| 1. |
- Englund, Hillevi, 1980-, et al.
(författare)
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Sensitive ELISA detection of amyloid-β protofibrils in biological samples
- 2007
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Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 103:1, s. 334-345
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid-β (Aβ) protofibrils are known intermediates of the in vitro Aβ aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Aβ protofibrils in Alzheimer's disease (AD). To verify their in vivo relevance and to establish a quantitative Aβ protofibril immunoassay, Aβ conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Aβ protofibrils without interference from Aβ monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of Aβ protofibrils in both cell and animal models, proving that Aβ protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated Aβ protofibril levels in the Arctic-Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of Aβ protofibrils in AD and has the potential of becoming an important diagnostic assay.
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| 2. |
- Huang, Jenny, et al.
(författare)
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ELISpot and ELISA analyses of human IL-21-secreting cells : Impact of blocking IL-21 interaction with cellular receptors
- 2015
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 417, s. 60-66
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Tidskriftsartikel (refereegranskat)abstract
- Interleukin (IL)-21 is crucial for the regulation of lymphocytes and is implicated in autoimmune and other diseases. The relevance of being able to measure human IL-21 prompted us to develop ELISA and ELISpot assays for analysis of IL-21 levels and IL-21-producing cells, respectively. Monoclonal antibodies (mAbs) to IL-21 were made and ELISA and ELISpot assays were developed. The selected detection mAb also neutralized IL-21-mediated activation of human cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors (n = 24) were stimulated polyclonally (phytohemagglutinin; PHA) or with antigen (Candida albicans extract and tetanus toxoid). Using ELISpot, high numbers of IL-21-producing cells were detected after PHA activation; lower but positive responses to antigen were seen in approximately 50% of the donors. In contrast, the ELISA detected IL-21 in supernatants from PHA-activated cells but not from antigen-stimulated cells. When analyzing IL-17A in parallel, PHA and antigens induced detectable responses in ELISpot as well as in ELISA. Hypothesizing that the lack of detectable IL-21 levels after antigenic stimulation was due to a combination of low frequencies of IL-21-secreting cells and consumption of IL-21 by cellular receptors during cell culture, PBMCs (n = 18) were stimulated in the presence of the neutralizing detection mAb. When preventing IL-21 from interacting with its receptor, increased IL-21 levels were found by ELISA after PHA activation and IL-21 could also be measured after antigen stimulation. ELISpot results were unaffected by the addition of the neutralizing mAb. In conclusion, IL-21 secreted by low frequencies of antigen-specific ex vivo-stimulated PBMC can be difficult to detect by ELISA but prevention of IL-21 interaction with its receptor leads to detectable IL-21 levels. In ELISpot, where the cytokine is captured by mAbs on a solid phase immediately upon secretion, blocking the receptor interaction does not affect the detection of IL-21-secreting cells.
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| 3. |
- Patlaka, Christina, et al.
(författare)
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Intensive weight gain therapy in patients with anorexia nervosa results in improved serum tartrate-resistant acid phosphatase (TRAP) 5a and 5b isoform protein levels
- 2020
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Ingår i: Eating and Weight Disorders. - : SPRINGER. - 1124-4909 .- 1590-1262. ; 25:5, s. 1387-1397
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Tidskriftsartikel (refereegranskat)abstract
- Aim Tartrate-resistant acid phosphatase (TRAP) exists as isoforms 5a and 5b. TRAP 5a is a biomarker of chronic inflammation and influences adipose tissue and 5b associates with bone metabolism/pathologies. The aim was to investigate the association of serum TRAP 5a/5b isoforms with fat and bone markers and anthropometric parameters in patients with anorexia nervosa (AN) during weight gain therapy. Methods Twenty-five Swedish female AN patients, age 16-24 years, were treated for 12 weeks with a high-energy diet with six meals daily. Serum TRAP 5a/5b, markers of fat/glucose metabolism, markers of bone resorption and formation were measured. Parameters of bone and body composition were assessed by dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. Results BMI increased from median 15.4 kg/m(2)to 19.0 kg/m(2),p < 0.0001. TRAP 5a and 5a/5b ratio increased but TRAP 5b decreased during the study. TRAP Delta 5a and Delta 5b correlated with Delta insulin and Delta adiponectin, respectively. TRAP 5b correlated with trabecular density at start but not at week 12. At 12 weeks, TRAP 5b correlated with CTX, and Delta decrease in TRAP 5b correlated to Delta increase in bone-specific alkaline phosphatase. Conclusions This clinical interventional study resulted in increased BMI in patients with AN. The decreased TRAP 5b protein levels confirm a role for TRAP 5b as a marker of bone resorption, whereas increased TRAP 5a seemed to derive from systemic changes in bone as well as metabolic changes. The combined detection of TRAP 5a and TRAP 5b in serum could be an indicator of improved bone metabolism.
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| 4. |
- Paulie, Staffan, 1951-
(författare)
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Immunological analysis of the human tumor cell surface : characterization of polypeptides associated with urinary bladder carcinoma
- 1985
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Tumors express antigens characteristic of their malignant phenotype, a fact that has become exploited for the diagnosis of certain types of cancer. As these tumor-associated antigens (TAA) may also provide Information on the molecular mechanisms of tumorogenesls as well as constituting the basis for immunotherapeutlc approaches, much Interest has been focused on the Identification and characterization of TAAs.In the present study we have Investigated the cell surface of human urinary bladder carcinomas (TCC) for the presence of TAAs. Three different approaches were used: 1) lectins of various sugar specificities were employed for the Isolation and comparison of the glycoprotein patterns of different normal and malignant cells, 2) mouse monoclonal antibodies were raised against Intact TCC cells and a large number of hybridomas were searched for tumor-related reactivity, 3) antibody producing B cell cultures from TCC- patients were established by Infection with Epstein Barr virus (EBV).Totally six distinct antigens of protein or glycoprotein nature have 1n this way been Identified as being more or less restricted to TCC cells. Five of these were Identified with mouse monoclonal antibodies and have been subjected to serological analysis against a large number of normal and malignant cell types derived from cell cultures or from fresh tissue Isolates. Although showing a high degree of specificity with both cell lines and tissue the TCC-restr1cted expression was generally more pronounced in the histological samples. Thus, two antigens (plOO and the pl90, pl70 complex) were found on almost all TCC specimens but not on any other cell type tested Including normal uroepithel1um. A third antigen (p92, p23) showed a similar distributional pattern but was to some extent also found on certain normal cells. With the antibody S2C6 an interesting antigenic relationship between TCC tumors and B lymphocytes was revealed. In addition to being selectively expressed by TCC cells the S2C6 antigen (p50) appeared to represent a novel B cell marker of high restriction within the hematopoietic system. Although found on both normal and malignant B cells antigen expression was highly Increased 1n rapidly proliferating B cell cultures suggesting that expression may be growth related. Two other antigens, gp115 Identified by Its affinity for leukoagglutinln and the pl40, p85 complex defined by a mouse monoclonal antibody, have been less well established in terms of tissue type distribution but the available data suggest a close TCC-assoc1at1on also of these molecules.With hopes of finding antibody reactivities reflecting the humoral antitumor response of TCC-patients, a series of EBV-transformed B cell cultures were also established. Although no tumor-related antibodies were found 1n this Initial study the observation that B cell cultures producing antibodies to a variety of cellular antigens could be Isolated from most patients confirms that the method may be of value for the delineation of the Immune response to tumors.In conclusion, the present study has led to the Identification and preliminary characterization of several antigenic components associated with urinary bladder cancer. The restricted distribution of these molecules make them interesting as markers of this disease and their potential use for clinical applications as well as possible Implications 1n tumorogenesis 1s discussed.
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| 5. |
- Sehlin, Dag, et al.
(författare)
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Heavy-Chain Complementarity-Determining Regions Determine Conformation Selectivity of Anti-A beta Antibodies
- 2011
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Ingår i: Neurodegenerative Diseases. - : S. Karger AG. - 1660-2854 .- 1660-2862. ; 8:3, s. 117-123
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Tidskriftsartikel (refereegranskat)abstract
- Background/Aims: Amyloid-beta (Abeta) protofibrils are neurotoxic soluble intermediates in the Abeta aggregation process eventually forming senile plaques in Alzheimer's disease. This Abeta species is a potential biomarker for Alzheimer's disease and also a promising target for immunotherapy. In this study, we investigated the characteristics of conformation-dependent Abeta antibodies specific for Abeta protofibrils. Methods: Mice were immunized with Abeta protofibrils to generate hybridomas producing Abeta-specific monoclonal antibodies. Binding of antibodies to different Abeta conformations was investigated with inhibition ELISA. The antibodies' complementarity-determining region (CDR) sequences were determined and compared. Results: A majority of the antibodies were of the IgM class, all selectively binding to aggregated Abeta. Two IgG antibodies were generated: one with selective affinity for Abeta protofibrils and the other bound Abeta in all conformations. A high degree of similarity between the heavy-chain CDRs of the conformation-dependent antibodies was found, and all high-affinity Abeta antibodies displayed a high degree of sequence similarity in the light-chain CDRs. Conclusion: Sequence similarity in the heavy-chain CDRs is associated with conformation selectivity of the antibodies, while sequence similarity in the light-chain CDRs correlates with the affinity for Abeta.
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| 6. |
- Sehlin, Dag, et al.
(författare)
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Interference from Heterophilic Antibodies in Amyloid-beta Oligomer ELISAs
- 2010
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Ingår i: Journal of Alzheimer's Disease. - : IOS Press. - 1387-2877 .- 1875-8908. ; 21:4, s. 1295-1301
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid-beta (Abeta) oligomers of different sizes and forms have recently been the focus for many Alzheimer's disease (AD) researchers. Various immunoassays have been used to detect low concentrations of these elusive Abeta species in different forms of human samples using little or no sample dilutions. However, the possibility that positive results may be caused by interference from heterophilic antibodies (HA) is often overlooked. HA, which recognize immunoglobulins from other species, are present in human plasma and cerebrospinal fluid (CSF) and may cause interference in sandwich immunoassays like enzyme-linked immunosorbent assays (ELISAs) by cross-binding the capture and detection antibodies of the assay. They thus may generate a false positive signal. Here we show that when assessing the Abeta oligomer content in plasma samples from 44 individuals with a sandwich ELISA, none of the 21 positive signals remained when the assay was repeated in the presence of factors blocking HA. Similarly, in CSF samples from 104 individuals, the signals from the 22 positive samples were strongly reduced when analyzed after anti-HA treatment. Taken together, HA interference is a problem that needs to be addressed when measuring low levels of an antigen in human plasma and CSF samples.
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| 7. |
- Söllvander, Sofia, et al.
(författare)
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Increased Number of Plasma B Cells Producing Autoantibodies Against A beta(42) Protofibrils in Alzheimer's Disease
- 2015
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Ingår i: Journal of Alzheimer's Disease. - 1387-2877 .- 1875-8908. ; 48:1, s. 63-72
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Tidskriftsartikel (refereegranskat)abstract
- The Alzheimer's disease (AD)-related peptide amyloid-beta (A beta) has a propensity to aggregate into various assemblies including toxic soluble A beta protofibrils. Several studies have reported the existence of anti-A beta antibodies in humans. However, it is still debated whether levels of anti-A beta antibodies are altered in AD patients compared to healthy individuals. Formation of immune complexes with plasma A beta makes it difficult to reliably measure the concentration of circulating anti-A beta antibodies with certain immunoassays, potentially leading to an underestimation. Here we have investigated anti-A beta antibody production on a cellular level by measuring the amount of anti-A beta antibody producing cells instead of the plasma level of anti-A beta antibodies. To our knowledge, this is the first time the anti-A beta antibody response in plasma has been compared in AD patients and age-matched healthy individuals using the enzyme-linked immunospot (ELISpot) technique. Both AD patients and healthy individuals had low levels of B cells producing antibodies binding A beta(40) monomers, whereas the number of cells producing antibodies toward A beta(42) protofibrils was higher overall and significantly higher in AD compared to healthy controls. This study shows, by an alternative and reliable method, that there is a specific immune response to the toxic A beta protofibrils, which is significantly increased in AD patients.
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| 8. |
- Vallhov, Helen, et al.
(författare)
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Adjuvant Properties of Mesoporous Silica Particles Tune the Development of Effector T Cells
- 2012
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Ingår i: Small. - : John Wiley & Sons. - 1613-6810 .- 1613-6829. ; 8:13, s. 2116-2124
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Tidskriftsartikel (refereegranskat)abstract
- Alum is the most frequently used adjuvant today, primarily inducing Th2 responses. However, Th1-type responses are often desirable within immune therapy, and therefore the development of new adjuvants is greatly needed. Mesoporous silica particles with a highly ordered pore structure have properties that make them very interesting for future controlled drug delivery systems, such as controllable particle and pore size; they also have the ability to induce minor immune modulatory effects, as previously demonstrated on human-monocyte-derived dendritic cells (MDDCs). In this study, mesoporous silica particles are shown to be efficiently engulfed by MDDCs within 2 h, probably by phagocytic uptake, as seen by confocal microscopy and transmission electron microscopy. A co-culture protocol is developed to evaluate the capability of MDDCs to stimulate the development of naive CD4+ T cells in different directions. The method, involving ELISpot as a readout system, demonstrates that MDDCs, after exposure to mesoporous silica particles (AMS-6 and SBA-15), are capable of tuning autologous naive T cells into different effector cells. Depending on the size and functionalization of the particles added to the cells, different cytokine patterns are detected. This suggests that mesoporous silica particles can be used as delivery vehicles with tunable adjuvant properties, which may be of importance for several medical applications, such as immune therapy and vaccination.
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| 9. |
- Witasp, Erika, et al.
(författare)
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Efficient internalization of mesoporous silica particles of different sizes by primary human macrophages without impairment of macrophage clearance of apoptotic or antibody-opsonized target cells
- 2009
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Ingår i: Toxicology and Applied Pharmacology. - : Elsevier BV. - 0041-008X .- 1096-0333. ; 239:3, s. 306-319
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Tidskriftsartikel (refereegranskat)abstract
- Macrophage recognition and ingestion of apoptotic cell corpses, a process referred to as programmed cell clearance, is of considerable importance for the maintenance of tissue homeostasis and in the resolution of inflammation. Moreover, macrophages are the first line of defense against microorganisms and other foreign materials including particles. However, there is sparse information on the mode of uptake of engineered nanomaterials by primary macrophages. In this study, mesoporous silica particles with cubic pore geometries and covalently fluorescein-grafted particles were synthesized through a novel route, and their interactions with primary human monocyte-derived macrophages were assessed. Efficient and active internalization of mesoporous silica particles of different sizes was observed by transmission electron microscopic and flow cytometric analysis and studies using pharmacological inhibitors suggested that uptake occurred through a process of endocytosis. Moreover, uptake of silica particles was independent of serum factors. The silica particles with very high surface areas due to their porous structure did not impair cell viability or function of macrophages, including the ingestion of different classes of apoptotic or opsonized target cells. The current findings are relevant to the development of mesoporous materials for drug delivery and other biomedical applications.
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