Association Between Low HIV-1 DNA and Western Blot
Brief Report: Association Between Low HIV-1 DNA and Western Blot Reactivity to HIV-1 Pol in Chronically Infected Individuals.
The aim of the study was to evaluate whether negative HIV-1 pol on Western blot (WB) was associated with low HIV-DNA in adults with chronic HIV-1 infection and suppressive antiretroviral therapy.Cross-sectional parent study of the APACHE trial, conducted in subjects with chronic infection, HIV-1 RNA <50 copies/mL for ≥10 years, no residual viremia for ≥5 years and CD4 >500 cells/µL screened for HIV-1 DNA. HIV-1 DNA was quantified in peripheral blood mononuclear cells (PBMCs) by real-time polymerase chain reaction and HIV-1 serostatus was tested by HIV Blot 2.2 WB assay.
Multivariate logistic regression was used to determine factors associated with low HIV-1 DNA.We evaluated 96 patients: 78 (81%) and 18 (19%) subjects with HIV-1 DNA ≥100 copies/10 PBMCs and with HIV-1 DNA <100 copies/10 PBMCs, respectively. Median age was 32.5 (25.3-38.9), and 61 (64%) were men; moreover, we reported that nadir CD4 was 253 (167-339) cells/µL and HIV-RNA <50 copies/mL for 11.7 (10.6-14.0) years.
At multivariate analysis, higher nadir CD4 [adjusted odds ratio (AOR) [95% confidence interval (CI) 1.35 (95% CI: 1.03 to 1.76), P = 0.029], longer years of HIV-1 RNA <50 copies/mL [AOR (95% CI) 2.98 (95% CI: 1.25 to 7.10), P = 0.014], a R5-tropic virus [AOR (R5 vs. non-R5) 0.20 (95% CI: 0.04 to 0.96), P = 0.044], and negative HIV-1 pol [AOR 6.59 (95% CI: 1.47 to 29.54), P = 0.014] were associated with low HIV-1 DNA.In patients with chronic HIV-1 infection and suppressive antiretroviral therapy, negative HIV-1 pol on WB was associated with low HIV-1 DNA as well as higher nadir CD4, longer years of HIV-1 RNA <50 copies/mL, and a R5-tropic virus.
Inactivation of Horseradish Peroxidase by Acid for Sequential Chemiluminescent Western Blot.
Chemiluminescent western blot (WB) is often performed sequentially for detection of overlapping proteins; in between, prior antibodies must be stripped or the conjugated horseradish peroxidase (HRP) inactivated. However, often, stripping either is insufficient to remove all the bound antibodies or causes protein loss, whereas treatment with hydrogen peroxide (H2 O2 ), a popular way to inactivate HRP, may affect epitope recognition as our recent work revealed.
To date, an ideal method for sequential chemiluminescent WB was still missing. Here we demonstrate that acid equivalent to 10% acetic acid can efficiently inactivate HRP, allowing sequential probing without protein loss or epitope damage. This article is protected by copyright. All rights reserved.
Detection of wheat allergens using 2D Western blot and mass spectrometry.
Wheat allergy is relatively common and the associated clinical manifestations depend on the involved molecular allergens as well as on the way of exposure. Different symptoms have been described: wheat-dependent exercise-induced anaphylaxis (WDEIA), atopic dermatitis (AD) and pollen rhinitis (PR). Traditional diagnostic methods do not allow accurate molecular identification of the allergens that are essential for risk assessment and for the choice of the most adapted treatment.Standardized total protein extracts obtained from wheat seeds were separated by 2D electrophoresis.
Twenty-one sera with high wheat-specific immunoglobulin E (sIgE) levels were classified into three patients groups based on their clinical profile. These sera were tested by Western blot on 2D separated standardized wheat protein extract and their sIgE sensitization profiles were compared.Specific sensitization profiles were identified for each phenotype group. For WDEIA, protein spots around 37 kDa (pH 6-9) and 37-50 kDa (pH 5-6) were identified. For AD, spots were observed around 50 kDa (pH 9), 10 kDa (pH 9) and 20 to 75 kDa (pH3).
For PR, specific spots were located around 90 kDa (pH 9). The mass spectrometry (UHPLC-MS/MS) analysis of these identified spots pointed out several potential interesting allergens: Tri a 26, Tri a bA, Tri a 34, Tri a tritin.The present study allowed the identification of different protein areas specific to these studied groups. The protein spots of interest were identified by UHPLC-MS/MS. It has been possible to establish a link between a specific symptomatology and the newly identified responsible allergens.
LINE IMMUNOASSAY FOR CONFIRMATION AND DISCRIMINATION OF HUMAN T-CELL LYMPHOTROPIC VIRUS INFECTIONS IN INCONCLUSIVE WESTERN BLOT SERUM SAMPLES FROM BRAZIL.
Difficulties to confirm and discriminate human T-cell lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2) infections by serological Western Blotting (WB) assay (HTLV Blot 2.4, MP Biomedicals) has been reported in Brazil, mainly in HIV/AIDS patients, with a large number of WB-indeterminate and WB-positive but HTLV untypeable results. Nonetheless, the line immunoassay (LIA) (INNO-LIA HTLV-I/II, Fujirebio) was pointed to enhance specificity and sensitivity for confirming HTLV-1/2 infections. To add information concerning the improved ability of LIA in relation to WB when applied in samples of individuals from different risk-groups from Brazil, we performed the present study.
Three groups were analyzed: group 1 [G1], 62 samples from HIV/AIDS patients from São Paulo-SP (48 WB-indeterminate + 14 HTLV); group 2 [G2], 24 samples from patients with hepatitis B or hepatitis C from São Paulo (21 WB-indeterminate + 3 HTLV; 17 HIV-seropositive), and group 3 [G3], 25 samples from HTLV out-patients clinic from Salvador-Bahia (16 WB-indeterminate + 9 HTLV; all HIV-seronegative). Overall, the LIA confirmed HTLV-1/2 infection (HTLV-1, HTLV-2 or HTLV) in 66.1% [G1], 83.3% [G2], and 76.0% [G3] of samples.
Interestingly, the majority of WB-indeterminate results were confirmed by LIA as HTLV-2 in G1 and G2, but not in G3, in which the samples were defined as HTLV-1 or HTLV positives. These results agree with the virus types that circulate in such patients of different regions in Brazil, and emphasize the LIA as the best serological test for confirming HTLV-1 and HTLV-2 infections, independently of being applied in HTLV-monoinfected or HTLV-coinfected individuals.
The answer depends on the question: Optimal conditions for western blot characterization of muscle collagen type 1 depends on desired isoform.
Fibrillar collagen type 1 is the most abundant type of collagen within the body and is a critical component of extracellular infrastructure. In order to assess collagen synthesis and extracellular accumulation in fibrotic disorders, improved methods are needed to detect changes in procollagen versus mature collagen at the protein level. Using Western blot methodology, we systematically examined: (1) gel composition (Tris-glycine vs. bis-Tris, gradient vs. non-gradient, sodium dodecyl sulfate (SDS) vs. no SDS); (2) sample preparation (SDS vs. no SDS, β-mercaptoethanol (BME) vs. no BME, boiling vs. no boiling); and (3) running buffer composition (SDS vs. no SDS).
Our results indicate full native gel conditions prevent resolution of all collagen type 1 bands. The best resolution of type 1 procollagens is achieved using 4%-12% Tris-glycine gels without the presence of SDS in the gel itself, although SDS in the running and sample buffers are needed. Also, BME must not be added to the sample buffer and samples should not be boiled. For characterization of mature collagen 1(I), both 8% and gradients type gels are appropriate, although still without SDS, yet with SDS included in both running and sample buffers, BME must be added to the sample buffer, and samples should not be boiled. Boiling is to be avoided as the antigenic site recognized by the monoclonal antibody used is sensitive to thermal denaturation, as is the case with many monoclonal antibodies available on the market. Thus, the exact parameters employed are dependent upon the collagen protein product that the scientist desires to identify.
