Methodological aspects of ELISA analysis of thioredoxin 1 in human plasma and cerebrospinal fluid.

Thioredoxin-1 (Trx1) is a protein antioxidant involved in major cellular processes. Increased plasma levels of Trx1 have been associated with human diseases suggesting that Trx1 is a marker for oxidative stress with putative clinical use. However, the reported mean levels of Trx1 in the control cohorts vary a hundred-fold between studies (0.8-87 ng/ml), possibly due to methodological differences between the capture ELISA used in the different studies. The aim of this study was to investigate methodological aspects related to the ELISA measurement of Trx1. ELISAs utilizing different capture and detection combinations of antibodies to Trx1 and as well as recombinant human (rh) Trx1 standards from two sources were characterized. The different ELISAs were subsequently used to measure Trx1 in human plasma and cerebrospinal fluid samples (CSF) from healthy donors and from patients with various neurological diagnoses. The Trx1 standards differed in their content of monomeric and oligomeric Trx1, which affected the ELISAs composed of different antibody combinations. Thus, the levels of Trx1 determined in human plasma and CSF samples varied depending on the antibody used in the ELISAs and on the rhTrx1 standard. Furthermore, the relevance of preventing interference by heterophilic antibodies (HA) in human plasma and CSF was investigated. The addition of a HA blocking buffer to human samples drastically reduced the ELISA signals in many samples showing that HA are likely to cause false positive results unless they are blocked. In conclusion, the study shows that the design of a Trx1 ELISA in regards to antibodies and standards used has an impact on the measured Trx1 levels. Importantly, analyses of human plasma and CSF without preventing HA interference may obscure the obtained data. Overall, the results of this study are crucial for the improvement of future studies on the association of Trx1 levels with various diseases.

Alteration of thioredoxin and glutaredoxin in the progression of Alzheimer's disease.

Oxidative stress has an important role in the pathological process of most neurodegenerative disorders, including Alzheimer's disease (AD). The glutaredoxin (Grx) and thioredoxin (Trx) systems are central in maintaining a reduced environment in the cell and thus render protection against oxidative stress. Here, we show that Trx1 and Grx1 were released to the cerebrospinal fluid in 120 cases examined, and that the levels of these proteins increased significantly in the early stages of AD in comparison to mild cognitive impairment (MCI). Trx1 and Grx1 levels correlated with the established AD biomarkers tau and phospho-tau (p-tau). Moreover, by determining the levels of Trx1 and Grx1, discrimination between MCI converters and patients with stable MCI were possible. By applying the protein levels of Trx1 together with conventional diagnostic markers (Mini-Mental State Examination, tau, and p-tau) to a stepwise regression model, MCI stable, MCI converter, mild AD, and moderate AD was correctly diagnosed in 32 out of 33 cases. In order to further evaluate the involvement of these systems in AD, the immunoreactivity of Trx1, Trx2, Grx1, and Grx2 were investigated and the expression pattern was shown to be altered in hippocampus tissue sections from AD patients compared to controls. In conclusion, we introduce members of the thioredoxin super family of proteins as promising early biomarkers in the diagnosis of AD, suggesting their potential involvement in the pathogenesis of the disease.

Proteomic biomarkers associated with Streptococcus agalactiae invasive genogroups.

Group B streptococcus (GBS, Streptococcus agalactiae) is a leading cause of meningitis and sepsis in newborns and an etiological agent of meningitis, endocarditis, osteoarticular and soft tissue infections in adults. GBS isolates are routinely clustered in serotypes and in genotypes. At present one GBS sequence type (i.e. ST17) is considered to be closely associated with bacterial invasiveness and novel proteomic biomarkers could make a valuable contribution to currently available GBS typing data. For that purpose we analyzed the protein profiles of 170 genotyped GBS isolates by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI). Univariate statistical analysis of the SELDI profiles identified four protein biomarkers significantly discriminating ST17 isolates from those of the other sequence types. Two of these biomarkers (MW of 7878 Da and 12200 Da) were overexpressed and the other two (MW of 6258 Da and 10463 Da) were underexpressed in ST17. The four proteins were isolated by mass spectrometry-assisted purification and their tryptic peptides analyzed by LC-MS/MS. They were thereby identified as the small subunit of exodeoxyribonuclease VII, the 50S ribosomal protein L7/L12, a CsbD-like protein and thioredoxin, respectively. In conclusion, we identified four candidate biomarkers of ST17 by SELDI for high-throughput screening. These markers may serve as a basis for further studies on the pathophysiology of GBS infection, and for the development of novel vaccines.