development for routine use. Multiplex technology can be used to simultaneously detect multiple microorganisms as well as resistance genes. Using super-convection with ultracentrifugation high-speed PCR, results can be obtained in 10 minutes and the amplificate can also be analyzed by DNA-sequencing to achieve species identification as well as detection of resistance gene mutations. The technique has mainly been applied to viruses, but is now slowly adapted to bacteria, fungi, protozoa and helminths. PCR is especially well suited for slow growing bacteria like Mycobacteria, fastidious organisms like Bartonella and contagious agents like tularemia, but also for malaria and fungi, where the advantages in sensitivity and speed can be exploited. The limit for application to routine analysis will depend on the development of simple and fast procedures for nucleic acid extraction, as well as interpretation of the PCR analysis per se, since highly efficient thermocyclers now are on the markets.

Rat mast cell protease 4 is a beta-chymase with unusually stringent substrate recognition profile.

Activated mast cells release a variety of potent inflammatory mediators including histamine, cytokines, proteoglycans, and serine proteases. The serine proteases belong to either the chymase (chymotrypsin-like substrate specificity) or tryptase (trypsin-like specificity) family. In this report we have investigated the substrate specificity of a recently identified mast cell protease, rat mast cell protease-4 (rMCP-4). Based on structural homology, rMCP-4 is predicted to belong to the chymase family, although rMCP-4 has previously not been characterized at the protein level. rMCP-4 was expressed with an N-terminal His tag followed by an enterokinase site substituting for the native activation peptide. The enterokinase-cleaved fusion protein was labeled by diisopropyl fluorophosphate, demonstrating that it is an active serine protease. Moreover, rMCP-4 hydrolyzed MeO-Suc-Arg-Ala-Tyr-pNA, thus verifying that this protease belongs to the chymase family. rMCP-4 bound to heparin, and the enzymatic activity toward MeO-Suc-Arg-Ala-Tyr-pNA was strongly enhanced in the presence of heparin. Detailed analysis of the substrate specificity was performed using peptide phage display technique. After six rounds of amplification a consensus sequence, Leu-Val-Trp-Phe-Arg-Gly, was obtained. The corresponding peptide was synthesized, and rMCP-4 was shown to cleave only the Phe-Arg bond in this peptide. This demonstrates that rMCP-4 displays a striking preference for bulky/aromatic amino acid residues in both the P1 and P2 positions.