Background-free Detection of Mouse Antibodies on Mouse Tissue by Anti-isotype Secondary Antibodies.
Ontology highlight
ABSTRACT:
SUBMITTER: Mascadri F
PROVIDER: S-EPMC8329438 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Early detection of infection is crucial to limit the spread of coronavirus disease 2019 (COVID-19). Here we develop a flow cytometry-based assay to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein antibodies in individuals with COVID-19. The assay detects specific immunoglobulin M (IgM), IgA, and IgG in individuals with COVID-19 and also acquisition of all IgG subclasses, with IgG1 being the most dominant. The antibody response is significantly higher at a later stage of infection. Furthermore, asymptomatic individuals with COVID-19 also develop specific IgM, IgA, and IgG, with IgG1 being the most dominant subclass. Although the antibody levels are lower in asymptomatic infection, the assay is highly sensitive and detects 97% of asymptomatic infections. These findings demonstrate that the assay can be used for serological analysis of symptomatic and asymptomatic infections, which may otherwise remain undetected.
| S-EPMC7816583 | biostudies-literature
Project description:BACKGROUND:The aim of this study was to determine the frequency of anti-cardiolipin antibodies (aCL) and anti-?2 glycoprotein I antibodies (a?2GPI) among Tunisian patients with rheumatoid arthritis (RA). METHODS:Ninety RA patients with positive anti-cyclic citrullinated antibodies (anti-CCP) and 90 healthy blood donors (HBD) were studied. aCL and a?2GPI of isotype IgG, IgA and IgM were detected by ELISA. RESULT:The frequency of antiphopholipid antibodies (aPL) (aCL and/or a?2GPI) was significantly higher in patients with RA than in HBD (35.5% vs 11.1%, P = .0001). The frequencies of aCL and a?2GPI were significantly higher in patients than in healthy subjects (15.5% vs 5.5%, P = .04 and 32.2% vs 11.1%, P = .0005 respectively). a?2GPI-IgA were significantly more frequent in patients than in the control group (26.7% vs 7.8%, P = .0007). In patients, a?2GPI-IgA were significantly more frequent than a?2GPI-IgG (26.7% vs. 6.7%, P = .0003) and a?2GPI-IgM (26.7% vs 5.6%, P = .0001). In RA patients, the frequency of a?2GPI was significantly higher than that of aCL (32.2% vs 15.5%, P = .008). a?2GPI-IgA was significantly more frequent than aCL-IgA (26.7% vs 4.4%, P = .00005). The average titer of anti-CCP in aPL positive patients was significantly higher than in aPL negative patients (170.6 ± 50 RU/mL vs 147.7 ± 51 RU/mL, P = .04). Significant correlation was found between a?2GPI-IgA and anti-CCP (r = .235, P = .026). CONCLUSIONS:aPL and particularly a?2GPI-IgA are frequent in RA and are correlated with anti-CCP.
| S-EPMC7307372 | biostudies-literature
Project description:Pleckstrin homology domain and leucine rich repeat protein phosphatase 1 (PHLPP1) is a member of the serine/threonine family of phosphatases. It has been studied in organs including brain, heart, pancreas, adipose, breast, and prostate. Human PHLPP1 encodes two splice variants - PHLPP1? (~140-150?kDa) and PHLPP1? (~180-190?kDa). Commercial antibodies are widely used to characterize PHLPP1 proteins in cells/tissues. Here we validate five different antibodies to detect PHLPP1?/? by Western blot using PHLPP1 WT/KO mice. All antibodies recognize PHLPP1? in brain. Only a single antibody (Cosmo Bio Co) detects PHLPP1? (~145-150?kDa). The other four antibodies detect a non-specific signal at ~150?kDa as evidenced by its abundance in PHLPP1 KO tissues. Results suggest Cosmo antibody is a better reagent to detect PHLPP1? by Western blot. In contrast, we found it unsuitable for immunofluorescence applications in brain. Our findings caution interpretation of the ~150?kDa band detected by some PHLPP1 antibodies in rodent and human tissues. Results also recapitulate the importance of including molecular weight standards in Western blot data to simplify retrospective analysis.
| S-EPMC4649889 | biostudies-literature
Project description:BackgroundAnti-carbamylated protein (anti-CarP) antibodies have recently been reported to occur in around 45% of rheumatoid arthritis (RA) patients and to have prognostic and diagnostic properties. At present, the breadth and molecular make-up of the anti-CarP antibody response is ill defined. To understand the anti-CarP antibody immune response and potential immune effector mechanisms it can recruit, we determined the anti-CarP antibody isotype and IgG-subclass usage in RA patients.MethodsAnti-CarP antibody IgM, IgA, and IgG or IgG subclasses were detected by enzyme-linked immunosorbent assay (ELISA) in sera from 373 unselected RA patients and 196 healthy controls. An additional 114 anti-citrullinated protein antibody (ACPA) and anti-CarP IgG double-positive patients were selected to study the concomitant presence of both antibody systems.ResultsAnti-CarP IgG was present in around 45% of the patients and comprised all anti-CarP IgG subclasses. The presence of anti-CarP IgG1 particularly associates with radiological damage. Anti-CarP IgM was detected in 16% of RA patients, even in anti-CarP IgG-positive individuals, and is indicative of an actively ongoing immune response. Around 45% of the patients were positive for IgA which included ACPA-positive cases but also 24% of the ACPA-negative cases. In ACPA and anti-CarP double-positive patients, the distribution and number of isotypes and IgG subclasses was similar for both autoantibodies at the group level, but substantial variation was observed within individual patient samples.ConclusionsIn RA, the anti-CarP antibody response uses a broad spectrum of isotypes and seems to be an actively ongoing immune reaction. Furthermore, the anti-CarP and ACPA autoantibody responses seems to be differentially regulated.
| S-EPMC5558706 | biostudies-literature
Project description:In the last decades, antibody-based tumor therapy has fundamentally improved the efficacy of treatment for patients with cancer. Currently, almost all tumor antigen-targeting antibodies approved for clinical application are of IgG1 Fc isotype. Similarly, the mouse homolog mIgG2a is the most commonly used in tumor mouse models. However, in mice, the efficacy of antibody-based tumor therapy is largely restricted to a prophylactic application. Direct isotype comparison studies in mice in a therapeutic setting are scarce. In this study, we assessed the efficacy of mouse tumor-targeting antibodies of different isotypes in a therapeutic setting using a highly systematic approach. To this end, we engineered and expressed antibodies of the same specificity but different isotypes, targeting the artificial tumor antigen CD90.1/Thy1.1 expressed by B16 melanoma cells. Our experiments revealed that in a therapeutic setting mIgG2a was superior to both mIgE and mIgG1 in controlling tumor growth. Furthermore, the observed mIgG2a antitumor effect was entirely Fc mediated as the protection was lost when an Fc-silenced mIgG2a isotype (LALA-PG mutations) was used. These data confirm mIgG2a superiority in a therapeutic tumor model.SignificanceDirect comparisons of different antibody isotypes of the same specificity in cancer settings are still scarce. Here, it is shown that mIgG2a has a greater effect compared with mIgG1 and mIgE in controlling tumor growth in a therapeutic setting.
| S-EPMC10035513 | biostudies-literature
Project description:Anti-CD40 monoclonal antibodies (mAbs) that promote or inhibit receptor function hold promise as therapeutics for cancer and autoimmunity. Rules governing their diverse range of functions, however, are lacking. Here we determined characteristics of nine hCD40 mAbs engaging epitopes throughout the CD40 extracellular region expressed as varying isotypes. All mAb formats were strong agonists when hyper-crosslinked; however, only those binding the membrane-distal cysteine-rich domain 1 (CRD1) retained agonistic activity with physiological Fc gamma receptor crosslinking or as human immunoglobulin G2 isotype; agonistic activity decreased as epitopes drew closer to the membrane. In addition, all CRD2-4 binding mAbs blocked CD40 ligand interaction and were potent antagonists. Thus, the membrane distal CRD1 provides a region of choice for selecting CD40 agonists while CRD2-4 provides antagonistic epitopes.
| S-EPMC5896247 | biostudies-literature
Project description:MicroRNAs, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA detection method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design a DNA circuit that converts, thresholds, amplifies, and reports the presence of a specific microRNA, down to the femtomolar concentration. Using a leak absorption mechanism, we were able to suppress nonspecific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNAs: By partitioning the reaction mixture into water-in-oil droplets, resulting in single microRNA encapsulation and amplification, the method provides absolute target quantification. The modularity of our approach enables to repurpose the assay for various microRNA sequences.
| S-EPMC6976291 | biostudies-literature
Project description:ObjectiveAnti-topoisomerase I (anti-topo I) autoantibodies in systemic sclerosis (SSc) are associated with diffuse skin involvement and interstitial lung fibrosis. Thus far, however, the relationship between anti-topo I antibody response and disease course has not yet been fully evaluated. This study was undertaken to gain insight into the association between characteristics of the anti-topo I antibody response and clinical disease course in SSc patients positive for anti-topo I antibodies.MethodsLevels of anti-topo I IgG, anti-topo I IgM, and anti-topo I IgA were assessed in consecutive serum samples obtained from patients at baseline who were positive for anti-topo I IgG in the Leiden Combined Care In Systemic Sclerosis (CCISS) cohort. One-year disease progression was defined by a relevant increase in modified Rodnan skin thickness score (MRSS), decline in pulmonary function, development of digital ulcers, renal crisis, and pulmonary hypertension, and/or mortality. Validation was performed in SSc patients who were positive for anti-topo I from the Oslo University Hospital and University Hospital Zurich.ResultsOf the 103 patients with anti-topo I IgG in the CCISS cohort, clinical data were available to assess 1-year disease progression in 81 patients. Of these 81 patients, 23 (28%) had disease progression. At baseline, patients with disease progression were significantly more often anti-topo I IgM-positive than those who did not experience disease progression (21 [91%] of 23 versus 33 [57%] of 58; P < 0.01). This finding was confirmed in the independent validation samples.ConclusionIn SSc patients who were anti-topo I IgG-positive, presence of anti-topo I IgM, which might be considered as a surrogate for an ongoing autoreactive B cell immune response, is associated with disease progression.
| S-EPMC7702063 | biostudies-literature
Project description:Förster or fluorescence resonance energy transfer (FRET) enables to probe biomolecular interactions, thus playing a vital role in bioassays. However, conventional FRET platforms suffer from limited sensitivity due to the low FRET efficiency and poor anti-interference of existing FRET pairs. Here we report a NIR-II (1000-1700 nm) FRET platform with extremely high FRET efficiency and exceptional anti-interference capability. This NIR-II FRET platform is established based on a pair of lanthanides downshifting nanoparticles (DSNPs) by employing Nd3+ doped DSNPs as an energy donor and Yb3+ doped DSNPs as an energy acceptor. The maximum FRET efficiency of this well-engineered NIR-II FRET platform reaches up to 92.2%, which is much higher than most commonly used ones. Owing to the all-NIR advantage (λex = 808 nm, λem = 1064 nm), this highly efficient NIR-II FRET platform exhibits extraordinary anti-interference in whole blood, and thus enabling background-free homogeneous detection of SARS-CoV-2 neutralizing antibodies in clinical whole blood sample with high sensitivity (limit of detection = 0.5 μg/mL) and specificity. This work opens up new opportunities for realizing highly sensitive detection of various biomarkers in biological samples with severe background interference.
| S-EPMC10195770 | biostudies-literature
Project description:Quantitation of ?-tubulin isotype expression in taxane resistant human tumor tissue has been difficult to achieve because of the limited availability of validated antibodies. Here we present a label-free MS method to quantitate relative expression levels of ?-tubulin isotypes.Using isotype-specific reporter peptides, we determined relative ?-tubulin isotype expression levels in human lung tumor tissue.Four reporter peptides were chosen to quantitate the ?I/?II, ?IV, ?III, and ?V tubulin isotypes. These peptides were validated using human cancer cell lines. The label-free method was then used to determine ?-tubulin isotype expression in nine human lung tumor samples, which had been described as high or low ?III-tubulin expressing using immunohistochemistry. It was found that ?I/?II (accounting for 18.7-65.7% of total ?-tubulin) and ?IVa/?IVb (26.3-79.1%) were the most abundant isotypes and that the ?III (0-8.9%) and ?V (1.0-10.4%) were less abundant in the tissue. We also categorized the samples as high or low ?III-tubulin expressing.With this method we can determine the relative expression levels of ?-tubulin isotypes in human tumor tissue. This method will facilitate studies assessing the use of tubulin isotypes as biomarkers of taxane resistance.
| S-EPMC3484181 | biostudies-literature