Performance characteristic of anti-cyclic citrullinated peptide (CCP) assay on Korean rheumatoid arthritis (RA) patients and healthy controls.

The objective of this study was to evaluate the performance of the established anti-cyclic citrullinated peptide (anti-CCP) enzyme-linked immunosorbent assay (ELISA) compared to Rheumatoid Factor-Immunoglobulin M (RF-IgM) and C - reactive protein (CRP). Serum samples of 176 patients were analyzed with the anti-CCP ELISA assay method established in our laboratory. The results of rheumatoid arthritis (RA) patients, the other inflammatory patients, and healthy controls were compared using MedCalc (version 7.0). The anti-CCP assay results were compared with RF-IgM and CRP concentration analyzed in Catholic University. The specificity of ELISA test results of RA patients showed 91% and 87%, when healthy controls or osteoarthritis patients were considered as negative. Thus, the established ELISA method was RA specific, but its sensitivity was low. To see the low sensitivity may from aging effect, the concentration of anti-CCP was analyzed for different aging group. We tested 110 healthy controls' sera using the same method. The statistical results of young subjects (<45 years old) showed significantly lower anti-CCP concentrations than those of older subjects (>65 years old, p<0.0001). The citrullination might also be occurring during aging process in healthy populations. The validation results imply that the established method could be used as a clinical diagnostic for RA together with RF-IgM.

Immobilization of E. coli with autodisplayed Z-domains to a surface-modified microplate for immunoassay.

Escherichia coli with autodisplayed Z-domains was reported to improve the sensitivity of immunoassays by the orientation control of antibodies. In this work, a sensitive microplate-based immunoassay is presented by immobilizing E. coli cells to a surface-modified microplate. The microplate was prepared by coating parylene-H film with formyl groups, and then covalently coupling poly-L-lysine to the parylene-H film. The E. coli cells were bound to the microplate by charge interactions between the negatively charged E. coli outer membrane and the positively charged microplate surface. In this work, the preparation of the microplate coated with poly-L-lysine is presented. The immobilization efficiency of E. coli to the modified surface was estimated to be far higher than non-specific interaction by fluorescence microscope and the optical transmittance of the modified microplate was measured to be feasible for immunoassay. The microplate-based immunoassay is demonstrated to be feasible for medical diagnosis of inflammatory diseases by using C-reactive protein as a target analyte for the medical diagnosis of inflammatory diseases.

One step immobilization of peptides and proteins by using modified parylene with formyl groups.

One-step immobilization method for peptides and proteins is developed by using modified parylene film with formyl groups which is suitable for microplate-based immunoassay and SPR biosensor application. The immobilization of peptides and proteins is achieved through the covalent bonding of the formyl group with the primary amine groups of peptides and proteins, which no additional activation step is required. In this work, the immobilization efficiency of parylene-H is estimated in comparison with parylene-A and physical adsorption, using biotinylated-cyclic citrullinated peptide (biotinylated-CCP), human chorionic gonadotropin (hCG) and horseradish peroxidase (HRP) as model proteins. The applicability of parylene-H film to SPR biosensor is demonstrated by estimating the detection range and sensitivity of SPR biosensor at various thicknesses. The immobilization efficiency of parylene-H film for SPR biosensor was compared with physical adsorption by using HRP as a model protein.

The differential protein expression profiles and immunogenicity of tachyzoites and bradyzoites of in vitro cultured Neospora caninum.

We report a study on the variations in the protein expression profiles of tachyzoites and bradyzoites of Neospora caninum. The in vitro stage conversion of N. caninum-infected Vero cells was induced by continuous treatment of infected cultures with 70 muM sodium nitroprusside (SNP) for up to 9 days. The stage conversion indicated by the expression of the bradyzoite-specific antigen BAG1 was analyzed by immunofluoresence assay. Morphological changes between tachyzoites and bradyzoites and localization of nuclei were demonstrated by transmission electron microscopy. Notably, we showed the differential protein expression profiles of tachyzoites and bradyzoites of N. caninum upon treatment with SNP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated different protein patterns between tachyzoites and bradyzoites. Furthermore, Western blotting using rabbit polyclonal antibodies directed against tachyzoites revealed several reactive bands, one of which represented a tachyzoite-specific antigen of approximately 40 kDa remarkably expressed in the tachyzoite stage, but was absent from bradyzoites. Moreover, rabbit polyclonal serum raised against bradyzoites recognized a significant increased expression of an antigen with a MW of approximately 25 kDa in bradyzoites by Western blotting, suggesting that this protein is specifically expressed at the bradyzoite stage. Taken together, our data showed that differential protein expression profiling is a useful tool for discriminating between the two stages during tachyzoite-bradyzoite interconversion in N. caninum infections.

The differentiation of transcription between tachyzoites and bradyzoites of in vitro cultured Neospora caninum.

Interconversion between tachyzoites and bradyzoites of Neospora caninum plays a pivotal role in transmission of the parasite. Although significant efforts have been made toward understanding the mechanisms that trigger and control stage conversion of the parasite, little is known about this process. We used annealing control primer (ACP)-based polymerase chain reaction (PCR) technique to characterize the differences in transcription between tachyzoites and bradyzoites of N. caninum. The in vitro stage conversion of N. caninum-infected Vero cells was induced by treatment of infected cultures with 70 microM sodium nitroprusside. Subsequently, the gene expression profiles of the tachyzoites and bradyzoites were analyzed through comparison of the level of messenger RNA expression. ACP-based PCR revealed 85 amplicons that were consistently differentially expressed between tachyzoite and bradyzoite stages. Of the 85 differentially expressed transcripts identified, ten were cloned into Topo TA cloning vector, sequenced, and further analyzed by the Basic Local Alignment Search Tool. These differentially expressed transcripts include a combination of known genes and as yet unidentified genes. The present work provides candidate genes for further investigation on molecular basis of stage conversion from tachyzoites to bradyzoites of N. caninum.