| 1. |
- Edfors, Fredrik, et al.
(författare)
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Enhanced validation of antibodies for research applications
- 2018
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.
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| 2. |
- Lauren, Ida, et al.
(författare)
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Long-term SARS-CoV-2-specific and cross-reactive cellular immune responses correlate with humoral responses, disease severity, and symptomatology
- 2022
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Ingår i: Immunity, Inflammation and Disease. - : John Wiley & Sons. - 2050-4527. ; 10:4
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Tidskriftsartikel (refereegranskat)abstract
- Background: Cellular immune memory responses post coronavirus disease 2019 (COVID-19) have been difficult to assess due to the risks of contaminating the immune response readout with memory responses stemming from previous exposure to endemic coronaviruses. The work herein presents a large-scale long-term follow-up study investigating the correlation between symptomology and cellular immune responses four to five months post seroconversion based on a unique severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific peptide pool that contains no overlapping peptides with endemic human coronaviruses. Methods: Peptide stimulated memory T cell responses were assessed with dual interferon-gamma (IFN gamma) and interleukin (IL)-2 Fluorospot. Serological analyses were performed using a multiplex antigen bead array. Results: Our work demonstrates that long-term SARS-CoV-2-specific memory T cell responses feature dual IFN gamma and IL-2 responses, whereas cross-reactive memory T cell responses primarily generate IFN gamma in response to SARS-CoV-2 peptide stimulation. T cell responses correlated to long-term humoral immune responses. Disease severity as well as specific COVID-19 symptoms correlated with the magnitude of the SARS-CoV-2-specific memory T cell response four to five months post seroconversion. Conclusion: Using a large cohort and a SARS-CoV-2-specific peptide pool we were able to substantiate that initial disease severity and symptoms correlate with the magnitude of the SARS-CoV-2-specific memory T cell responses.
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| 3. |
- Tegel, Hanna, et al.
(författare)
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High throughput generation of a resource of the human secretome in mammalian cells
- 2020
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 58, s. 45-54
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Tidskriftsartikel (refereegranskat)abstract
- The proteins secreted by human tissues and blood cells, the secretome, are important both for the basic understanding of human biology and for identification of potential targets for future diagnosis and therapy. Here, a high-throughput mammalian cell factory is presented that was established to create a resource of recombinant full-length proteins covering the majority of those annotated as 'secreted' in humans. The full-length DNA sequences of each of the predicted secreted proteins were generated by gene synthesis, the constructs were transfected into Chinese hamster ovary (CHO) cells and the recombinant proteins were produced, purified and analyzed. Almost 1,300 proteins were successfully generated and proteins predicted to be secreted into the blood were produced with a success rate of 65%, while the success rates for the other categories of secreted proteins were somewhat lower giving an overall one-pass success rate of ca. 58%. The proteins were used to generate targeted proteomics assays and several of the proteins were shown to be active in a phenotypic assay involving pancreatic beta-cell dedifferentiation. Many of the proteins that failed during production in CHO cells could be rescued in human embryonic kidney (HEK 293) cells suggesting that a cell factory of human origin can be an attractive alternative for production in mammalian cells. In conclusion, a high-throughput protein production and purification system has been successfully established to create a unique resource of the human secretome.
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| 4. |
- Uhlén, Mathias, et al.
(författare)
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The human secretome
- 2019
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Ingår i: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 12:609
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Tidskriftsartikel (refereegranskat)abstract
- The proteins secreted by human cells (collectively referred to as the secretome) are important not only for the basic understanding of human biology but also for the identification of potential targets for future diagnostics and therapies. Here, we present a comprehensive analysis of proteins predicted to be secreted in human cells, which provides information about their final localization in the human body, including the proteins actively secreted to peripheral blood. The analysis suggests that a large number of the proteins of the secretome are not secreted out of the cell, but instead are retained intracellularly, whereas another large group of proteins were identified that are predicted to be retained locally at the tissue of expression and not secreted into the blood. Proteins detected in the human blood by mass spectrometry-based proteomics and antibody-based immuno-assays are also presented with estimates of their concentrations in the blood. The results are presented in an updated version 19 of the Human Protein Atlas in which each gene encoding a secretome protein is annotated to provide an open-access knowledge resource of the human secretome, including body-wide expression data, spatial localization data down to the single-cell and subcellular levels, and data about the presence of proteins that are detectable in the blood.
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| 5. |
- Abouzayed, Ayman, et al.
(författare)
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The GRPR Antagonist [Tc-99m]Tc-maSSS-PEG(2)-RM26 towards Phase I Clinical Trial : Kit Preparation, Characterization and Toxicity
- 2023
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Ingår i: Diagnostics. - : MDPI AG. - 2075-4418. ; 13:9, s. 1611-
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Tidskriftsartikel (refereegranskat)abstract
- Gastrin-releasing peptide receptors (GRPRs) are overexpressed in the majority of primary prostate tumors and in prostatic lymph node and bone metastases. Several GRPR antagonists were developed for SPECT and PET imaging of prostate cancer. We previously reported a preclinical evaluation of the GRPR antagonist [Tc-99m]Tc-maSSS-PEG2-RM26 (based on [D-Phe(6), Sta(13), Leu(14)-NH2]BBN(6-14)) which bound to GRPR with high affinity and had a favorable biodistribution profile in tumor-bearing animal models. In this study, we aimed to prepare and test kits for prospective use in an early-phase clinical study. The kits were prepared to allow for a one-pot single-step radiolabeling with technetium-99m pertechnetate. The kit vials were tested for sterility and labeling efficacy. The radiolabeled by using the kit GRPR antagonist was evaluated in vitro for binding specificity to GRPR on PC-3 cells (GRPR-positive). In vivo, the toxicity of the kit constituents was evaluated in rats. The labeling efficacy of the kits stored at 4 degrees C was monitored for 18 months. The biological properties of [Tc-99m]Tc-maSSS-PEG2-RM26, which were obtained after this period, were examined both in vitro and in vivo. The one-pot (gluconic acid, ethylenediaminetetraacetic acid, stannous chloride, and maSSS-PEG(2)-RM26) single-step radiolabeling with technetium-99m was successful with high radiochemical yields (>97%) and high molar activities (16-24 MBq/nmol). The radiolabeled peptide maintained its binding properties to GRPR. The kit constituents were sterile and non-toxic when tested in living subjects. In conclusion, the prepared kit is considered safe in animal models and can be further evaluated for use in clinics.
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| 6. |
- Adler, Belinda, et al.
(författare)
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Miniaturized and Automated High-Throughput Verification of Proteins in the ISET Platform with MALDI MS
- 2012
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 84:20, s. 8663-8669
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Tidskriftsartikel (refereegranskat)abstract
- A major bottleneck in high-throughput protein production is the validation step, which is why parallel and automated sample processing methods are highly desirable. Also, a miniaturized sample preparation format is preferred, as the reduction of reagent volumes significantly decreases the analysis cost per sample. We have developed an automated and miniaturized protein sequence verification protocol for recombinant proteins utilizing peptide mass fingerprinting and MS/MS analysis. The integrated selective enrichment target (ISET) platform, previously developed in our group, with its dual functionality, being both a sample preparation platform and a MALDI target plate, is employed. All steps including immobilized metal ion affinity chromatography of protein on cobalt-loaded beads, tryptic digestion, and MALDI MS analysis are performed in an array format, without any sample transfers, on the same ISET chip. The automated configuration reduced the sample preparation time significantly. Starting with crude lysate, a full plate of 48 purified, digested samples prepared for MALDI-MS can be generated in 4 h, with only 30 min of operator involvement. This paper demonstrates the utility of the method by parallel analysis of 45 His-tagged human recombinant proteins.
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| 7. |
- Agaton, Charlotta, et al.
(författare)
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Genome-based proteomics
- 2004
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 25:9, s. 1280-1288
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Tidskriftsartikel (refereegranskat)abstract
- Protein-protein interactions play crucial roles in various biological pathways and functions. Therefore, the characterization of protein levels and also the network of interactions within an organism would contribute considerably to the understanding of life. The availability of the human genome sequence has created a range of new possibilities for biomedical research. A crucial challenge is to utilize the genetic information for better understanding of protein distribution and function in normal as well as in pathological biological processes. In this review, we have focused on different platforms used for systematic genome-based proteome analyses. These technologies are in many ways complementary and should be seen as various ways to elucidate different functions of the proteome.
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| 8. |
- Agaton, Charlotta, et al.
(författare)
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Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts
- 2004
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1043, s. 33-40
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Tidskriftsartikel (refereegranskat)abstract
- A high stringency protocol, suitable for systematic purification of polyclonal antibodies, is described. The procedure is designed to allow the generation of target protein-specific antibodies suitable for functional annotation of proteins. Antibodies were generated by immunization with recombinantly produced affinity-tagged target proteins. To obtain stringent recovery of the antibodies, a two-step affinity chromatography principle was devised to first deplete the affinity tag-specific antibodies followed by a second step for affinity capture of the target protein-specific antibodies. An analytical dot-blot array system was developed to analyze the cross-reactivity of the affinity-purified antibodies. The results suggest that the protocol can be used in a highly parallel and automated manner to generate mono-specific polyclonal antibodies for large-scale, antibody-based proteomics efforts, i.e. affinity proteomics.
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| 9. |
- Ahmadian, Afshin, et al.
(författare)
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Pyrosequencing : History, biochemistry and future
- 2006
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Ingår i: Clinica Chimica Acta. - : Elsevier BV. - 0009-8981 .- 1873-3492. ; 363:02-jan, s. 83-94
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Forskningsöversikt (refereegranskat)abstract
- Background: Pyrosequencing is a DNA sequencing technology based on the sequencing-by-synthesis principle. Methods: The technique is built on a 4-enzyme real-time monitoring of DNA synthesis by bioluminescence using a cascade that upon nucleotide incorporation ends in a detectable light signal (bioluminescence). The detection system is based on the pyrophosphate released when a nucleotide is introduced in the DNA-strand. Thereby, the signal can be quantitatively connected to the number of bases added. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis and genotyping. Mutation detection and single-nucleotide polymorphisin genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. In order to expand the field for pyrosequencing, the read length needs to be improved. Conclusions: Th pyrosequencing system is based on an enzymatic system. There are different current and future applications of this technique.
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| 10. |
- Alkharaan, Hassan, et al.
(författare)
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Persisting Salivary IgG Against SARS-CoV-2 at 9 Months After Mild COVID-19 : A Complementary Approach to Population Surveys
- 2021
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Ingår i: Journal of Infectious Diseases. - : Oxford University Press (OUP). - 0022-1899 .- 1537-6613. ; 224:3, s. 407-414
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Tidskriftsartikel (refereegranskat)abstract
- Background. Declining humoral immunity in coronavirus disease 2019 (COVID-19) patients and possible reinfection have raised concern. Mucosal immunity, particularly salivary antibodies, may be short lived although long-term studies are lacking. Methods. Using a multiplex bead-based array platform, we investigated antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins in 256 saliva samples from convalescent patients 1-9 months after symptomatic COVID-19 (n = 74, cohort 1), undiagnosed individuals with self-reported questionnaires (n = 147, cohort 2), and individuals sampled prepandemic (n = 35, cohort 3). Results. Salivary IgG antibody responses in cohort 1 (mainly mild COVID-19) were detectable up to 9 months postrecovery, with high correlations between spike and nucleocapsid specificity. At 9 months, IgG remained in blood and saliva in most patients. Salivary IgA was rarely detected at this time point. In cohort 2, salivary IgG and IgA responses were significantly associated with recent history of COVID-19-like symptoms. Salivary IgG tolerated temperature and detergent pretreatments. Conclusions. Unlike SARS-CoV-2 salivary IgA that appeared short lived, specific saliva IgG appeared stable even after mild COVID-19, as for blood serology. This noninvasive saliva-based SARS-CoV-2 antibody test with home self-collection may be a complementary alternative to conventional blood serology.
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| 11. |
- Alm, Tove, et al.
(författare)
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A small bispecific protein selected for orthogonal affinity purification
- 2010
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Ingår i: BIOTECHNOL J. - : Wiley. - 1860-6768. ; 5:6, s. 605-617
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Tidskriftsartikel (refereegranskat)abstract
- A novel protein domain with dual affinity has been created by randomization and selection. The small alkali-stabilized albumin-binding domain (ABD(star)), used as scaffold to construct the library, has affinity to human serum albumin (HSA) and is constituted of 46 amino acids of which 11 were randomized. To achieve a dual binder, the binding site of the inherent HSA affinity was untouched and the randomization was made on the opposite side of the molecule. Despite its small size and randomization of almost a quarter of its amino acids, a bifunctional molecule, ABDz1, with ability to bind to both HSA and the Z(2) domain/protein A was successfully selected using phage display. Moreover, the newly selected variant showed improved affinity for HSA compared to the parental molecule. This novel protein domain has been characterized regarding secondary structure and affinity to the two different ligands. The possibility for affinity purification on two different matrices has been investigated using the two ligands, the HSA matrix and the protein A-based, MabSelect SuRe matrix, and the new protein domain was purified to homogeneity. Furthermore, gene fusions between the new domain and three different target proteins with different characteristics were made. To take advantage of both affinities, a purification strategy referred to as orthogonal affinity purification using two different matrices was created. Successful purification of all three versions was efficiently carried out using this strategy.
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| 12. |
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| 13. |
- Alm, Tove, et al.
(författare)
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High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography
- 2007
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Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 2, s. 709-716
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Tidskriftsartikel (refereegranskat)abstract
- A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.
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| 14. |
- Alm, Tove, 1977-
(författare)
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Interaction engineered three-helix bundle domains for protein recovery and detection
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- HTML clipboard The great advances in DNA technology, e.g. sequencing and recombinant DNA techniques, have given us the genetic information and the tools needed to effectively produce recombinant proteins. Recombinant proteins are valuable means in biotechnological applications and are also emerging as alternatives in therapeutic applications. Traditionally, monoclonal antibodies have been the natural choice for biotechnological and therapeutic applications due to their ability to bind a huge range of different molecules and their natural good affinity. However, the large size of antibodies (150 kDa) limits tissue penetration and the recombinant expression is complicated. Therefore, alternative binders with smaller sizes have been derived from antibodies and alternative scaffolds.In this thesis, two structurally similar domains, Zbasic and ABDz1, have been used as purification tags in different contexts. They are both three-helical bundles and derived from bacterial surface domains, but share no sequence homology. Furthermore, by redesign of the scaffold used for ABDz1, a molecule intended for drug targeting with extended in-vivo half-life has been engineered. In Papers I and II, the poly-cationic tag Zbasic is explored and evaluated. Paper I describes the successful investigation of Zbasic as a purification handle under denaturating conditions. Moreover, Zbasic is evaluated as an interaction domain in matrixassisted refolding. Two different proteins were successfully refolded using the same setup without individual optimization. In Paper II, Zbasic is further explored as a purification handle under non-native conditions in a multi-parallel setup. In total, 22 proteins with varying characteristics are successfully purified using a multi-parallel protein purification protocol and a robotic system. Without modifications, the system can purify up to 60 proteins without manual handling. Paper I and II clearly demonstrate that Zbasic can be used as an interaction domain in matrix-assisted refolding and that it offers a good alternative to the commonly used His6-tag under denaturating conditions. In paper III, the small bifunctional ABDz1 is selected from a phage display library. Endowed with two different binding interfaces, ABDz1 is capable of binding both the HSA-sepharose and the protein A-derived MabSelect SuRe-matrix. The bifunctionality of the domain is exploited in an orthogonal affinity setup. Three target proteins are successfully purified using the HSA-matrix and the MabSelect SuRe-matrix. Furthermore, the purity of the target proteins is effectively improved by combining the two chromatographic steps. Thus, paper III shows that the small ABDz1 can be used as an effective purification handle and dual affinity tag without target specific optimization. Paper IV describes the selection and affinity maturation of small bispecific drug-targeting molecules. First generation binders against tumor necrosis factor-α are selected using phage display. Thereafter on-cell surface display and flow cytometry is used to select second-generation binders. The binding to tumor necrosis factor-α is improved up to 30 times as compared to the best first generation binder, and a 6-fold improvement of the binding strength was possible with retained HSA affinity. Paper III and IV clearly demonstrate that dual interaction surfaces can successfully be grafted on a small proteinaceous domain, and that the strategy in paper IV can be used for dual selection of bifunctional binders.
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| 15. |
- Andersson, K, et al.
(författare)
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Kinetic characterization of the interaction of the Z-fragment of protein A with mouse-IgG3 in a volume in chemical space.
- 1999
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Ingår i: Proteins. - 0887-3585 .- 1097-0134. ; 37:3
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Tidskriftsartikel (refereegranskat)abstract
- The kinetic rate parameters for the interaction between a single domain analogue of staphylococcal protein A (Z) and a mouse-IgG3 monoclonal antibody (MAb) were measured in Hepes buffer with different chemical additives. Five buffer ingredients (pH, NaCl, DMSO, EDTA, and KSCN) were varied simultaneously in 16 experiments following a statistical experimental plan. The 16 buffers thus spanned a volume in chemical space. A mathematical model, using data from the buffer composition, was developed and used to predict apparent kinetic parameters in five new buffers within the spanned volume. Association and dissociation parameters were measured in the new buffers, and these agreed with the predicted values, indicating that the model was valid within the spanned volume. The pattern of variation of the kinetic parameters in relation to buffer composition was different for association and dissociation, such that pH influenced both association and dissociation and NaCl influenced only dissociation. This indicated that the recognition mechanism (association) and the stability of the formed complex (dissociation) involve different binding forces, which can be further investigated by kinetic studies in systematically varied buffers.
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| 16. |
- Andersson, Sandra, et al.
(författare)
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Antibodies Biotinylated Using a Synthetic Z-domain from Protein A Provide Stringent In Situ Protein Detection
- 2013
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Ingår i: Journal of Histochemistry and Cytochemistry. - : SAGE Publications. - 0022-1554 .- 1551-5044. ; 61:11, s. 773-784
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Tidskriftsartikel (refereegranskat)abstract
- Antibody-based protein profiling on a global scale using immunohistochemistry constitutes an emerging strategy for mapping of the human proteome, which is crucial for an increased understanding of biological processes in the cell. Immunohistochemistry is often performed indirectly using secondary antibodies for detection, with the benefit of signal amplification. Direct immunohistochemistry instead brings the advantage of multiplexing; however, it requires labeling of the primary antibody. Many antibody-labeling kits do not specifically target IgG and may therefore cause labeling of stabilizing proteins present in the antibody solution. A new conjugation method has been developed that utilizes a modified Z-domain of protein A (ZBPA) to specifically target the Fc part of antibodies. The aim of the present study was to compare the ZBPA conjugation method and a commercially available labeling kit, Lightning-Link, for in situ protein detection. Fourteen antibodies were biotinylated with each method and stained using immunohistochemistry. For all antibodies tested, ZBPA biotinylation resulted in distinct immunoreactivity without off-target staining, regardless of the presence of stabilizing proteins in the buffer, whereas the majority of the Lightning-Link biotinylated antibodies displayed a characteristic pattern of nonspecific staining. We conclude that biotinylated ZBPA domain provides a stringent method for antibody biotinylation, advantageous for in situ protein detection in tissues.
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| 17. |
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| 18. |
- Appelberg, S., et al.
(författare)
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A universal SARS-CoV DNA vaccine inducing highly cross-reactive neutralizing antibodies and T cells
- 2022
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Ingår i: EMBO Molecular Medicine. - : EMBO. - 1757-4676 .- 1757-4684. ; 14:10
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Tidskriftsartikel (refereegranskat)abstract
- New variants in the SARS-CoV-2 pandemic are more contagious (Alpha/Delta), evade neutralizing antibodies (Beta), or both (Omicron). This poses a challenge in vaccine development according to WHO. We designed a more universal SARS-CoV-2 DNA vaccine containing receptor-binding domain loops from the huCoV-19/WH01, the Alpha, and the Beta variants, combined with the membrane and nucleoproteins. The vaccine induced spike antibodies crossreactive between huCoV-19/WH01, Beta, and Delta spike proteins that neutralized huCoV-19/WH01, Beta, Delta, and Omicron virus in vitro. The vaccine primed nucleoprotein-specific T cells, unlike spike-specific T cells, recognized Bat-CoV sequences. The vaccine protected mice carrying the human ACE2 receptor against lethal infection with the SARS-CoV-2 Beta variant. Interestingly, priming of cross-reactive nucleoprotein-specific T cells alone was 60% protective, verifying observations from humans that T cells protect against lethal disease. This SARS-CoV vaccine induces a uniquely broad and functional immunity that adds to currently used vaccines.
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| 19. |
- Asplund Högelin, K., et al.
(författare)
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B-cell repopulation dynamics and drug pharmacokinetics impact SARS-CoV-2 vaccine efficacy in anti-CD20-treated multiple sclerosis patients
- 2022
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Ingår i: European Journal of Neurology. - : Wiley. - 1351-5101 .- 1468-1331. ; 29:11, s. 3317-3328
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Tidskriftsartikel (refereegranskat)abstract
- Background and purpose: Recent findings document a blunted humoral response to SARS-CoV-2 vaccination in patients on anti-CD20 treatment. Although most patients develop a cellular response, it is still important to identify predictors of seroconversion to optimize vaccine responses. Methods: We determined antibody responses after SARS-CoV-2 vaccination in a real-world cohort of multiple sclerosis patients (n = 94) treated with anti-CD20, mainly rituximab, with variable treatment duration (median = 2.9, range = 0.4–9.6 years) and time from last anti-CD20 infusion to vaccination (median = 190, range = 60–1032 days). Results: We find that presence of B cells and/or rituximab in blood predict seroconversion better than time since last infusion. Using multiple logistic regression, presence of >0.5% B cells increased probability of seroconversion with an odds ratio (OR) of 5.0 (95% confidence interval [CI] = 1.0–28.1, p = 0.055), whereas the corresponding OR for ≥6 months since last infusion was 1.45 (95% CI = 0.20–10.15, p = 0.705). In contrast, detectable rituximab levels were negatively associated with seroconversion (OR = 0.05, 95% CI = 0.002–0.392, p = 0.012). Furthermore, naïve and memory IgG+ B cells correlated with antibody levels. Although retreatment with rituximab at 4 weeks or more after booster depleted spike-specific B cells, it did not noticeably affect the rate of decline in antibody titers. Interferon-γ and/or interleukin-13 T-cell responses to the spike S1 domain were observed in most patients, but with no correlation to spike antibody levels. Conclusions: These findings are relevant for providing individualized guidance to patients and planning of vaccination schemes, in turn optimizing benefit–risk with anti-CD20.
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| 20. |
- Berglund, Lisa, et al.
(författare)
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A genecentric Human Protein Atlas for expression profiles based on antibodies
- 2008
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 7:10, s. 2019-2027
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Forskningsöversikt (refereegranskat)abstract
- An attractive path forward in proteomics is to experimentally annotate the human protein complement of the genome in a genecentric manner. Using antibodies, it might be possible to design protein-specific probes for a representative protein from every protein-coding gene and to subsequently use the antibodies for systematical analysis of cellular distribution and subcellular localization of proteins in normal and disease tissues. A new version (4.0) of the Human Protein Atlas has been developed in a genecentric manner with the inclusion of all human genes and splice variants predicted from genome efforts together with a visualization of each protein with characteristics such as predicted membrane regions, signal peptide, and protein domains and new plots showing the uniqueness (sequence similarity) of every fraction of each protein toward all other human proteins. The new version is based on tissue profiles generated from 6120 antibodies with more than five million immunohistochemistry-based images covering 5067 human genes, corresponding to approximately 25% of the human genome. Version 4.0 includes a putative list of members in various protein classes, both functional classes, such as kinases, transcription factors, G-protein-coupled receptors, etc., and project-related classes, such as candidate genes for cancer or cardiovascular diseases. The exact antigen sequence for the internally generated antibodies has also been released together with a visualization of the application-specific validation performed for each antibody, including a protein array assay, Western blot analysis, immunohistochemistry, and, for a large fraction, immunofluorescence-based confocal microscopy. New search functionalities have been added to allow complex queries regarding protein expression profiles, protein classes, and chromosome location. The new version of the protein atlas thus is a resource for many areas of biomedical research, including protein science and biomarker discovery.
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| 21. |
- Berglund, Lisa, et al.
(författare)
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Generation of validated antibodies towards the human proteome
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Here we show the results from a large effort to generate antibodies towards the human proteome. A high-throughput strategy was developed based on cloning and expression of antigens as recombitant protein epitope signature tags (PrESTs) Affinity purified polyclonal antibodies were generated, followed by validation by protein microarrays, Western blotting and microarray-based immunohistochemistry. PrESTs were selected based on sequence uniqueness relative the proteome and a bioinformatics analysis showed that unique antigens can be found for at least 85% of the proteome using this general strategy. The success rate from antigen selection to validated antibodies was 31%, and from protein to antibody 55%. Interestingly, membrane-bound and soluble proteins performed equally and PrEST lengths between 75 and 125 amino acids were found to give the highest yield of validated antibodies. Multiple antigens were selected for many genes and the results suggest that specific antibodies can be systematically generated to most human proteibs.
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| 22. |
- Bergman, Peter, et al.
(författare)
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Safety and efficacy of the mRNA BNT162b2 vaccine against SARS-CoV-2 in five groups of immunocompromised patients and healthy controls in a prospective open-label clinical trial
- 2021
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Ingår i: EBioMedicine. - : Elsevier BV. - 2352-3964. ; 74
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Tidskriftsartikel (refereegranskat)abstract
- Background: Patients with immunocompromised disorders have mainly been excluded from clinical trials of vaccination against COVID-19. Thus, the aim of this prospective clinical trial was to investigate safety and efficacy of BNT162b2 mRNA vaccination in five selected groups of immunocompromised patients and healthy controls.Methods: 539 study subjects (449 patients and 90 controls) were included. The patients had either primary (n=90), or secondary immunodeficiency disorders due to human immunodeficiency virus infection (n=90), allogeneic hematopoietic stem cell transplantation/CAR T cell therapy (n=90), solid organ transplantation (SOT) (n=89), or chronic lymphocytic leukemia (CLL) (n=90). The primary endpoint was seroconversion rate two weeks after the second dose. The secondary endpoints were safety and documented SARS-CoV-2 infection.Findings: Adverse events were generally mild, but one case of fatal suspected unexpected serious adverse reaction occurred. 72.2% of the immunocompromised patients seroconverted compared to 100% of the controls (p=0.004). Lowest seroconversion rates were found in the SOT (43.4%) and CLL (63.3%) patient groups with observed negative impact of treatment with mycophenolate mofetil and ibrutinib, respectively.Interpretation: The results showed that the mRNA BNT162b2 vaccine was safe in immunocompromised patients. Rate of seroconversion was substantially lower than in healthy controls, with a wide range of rates and antibody titres among predefined patient groups and subgroups. This clinical trial highlights the need for additional vaccine doses in certain immunocompromised patient groups to improve immunity.
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| 23. |
- Björling, Erik, et al.
(författare)
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A web-based tool for in silico biomarker discovery based on tissue-specific protein profiles in normal and cancer tissues
- 2008
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 7:5, s. 825-844
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Tidskriftsartikel (refereegranskat)abstract
- Here we report the development of a publicly available Web-based analysis tool for exploring proteins expressed in a tissue- or cancer-specific manner. The search queries are based on the human tissue profiles in normal and cancer cells in the Human Protein Atlas portal and rely on the individual annotation performed by pathologists of images representing immunohistochemically stained tissue sections. Approximately 1.8 million images representing more than 3000 antibodies directed toward human proteins were used in the study. The search tool allows for the systematic exploration of the protein atlas to discover potential protein biomarkers. Such biomarkers include tissue-specific markers, cell type-specific markers, tumor type-specific markers, markers of malignancy, and prognostic or predictive markers of cancers. Here we show examples of database queries to generate sets of candidate biomarker proteins for several of these different categories. Expression profiles of candidate proteins can then subsequently be validated by examination of the underlying high resolution images. The present study shows examples of search strategies revealing several potential protein biomarkers, including proteins specifically expressed in normal cells and in cancer cells from specified tumor types. The lists of candidate proteins can be used as a starting point for further validation in larger patient cohorts using both immunological approaches and technologies utilizing more classical proteomics tools.
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