Evaluation of HER2 Protein Expression Using 2 New Monoclonal Antibodies.

This study describes the performance of 2 new mouse anti-HER2 monoclonal antibodies (Abs), clones 33F and 410G, in evaluating HER2 overexpression in a series of 123 invasive breast carcinoma cases. In-house immunohistochemistry (IHC) was performed and the results were compared with those for the SP3 and A0485 anti-HER2 Abs. Chromogenic in situ hybridization was used to detect ERBB2 amplification and its concordance with IHC was analyzed. Comparison of IHC results for 33F with SP3 and A0485 yielded concordance rates (K) of 0.81 and 0.75, respectively; the same concordance rates were found when comparing results for 410G with SP3 and A0485. Compared with SP3 and A0485, 33F and 410G specificities were 98.6% and 98.6%, and 100% and 100%, respectively, whereas the sensitivities were 80% and 74.1%, and 78% and 72.2%, respectively. The K values between 33F and 410G HER2+ expression and chromogenic in situ hybridization-positive amplification were 1 and 0.96, respectively. These concordance rates were reproduced in another production batch (K=0.96 and K=0.96). Together, these results show that the tested monoclonal Abs would be well suited for detecting HER2 protein overexpression by IHC.

Synthesis and antioxidant activity of 3-(Pyridin-2-ylmethyl)-1,3-thiazinan(thiazolidin)-4-ones.

The antioxidant properties of two series of thiazolidinones and thiazinanones were reported. The novel six-membered thiazinanones were synthesized from the efficient multicomponent reaction of 2-picolylamine (2-aminomethylpyridine), arenaldehydes, and the 3-mercaptopropionic acid in moderate to excellent yields. These novel compounds were fully identified and characterized by NMR and GC-MS techniques. In vitro antioxidant activities of all compounds were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) tests. The antioxidant assays of thiobarbituric acid reactive species and total thiol content levels in the cerebral cortex and liver of rats were also performed. Thiazinanone 5a showed the best radical scavenging activity in DPPH and ABTS tests, as well as reduced lipid peroxidation and increased total thiol group in biological systems. Altogether, the results may be considered a good starting point for the discovery of a new radical scavenger.

Monoclonal antibodies against the leptospiral immunoglobulin-like proteins A and B conserved regions.

Leptospirosis is an infectious disease caused by pathogenic spirochetes of the genus Leptospira that affects humans and a wide variety of animals. Recently the genomes of Leptospira interrogans, Leptospira borgpetersenii and Leptospira biflexa species were sequenced allowing the identification of new virulence factors involved in survival and pathogenesis of bacteria. LigA and LigB are surface-exposed bacterial adhesins whose expression is correlated with the virulence of Leptospira strains. In this study, we produced and characterized five monoclonal antibodies (MAbs) against a recombinant fragment of LigB (rLigBrep) with approximately 54kDa that comprise the portions of LigA and LigB (domains 2-7). The 5 MAbs obtained were of the IgG1 (2) and IgG2b (3) isotypes and their affinity constants for rLigBrep ranged from 7×10(7) M(-1) to 4×10(8) M(-1). The MAbs were able to react with the native antigen on the L. interrogans, L. borgpetersenii and Leptospira noguchii surfaces by indirect immunofluorescence, immunoblotting and immunoelectron microscopy. These results demonstrate that the MAbs anti-rLigBrep can be useful to complement genetic studies and to aid studies aiming understanding the role of Lig proteins in Leptospira pathogenesis and the development of Lig-based vaccines and improved diagnostic tests for leptospirosis.

Testing different antigen capture ELISA formats for detection of Leptospira spp. in human blood serum.

Leptospirosis is an infectious disease caused by pathogenic spirochetes of the genus Leptospira. The illness is characterized by an acute bacteremic phase followed by an immune phase, in which specific antibodies are found in blood and leptospires are eliminated in urine. Novel diagnostic strategies for use in the acute phase of leptospirosis are needed since clinical manifestations in this phase mimic other feverish tropical diseases. In the present study, mAbs and polyclonal IgY were used in the standardization of three different antigen capture ELISA formats for direct detection of leptospires in human blood during the acute phase of the disease. Detection limit of leptospires in experimentally contaminated human sera ranged from 10(5) to 10(7) cells ml(-1) in the different formats. The ELISA format with the best performance was able to detect 10(5) leptospires ml(-1) in human sera using a mAb against LipL32, the major outer membrane protein of pathogenic leptospires, as capture antibody, and a biotinylated polyclonal IgY against a pathogenic serovar of L. interrogans Icterohamorrhagiae as detection antibody. By increasing the degree of IgY biotinylation this detection limit could be improved to make the assay clinically useful.

Monoclonal antibodies against LipL32, the major outer membrane protein of pathogenic Leptospira: production, characterization, and testing in diagnostic applications.

Pathogenic serovars of Leptospira have a wide antigenic diversity attributed mainly to the lipopolysaccharide present in the outer membrane. In contrast, antigens conserved among pathogenic serovars are mainly represented by outer membrane proteins. Surface exposure of a major and highly conserved outer membrane lipoprotein (LipL32) was recently demonstrated on pathogenic Leptospira. LipL32 in its recombinant form (rLipL32) was used to immunize BALB/c mice to develop murine monoclonal antibodies (MAbs). Three MAbs against rLipL32 were produced, isotyped, and evaluated for further use in diagnostic tests of leptospirosis using different approaches. MAbs were conjugated to peroxidase and evaluated in a native protein enzyme-linked immunosorbent assay (ELISA) with intact and heat-treated leptospiral cells, conjugated to fluorescein isothiocyanate (FITC) for indirect immunofluorescence with intact and methanol fixed cells and were used for LipL32 immunoprecipitation from leptospiral cells. rLipL32 MAbs conjugated to peroxidase or used as primary antibody bound to intact and heat-treated cells in ELISA, proving that they could be used in enzyme immunoassays for detection of the native protein. In immunofluorescence assay, MAbs labeled bacterial cells either intact or methanol fixed. Two MAbs were able to immunoprecipitate the native protein from live and motile leptospiral cells and, adsorbed onto magnetic beads, captured intact bacteria from artificially contaminated human sera for detection by polymerase chain reaction (PCR) amplification. Results of this study suggest that the MAbs produced can be useful for the development of diagnostic tests based on detection of LipL32 leptospiral antigen in biological fluids.