Detection of anti-human serum albumin antibody using surface plasmon resonance biosensor

The biosensor based on surface plasmon resonance(SPR) technology is a very useful tool to study the interaction between biomolecles. The main advantages of this technique is to "visualize" macromolecular interactions directly in real time, and in a label-free mode rather than indirect methods like enzyme-linked immunosorbent assays (ELISAs). We immobilize human serum albumin (HSA) to the carboxymethyldextran-modified sensor chip surface covalently to detect the activity of anti-HSA in serum, and regenerate the surface with .1 mol/L phosphoric acid. The results show that SPR biosensor can detect the activity of anti-HSA in real-time quickly and the sensor chip can be used over 100 cycles.

Effect of Mikania glomerata (Asteraceae) leaf extract combined with anti-venom serum on experimental Crotalus durissus (Squamata: Viperidae) envenomation in rats

Crotalic envenomation represents the highest number of deaths when compared to other snakebite envenomations of medical interest. Crotalic venom has important characteristics such as neurotoxicity, myotoxicity, nephrotoxicity, and clotting and hemolytic action. We evaluated the clinical and laboratory aspects of Crotalus durissus terrificus experimental envenomation in Wistar rats treated with antivenom and the aqueous extract of the plant mikania glomerata. The animals were divided into three groups: Group C (control); Group VS-venom and antivenom; Group VSM-venom, antivenom and aqueous extract of M glomerata. Crotalic poison caused clinical and laboratory alterations in Wistar mice. Significant clinical alterations were: temperature decrease, edema in the venom inoculated member, sedation and a locomotion decrease in groups VS and VSM when compared with group C. A faster recovery from sedation was observed only for animals of group VSM when compared to VS. There was an increase in the number of leukocytes, neutrophils and creatine kinase in the VS and VSM groups, compared to group C. Wistar rats showed a high resistance to crotalic venom. Additional studies with different doses, time of treatment, different administration methods and histopathological and immunological studies are necessary to understand the action of M glomerata in crotalic accidents. Rev. Biol. Trop. 57 (4): 929-937. Epub 2009 December 01.

Electrophoretic patterns of lipopolysaccharides and antigenic analysis of soybean bradyrhizobia

Several serological methods have been used for the characterization and identification of soybean bradyrhizobia. However, some problem were non-reactivity of certain strains and cross-reactivity among others. Since lipopolysaccharide (LPS) can often be used in strain identification, the objective was to investigate the antigenic properties and polyacrylamide gel electrophoretic pattern of 12 Brazilian strains of Bradyrhizobium japonicum that nodulate soybean and to compare them to standard strains. The close correlation between the LPS patterns obtained by SDS-PAGE and the serological analysis permitted us to assign the strains to nine groups different or the same as the standard strains.

Trypanosoma cruzi: identification and characterization of a novel ribosomal protein L27 (TcrL27) that cross-reacts with an affinity-purified anti-Sm antibody

Small nuclear ribonucleoproteins (snRNPs)are involved in trans-splicing processing of pre-mRNA in Trypanosoma cruzi. To clone T. cruzi snRNPs we screened an epimastigote cDNA library with a purified antibody raised against the Sm-binding site of a yeast sequence. A clone was obtained containing a 507 bp-insert with an ORF of 399 bp and coding for a protein of 133 amino acids. Sequence analysis revealed high identity with the L27 ribosomal proteins from different species including: Canis familiaris, Homo sapiens, Schizosaccharomyces pombe and Saccharomyces cerevisiae. This protein has not been previously described in the literature and seems to be a new ribosomal protein in T. cruzi and was given the code TcrL27. To express this recombinant T. cruzi L27 ribosomal protein in E. coli, the insert was subcloned into the pET32a vector and a 26 kDa recombinant protein was purified. Immunoblotting studies demonstrated that this purified recombinant protein was recognized by the same anti-Sm serum used in the library screening as well as by chagasic and systemic lupus erythemathosus (SLE) sera. Our results suggest that the T. cruzi L27 ribosomal protein may be involved in autoimmunity of Chagas disease.

Promotion of fibronectin independent invasion by C5a peptidase into epithelial cells in group A Streptococcus

Background & objectives: Group A Streptococcus, causative agent of several clinical manifestations codes for multiple protein invasins which help the bacterium to enter non-phagocytic cells. C5a peptidase (SCPA) is a surface protein conserved among different serotypes of M1 strain. The present study was taken up to study SCPA promoted fibronectin independent entry of GAS into epithelial cells. Methods: An isogenic 90226 emm1DeltaAB (M1(-)) mutant was constructed, with thermosensitive pGhost vector. This isogenic M1(-) mutant expressed SCPA on the surface as determined by Western blotting and immunofluorescence. Results: On preincubation with anti-SCPA serum, the isogenic M1(-) strain exhibited 54 per cent decreased invasion as compared to the bacteria incubated with control serum. Also, purified recombinant SCPA proteins blocked internalization of M1(-) streptococci into HEp-2 cells. The M1(-) strain invaded at the same efficiency in the presence or absence of fibronectin. Interpretation & conclusion: These results suggested that SCPA acted as a potential invasin of group A streptococcus and promoted invasion independent of fibronectin.

A new fluorescent quantitative PCR-based in vitro neutralization assay for white spot syndrome virus

A fluorescent quantitative PCR (FQ-PCR) assay utilizing SYBR green I dye is described for quantitation of white spot syndrome virus (WSSV) particles isolated from infected crayfish, Cambarus clarkii. For this assay, a primer set was designed which amplifies, with high efficiency and specificity, a 129 bp target sequence within ORF167 of the WSSV genome. Conveniently, WSSV particles can be added into the FQ-PCR assay with a simple and convenient method to release its DNA. To establish the basis for an in vitro neutralization test, primary cultures of shrimp cells were challenged with WSSV that had been incubated with a polyclonal anti-WSSV serum or with control proteins. The number of WSSV particles released from the cells after these treatments were assayed by FQ-PCR. This test may serve as a method to screen monoclonal antibody pools or recombinant antibody pools for neutralizing activity prior to in vivo animal experiments. (c) 2007 Elsevier B.V. All rights reserved.

Molecular characterization of three Rana grylio virus (RGV) isolates and Paralichthys olivaceus lymphocystis disease virus (LCDV-C) in iridoviruses

Three Rana grylio virus (RGV) isolates and lymphocystis disease virus (LCDV-C) were molecularly characterized by antigenicity comparison, Western blot detection of viral polypeptides, restriction fragment length polymorphism analysis of viral genomes, and MCP sequence analysis. Significant antigenicity differences existed among the three RGV isolates and LCDV-C. Western blot detection indicated that the viral polypeptides of three RGV isolates could be recognized by the anti-RGV9807 serum, whereas no bands were observed in the LCDV-C, and significant differences exist among the band patterns of three RGV isolates. Restriction fragment length polymorphism (RFLP) analysis was performed by digesting genomic DNA of the four iridovirus isolates with restriction endonucleases HindIII, KpnI, XbaI and BamHI. On the whole, obvious discrepancies existed between LCDV-C and RGV isolates, and some significant band pattern differences were also revealed between RGV9808 and RGV9506 (or RGV9807) in the profiles of restriction endonucleases Xbal, Kpn I and BamHI. PCR amplification and sequence analysis of MCP gene sequence further revealed their phylogenetic relationship among the three RGV isolates, LCDV-C and other iridoviruses. RGV9506, RGV9807 and RGV9808 are clustered together with other ranaviruses, such as FV3, BIV, TFV and ENHV, although the RGV9808 is more close to EHNV than to other ranaviruses. Additionally, LCDV-C is clustered with LCDV-1, the type species of genus Lymphocystisvirus. The current study provides clear evidence that significant genetic difference exists among the three RGV isolates. Therefore, further work on comparative genomic studies will contribute significantly to understanding of their taxonomic position and pathological mechanism. (C) 2005 Elsevier B.V. All rights reserved.

Structural organization of alpha-subunit from purified and microsomal toad kidney (Na+ + K+)-ATPase as assessed by controlled trypsinolysis

The membrane organization of the alpha-subunit of purified (Na+ + K+)-ATPase ((Na+ + K+)-dependent adenosine triphosphate phosphorylase, EC 3.6.1.3) and of the microsomal enzyme of the kidney of the toad Bufo marinus was compared by using controlled trypsinolysis. With both enzyme preparations, digestions performed in the presence of Na+ yielded a 73 kDa fragment and in the presence of K+ a 56 kDa, a 40 kDa and small amounts of a 83 kDa fragment from the 96 kDa alpha-subunit. In contrast to mammalian preparations (Jørgensen, P.L. (1975) Biochim. Biophys. Acta 401, 399-415), trypsinolysis of the purified amphibian enzyme led to a biphasic loss of (Na+ + K+)-ATPase activity in the presence of both Na+ and K+. These data could be correlated with an early rapid cleavage of 3 kDa from the alpha-subunit in both ionic conditions and a slower degradation of the remaining 93 kDa polypeptide. On the other hand, in the microsomal enzyme, a 3 kDa shift of the alpha-subunit could only be produced in the presence of Na+. Our data indicate that (1) purification of the amphibian enzyme with detergent does not influence the overall topology of the alpha-subunit but produces a distinct structural alteration of its N-terminus and (2) the amphibian kidney enzyme responds to cations with similar conformational transitions as the mammalian kidney enzyme. In addition, anti alpha-serum used on digested enzyme samples revealed on immunoblots that the 40 kDa fragment was better recognized than the 56 kDa fragment. It is concluded that the NH2-terminal of the alpha-subunit contains more antigenic sites than the COOH-terminal domain in agreement with the results of Farley et al. (Farley, R.A., Ochoa, G.T. and Kudrow, A. (1986) Am. J. Physiol. 250, C896-C906).

Evaluation of a nucleoprotein-based enzyme-linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus

As an immunogen of the coronavirus, the nucleoprotein (N) is a potential antigen for the serological monitoring of infectious bronchitis virus (IBV). In this report, recombinant N protein from the Beaudette strain of IBV was produced and purified from Escherichia coli as well as Sf9 ( insect) cells, and used for the coating of enzyme-linked immunosorbent assay ( ELISA) plates. The N protein produced in Sf9 cells was phosphorylated whereas N protein from E. coli was not. Our data indicated that N protein purified from E. coli was more sensitive to anti-IBV serum than the protein from Sf9 cells. The recombinant N protein did not react with the antisera to other avian pathogens, implying that it was specific in the recognition of IBV antibodies. In addition, the data from the detection of field samples and IBV strains indicated that using the recombinant protein as coating antigen could achieve an equivalent performance to an ELISA kit based on infected material extracts as a source of antigen(s). ELISAs based on recombinant proteins are safe ( no live virus), clean ( only virus antigens are present), specific ( single proteins can be used) and rapid ( to respond to new viral strains and strains that cannot necessarily be easily cultured).

Antimicrobial activity in the tick Rhipicephalus (Boophilus) microplus eggs: Cellular localization and temporal expression of microplusin during oogenesis and embryogenesis

Arthropods display different mechanisms to protect themselves against infections, among which antimicrobial peptides (AMPs) play an important role, acting directly against invader pathogens. We have detected several factors with inhibitory activity against Candida albicans and Micrococcus luteus on the surface and in homogenate of eggs of the tick Rhipicephalus (Boophilus) microplus. One of the anti-M. luteus factors of the egg homogenate was isolated to homogeneity. Analysis by electrospray mass spectrometry (ESI-MS) revealed that it corresponds to microplusin, an AMP previously isolated from the cell-free hemolymph of X (B.) microplus. Reverse transcription (RT) quantitative polymerase chain reactions (qPCR) showed that the levels of microplusin mRNA gradually increase along ovary development, reaching an impressive highest value three days after the adult females have dropped from the calf and start oviposition. Interestingly, the level of microplusin mRNA is very low in recently laid eggs. An enhance of microplusin gene expression in eggs is observed only nine days after the onset of oviposition, achieving the highest level just before the larva hatching, when the level of expression decreases once again. Fluorescence microscopy analysis using an anti-microplusin serum revealed that microplusin is present among yolk granules of oocytes as well as in the connecting tube of ovaries. These results, together to our previous data. suggest that microplusin may be involved not only in protection of adult female hemocele, but also in protection of the female reproductive tract and embryos, what points this AMP as a considerable target for development of new methods to control R. (B.) microplus as well as the vector-borne pathogens. (c) 2009 Elsevier Ltd. All rights reserved.

Foot-and-mouth disease in red deer - experimental infection and test methods performance

Summary: The aim of this study was to evaluate a number of foot-and-mouth disease (FMD) test methods for use in red deer. Ten animals were intranasally inoculated with the FMD virus (FMDV) O UKG 11/2001, monitored for clinical signs, and samples taken regularly (blood, serum, oral swabs, nasal swabs, probang samples and lesion swabs, if present) over a 4-week period. Only one animal, deer 1103, developed clinical signs (lesions under the tongue and at the coronary band of the right hind hoof). It tested positive by 3D and IRES real-time reverse transcription polymerase chain reaction (rRT-PCR) in various swabs, lesion materials and serum. In a non-structural protein (NSP) in-house ELISA (NSP-ELISA-IH), one commercial ELISA (NSP-ELISA-PR) and a commercial antibody NSP pen side test, only deer 1103 showed positive results from day post-inoculation (dpi) 14 onwards. Two other NSP-ELISAs detected anti-NSP serum antibodies with lower sensitivity. It also showed rising antibody levels in the virus neutralization test (VNT), the in-house SPO-ELISA-IH and the commercial SPO-ELISA-PR at dpi 9, and in another two commercial SPO-ELISAs at dpi 12 (SPO-ELISA-IV) and dpi 19 (SPO-ELISA-IZ), respectively. Six of the red deer that had been rRT-PCR and antibody negative were re-inoculated intramuscularly with the same O-serotype FMDV at dpi 14. None of these animals became rRT-PCR or NSP-ELISA positive, but all six animals became positive in the VNT, the in-house SPO-ELISA-IH and the commercial SPO-ELISA-PR. Two other commercial SPO-ELISAs were less sensitive or failed to detect animals as positive. The rRT-PCRs and the four most sensitive commercial ELISAs that had been used for the experimentally inoculated deer were further evaluated for diagnostic specificity (DSP) using 950 serum samples and 200 nasal swabs from non-infected animals. DSPs were 100% for the rRT-PCRs and between 99.8 and 100% for the ELISAs.

Adherence and intracellular parasitism of Paracoccidioides brasiliensis in Vero cells

Paracoccidioides brasiliensis is a dimorphic fungus known to produce invasive systemic disease in humans. The 43-kDa glycoprotein of P, brasiliensis is the major diagnostic antigen of paracoccidioidomycosis and may act as a virulence factor, since it is a receptor for laminin. Very little is known about early interact-ions between this fungus and the host cells, so we developed in vitro a model system employing cultured mammalian cells (Vero cells), in order to investigate the factors and virulence mechanisms of P. brasiliensis related to the adhesion and invasion process. We found that there is a permanent interaction after 30 min of contact between the fungus and the cells. The yeasts multiply in the cells for between 5 and 24 h. Different strains of P, brasiliensis were compared, and strain 18 thigh virulence) was the most strongly adherent, followed by strain 113 (virulent), 265 (considered of low virulence) and 113M(mutant obtained by ultraviolet radiation, deficient in gp43). P. brasiliensis adhered to the epithelial cells by a narrow tube, while depressions were noticed in the cell surface, suggesting an active cavitation process. An inhibition assay was performed and it was verified that anti-gp43 serum and a pool of sera from individuals with paracoccidioidomycosis were able to inhibit the adhesion of P. brasiliensis to the Vero cells. Glycoprotein 43 (gp43) antiserum abolished 85 % of the binding activity of P. brasiliensis. This fungus can also invade the Vero cells, and intraepithelial parasitism could be an escape mechanism in paracoccidioidomycosis. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Sir2-Related Protein 1 from Leishmania amazonensis is a glycosylated NAD(+)-dependent deacetylase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Interference of vaccinal antibodies on serological diagnosis of leptospirosis in vaccinated buffalo using two types of commercial vaccines

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

THE LONG-TERM EVOLUTION OF IMMUNE DEPOSITS IN PASSIVE HEYMANN NEPHRITIS

1. To study the long term course of passive Heymann nephritis (PHN), 42 adult male Wistar rats were injected with rabbit anti-FX1A serum (PHN group) and 42 rats received normal rabbit serum (control group). Two animals from each group were sacrificed 2 weeks after the inoculation and 10 animals each from the control and PHN groups were sacrificed 4, 13, 25 and 53 weeks later.2. The PHN group exhibited a significant elevation in 20-h proteinuria which lasted from the first week (control group, 9.19 +/- 0.87; PHN group, 25.3 +/- 2.66) to the 25th week (control group, 22.6 +/- 2.15; PHN group, 66.7 +/- 10.4) except for week 17. From week 29 to week 53 there was no statistical difference between the 2 groups.3. Light microscopy showed no difference between the kidneys of PHN and control rats. Immunofluorescence microscopy in PHN rats showed granular deposition of autologous and heterologous IgG on the glomerular basement membrane (GBM), whose intensity and pattern did not change during 53 weeks of observation.4. When examined by electron microscopy the glomeruli of PHN rats showed: a) electron-dense deposits which were initially subepithelial and homogeneous and later intramembranous, granular and often surrounded by an electron-transparent halo; b) focal thickening of the GBM at the sites of intramembranous deposits; c) effacement of podocytes located close to the deposits; d) penetration of the podocytes into the GBM associated with the deposits; e) presence of osmiophilic granules in the cytoplasm of the podocyte located inside the GBM similar to the granules of the deposits next to them. The association of the penetration of the podocytes into the GBM with the deposits and the presence of the osmiophilic granules inside the foot process have not been described previously in PHN.5. The results suggest that the podocytes play a role in the clearing of intramembranous deposits in PHN.