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Andersson, A., Remnestål, J., Nellgård, B., Vunk, H., Kotol, D., Edfors, F., . . . Fredolini, C. (2019). Development of parallel reaction monitoring assays for cerebrospinal fluid proteins associated with Alzheimer's disease. Clinica Chimica Acta, 494, 79-93
Open this publication in new window or tab >>Development of parallel reaction monitoring assays for cerebrospinal fluid proteins associated with Alzheimer's disease
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2019 (English)In: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 494, p. 79-93Article in journal (Refereed) Published
Abstract [en]

Detailed knowledge of protein changes in cerebrospinal fluid (CSF) across healthy and diseased individuals would provide a better understanding of the onset and progression of neurodegenerative disorders. In this study, we selected 20 brain-enriched proteins previously identified in CSF by antibody suspension bead arrays (SBA) to be potentially biomarkers for Alzheimer's disease (AD) and verified these using an orthogonal approach. We examined the same set of 94 CSF samples from patients affected by AD (including preclinical and prodromal), mild cognitive impairment (MCI), non-AD dementia and healthy individuals, which had previously been analyzed by SBA. Twenty-eight parallel reaction monitoring (PRM) assays were developed and 13 of them could be validated for protein quantification. Antibody profiles were verified by PRM. For seven proteins, the antibody profiles were highly correlated with the PRM results (r > 0.7) and GAP43, VCAM1 and PSAP were identified as potential markers of preclinical AD. In conclusion, we demonstrate the usefulness of targeted mass spectrometry as a tool for the orthogonal verification of antibody profiling data, suggesting that these complementary methods can be successfully applied for comprehensive exploration of CSF protein levels in neurodegenerative disorders.

Place, publisher, year, edition, pages
Elsevier B.V., 2019
Keywords
AD, Alzheimer's disease, Biomarkers, Cerebrospinal fluid, Parallel reaction monitoring (PRM), Suspension bead array (SBA), alpha 1 aantitrypsin, alpha 1 antichymotrypsin, apolipoprotein, biological marker, cathepsin D, cholecystokinin, creatine kinase B type, dickkopf related protein 3, fibrinogen alpha, fructose bisphosphate aldolase C, glucose regulated protein 94, inter alpha trypsin inhibitor heavy chain H1, leucine rich alpha 2 glycoprotein, neurobeachin, neurofilament medium polypeptide, neuromodulin, plasminogen, prosaposin, protein S100B, SPARC like protein 1, unclassified drug, vascular cell adhesion protein 1, adult, aged, Alzheimer disease, Article, clinical article, cohort analysis, controlled study, correlational study, disease course, female, human, male, mass spectrometry, middle aged, mild cognitive impairment, multiple reaction monitoring, priority journal, protein blood level, protein cerebrospinal fluid level, protein microarray, suspension bead array, very elderly
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-252444 (URN)10.1016/j.cca.2019.03.243 (DOI)000470950400013 ()2-s2.0-85063002689 (Scopus ID)
Note

QC 20190715

Available from: 2019-07-15 Created: 2019-07-15 Last updated: 2019-07-15Bibliographically approved
Pernemalm, M., Sandberg, A., Zhu, Y., Boekel, J., Tamburro, D., Schwenk, J. M., . . . Lehtio, J. (2019). In-depth human plasma proteome analysis captures tissue proteins and transfer of protein variants across the placenta. eLIFE, 8, Article ID e41608.
Open this publication in new window or tab >>In-depth human plasma proteome analysis captures tissue proteins and transfer of protein variants across the placenta
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2019 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 8, article id e41608Article in journal (Refereed) Published
Abstract [en]

Here, we present a method for in-depth human plasma proteome analysis based on high-resolution isoelectric focusing HiRIEF LC-MS/MS, demonstrating high proteome coverage, reproducibility and the potential for liquid biopsy protein profiling. By integrating genomic sequence information to the MS-based plasma proteome analysis, we enable detection of single amino acid variants and for the first time demonstrate transfer of multiple protein variants between mother and fetus across the placenta. We further show that our method has the ability to detect both low abundance tissue-annotated proteins and phosphorylated proteins in plasma, as well as quantitate differences in plasma proteomes between the mother and the newborn as well as changes related to pregnancy.

Place, publisher, year, edition, pages
ELIFE SCIENCES PUBLICATIONS LTD, 2019
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-252639 (URN)10.7554/eLife.41608 (DOI)000468060900001 ()30958262 (PubMedID)2-s2.0-85066163570 (Scopus ID)
Note

QC 20190610

Available from: 2019-06-10 Created: 2019-06-10 Last updated: 2019-06-10Bibliographically approved
Edfors, F., Forsström, B., Vunk, H., Kotol, D., Fredolini, C., Maddalo, G., . . . Uhlén, M. (2019). Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics. Journal of Proteome Research, 18(7), 2706-2718
Open this publication in new window or tab >>Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics
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2019 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 18, no 7, p. 2706-2718Article in journal (Refereed) Published
Abstract [en]

The availability of proteomics resources hosting protein and peptide standards, as well as the data describing their analytical performances, will continue to enhance our current capabilities to develop targeted proteomics methods for quantitative biology. This study describes the analysis of a resource of 26,840 individually purified recombinant protein fragments corresponding to more than 16,000 human protein-coding genes. The resource was screened to identify proteotypic peptides suitable for targeted proteomics efforts, and we report LC-MS/MS assay coordinates for more than 25,000 proteotypic peptides, corresponding to more than 10,000 unique proteins. Additionally, peptide formation and digestion kinetics were, for a subset of the standards, monitored using a time-course protocol involving parallel digestion of isotope-labeled recombinant protein standards and endogenous human plasma proteins. We show that the strategy by adding isotope-labeled recombinant proteins before trypsin digestion enables short digestion protocols (<= 60 min) with robust quantitative precision. In a proof-of-concept study, we quantified 23 proteins in human plasma using assay parameters defined in our study and used the standards to describe distinct clusters of individuals linked to different levels of LPA, APOE, SERPINAS, and TFRC. In summary, we describe the use and utility of a resource of recombinant proteins to identify proteotypic peptides useful for targeted proteomics assay development.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2019
Keywords
targeted proteomics, stable isotope standards, mass spectrometry, protein quantification, recombinant proteins, protein fragment, trypsin digestion, spectral library, assay generation, peptide formation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-255390 (URN)10.1021/acs.jproteome.8b00924 (DOI)000474795500003 ()31094526 (PubMedID)2-s2.0-85067403932 (Scopus ID)
Note

QC 20190730

Available from: 2019-07-30 Created: 2019-07-30 Last updated: 2019-07-30Bibliographically approved
Fredolini, C., Byström, S., Sanchez-Rivera, L., Ioannou, M., Tamburro, D., Pontén, F., . . . Schwenk, J. M. (2019). Systematic assessment of antibody selectivity in plasma based on a resource of enrichment profiles. Scientific Reports, 9, Article ID 8324.
Open this publication in new window or tab >>Systematic assessment of antibody selectivity in plasma based on a resource of enrichment profiles
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 8324Article in journal (Refereed) Published
Abstract [en]

There is a strong need for procedures that enable context and application dependent validation of antibodies. Here, we applied a magnetic bead assisted workflow and immunoprecipitation mass spectrometry (IP-MS/MS) to assess antibody selectivity for the detection of proteins in human plasma. A resource was built on 414 IP experiments using 157 antibodies (targeting 120 unique proteins) in assays with heat-treated or untreated EDTA plasma. For each protein we determined their antibody related degrees of enrichment using z-scores and their frequencies of identification across all IP assays. Out of 1,313 unique endogenous proteins, 426 proteins (33%) were detected in >20% of IPs, and these background components were mainly comprised of proteins from the complement system. For 45% (70/157) of the tested antibodies, the expected target proteins were enriched (z-score >= 3). Among these 70 antibodies, 59 (84%) co-enriched other proteins beside the intended target and mainly due to sequence homology or protein abundance. We also detected protein interactions in plasma, and for IGFBP2 confirmed these using several antibodies and sandwich immunoassays. The protein enrichment data with plasma provide a very useful and yet lacking resource for the assessment of antibody selectivity. Our insights will contribute to a more informed use of affinity reagents for plasma proteomics assays.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Bioinformatics and Systems Biology
Identifiers
urn:nbn:se:kth:diva-254077 (URN)10.1038/s41598-019-43552-5 (DOI)000470243800005 ()31171813 (PubMedID)2-s2.0-85067067842 (Scopus ID)
Note

QC 20190624

Available from: 2019-06-24 Created: 2019-06-24 Last updated: 2019-06-24Bibliographically approved
Häussler, R. S., Bendes, A., Iglesias, M. J., Sanchez-Rivera, L., Dodig-Crnkovic, T., Byström, S., . . . Schwenk, J. M. (2019). Systematic Development of Sandwich Immunoassays for the Plasma Secretome. Proteomics, Article ID 1900008.
Open this publication in new window or tab >>Systematic Development of Sandwich Immunoassays for the Plasma Secretome
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2019 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, article id 1900008Article in journal (Refereed) Published
Abstract [en]

The plasma proteome offers a clinically useful window into human health. Recent advances from highly multiplexed assays now call for appropriate pipelines to validate individual candidates. Here, a workflow is developed to build dual binder sandwich immunoassays (SIA) and for proteins predicted to be secreted into plasma. Utilizing suspension bead arrays, ≈1800 unique antibody pairs are first screened against 209 proteins with recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies, dilution-dependent curves in plasma and concentration-dependent curves of full-length proteins for 102 (49%) of the targets are obtained. For 22 protein assays, the longitudinal, interindividual, and technical performance is determined in a set of plasma samples collected from 18 healthy subjects every third month over 1 year. Finally, 14 of these assays are compared with with SIAs composed of other binders, proximity extension assays, and affinity-free targeted mass spectrometry. The workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, and the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.

Place, publisher, year, edition, pages
Wiley, 2019
Keywords
antibodies, plasma, sandwich assays, screening, secreted proteins
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-255741 (URN)10.1002/pmic.201900008 (DOI)000477448900001 ()31278833 (PubMedID)
Note

QC 20190812

Available from: 2019-08-12 Created: 2019-08-12 Last updated: 2019-08-12Bibliographically approved
Drobin, K., Assadi, G., Hong, M.-G., Anggraeni Andersson, M., Fredolini, C., Forsström, B., . . . Halfvarson, J. (2019). Targeted Analysis of Serum Proteins Encoded at Known Inflammatory Bowel Disease Risk Loci. Inflammatory Bowel Diseases, 25(2), 306-316
Open this publication in new window or tab >>Targeted Analysis of Serum Proteins Encoded at Known Inflammatory Bowel Disease Risk Loci
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2019 (English)In: Inflammatory Bowel Diseases, ISSN 1078-0998, E-ISSN 1536-4844, Vol. 25, no 2, p. 306-316Article in journal (Refereed) Published
Abstract [en]

Few studies have investigated the blood proteome of inflammatory bowel disease (IBD). We characterized the serum abundance of proteins encoded at 163 known IBD risk loci and tested these proteins for their biomarker discovery potential. Based on the Human Protein Atlas (HPA) antibody availability, 218 proteins from genes mapping at 163 IBD risk loci were selected. Targeted serum protein profiles from 49 Crohns disease (CD) patients, 51 ulcerative colitis (UC) patients, and 50 sex- and age-matched healthy individuals were obtained using multiplexed antibody suspension bead array assays. Differences in relative serum abundance levels between disease groups and controls were examined. Replication was attempted for CD-UC comparisons (including disease subtypes) by including 64 additional patients (33 CD and 31 UC). Antibodies targeting a potentially novel risk protein were validated by paired antibodies, Western blot, immuno-capture mass spectrometry, and epitope mapping. By univariate analysis, 13 proteins mostly related to neutrophil, T-cell, and B-cell activation and function were differentially expressed in IBD patients vs healthy controls, 3 in CD patients vs healthy controls and 2 in UC patients vs healthy controls (q < 0.01). Multivariate analyses further differentiated disease groups from healthy controls and CD subtypes from UC (P < 0.05). Extended characterization of an antibody targeting a novel, discriminative serum marker, the laccase (multicopper oxidoreductase) domain containing 1 (LACC1) protein, provided evidence for antibody on-target specificity. Using affinity proteomics, we identified a set of IBD-associated serum proteins encoded at IBD risk loci. These candidate proteins hold the potential to be exploited as diagnostic biomarkers of IBD.

Place, publisher, year, edition, pages
Oxford University Press, 2019
Keywords
inflammatory bowel disease, affinity proteomics, LACC1
National Category
Gastroenterology and Hepatology
Identifiers
urn:nbn:se:kth:diva-249898 (URN)10.1093/ibd/izy326 (DOI)000462580900020 ()30358838 (PubMedID)2-s2.0-85059799081 (Scopus ID)
Note

QC 20190423

Available from: 2019-04-23 Created: 2019-04-23 Last updated: 2019-04-23Bibliographically approved
Byström, S., Eklund, M., Hong, M.-G., Fredolini, C., Eriksson, M., Czene, K., . . . Gabrielson, M. (2018). Affinity proteomic profiling of plasma for proteins associated to area-based mammographic breast density. Breast Cancer Research, 20, Article ID 14.
Open this publication in new window or tab >>Affinity proteomic profiling of plasma for proteins associated to area-based mammographic breast density
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2018 (English)In: Breast Cancer Research, ISSN 1465-5411, E-ISSN 1465-542X, Vol. 20, article id 14Article in journal (Refereed) Published
Abstract [en]

Background: Mammographic breast density is one of the strongest risk factors for breast cancer, but molecular understanding of how breast density relates to cancer risk is less complete. Studies of proteins in blood plasma, possibly associated with mammographic density, are well-suited as these allow large-scale analyses and might shed light on the association between breast cancer and breast density. Methods: Plasma samples from 1329 women in the Swedish KARMA project, without prior history of breast cancer, were profiled with antibody suspension bead array (SBA) assays. Two sample sets comprising 729 and 600 women were screened by two different SBAs targeting a total number of 357 proteins. Protein targets were selected through searching the literature, for either being related to breast cancer or for being linked to the extracellular matrix. Association between proteins and absolute area-based breast density (AD) was assessed by quantile regression, adjusting for age and body mass index (BMI). Results: Plasma profiling revealed linear association between 20 proteins and AD, concordant in the two sets of samples (p < 0.05). Plasma levels of seven proteins were positively associated and 13 proteins negatively associated with AD. For eleven of these proteins evidence for gene expression in breast tissue existed. Among these, ABCC11, TNFRSF10D, F11R and ERRF were positively associated with AD, and SHC1, CFLAR, ACOX2, ITGB6, RASSF1, FANCD2 and IRX5 were negatively associated with AD. Conclusions: Screening proteins in plasma indicates associations between breast density and processes of tissue homeostasis, DNA repair, cancer development and/or progression in breast cancer. Further validation and follow-up studies of the shortlisted protein candidates in independent cohorts will be needed to infer their role in breast density and its progression in premenopausal and postmenopausal women.

Place, publisher, year, edition, pages
BIOMED CENTRAL LTD, 2018
Keywords
Mammographic breast density, Plasma, Protein profiling, Suspension bead array, Affinity proteomics, KARMA cohort
National Category
Clinical Medicine
Identifiers
urn:nbn:se:kth:diva-223790 (URN)10.1186/s13058-018-0940-z (DOI)000425114200001 ()29444691 (PubMedID)2-s2.0-85042063089 (Scopus ID)
Funder
Swedish Research CouncilThe Kamprad Family FoundationKnut and Alice Wallenberg FoundationSwedish Cancer SocietyScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20180307

Available from: 2018-03-07 Created: 2018-03-07 Last updated: 2018-03-07Bibliographically approved
Chen, Z., Dodig-Crnkovic, T., Schwenk, J. M. & Tao, S.-c. (2018). Current applications of antibody microarrays. Clinical Proteomics, 15, Article ID 7.
Open this publication in new window or tab >>Current applications of antibody microarrays
2018 (English)In: Clinical Proteomics, ISSN 1542-6416, E-ISSN 1559-0275, Vol. 15, article id 7Article, review/survey (Refereed) Published
Abstract [en]

The concept of antibody microarrays is one of the most versatile approaches within multiplexed immunoassay technologies. These types of arrays have increasingly become an attractive tool for the exploratory detection and study of protein abundance, function, pathways, and potential drug targets. Due to the properties of the antibody microarrays and their potential use in basic research and clinical analytics, various types of antibody microarrays have already been developed. In spite of the growing number of studies utilizing this technique, few reviews about antibody microarray technology have been presented to reflect the quality and future uses of the generated data. In this review, we provide a summary of the recent applications of antibody microarray techniques in basic biology and clinical studies, providing insights into the current trends and future of protein analysis.

Place, publisher, year, edition, pages
BioMed Central, 2018
Keywords
Antibody microarray, Signalling, Drug mechanism, Clinical application, Systems biology, Technology advances
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-224692 (URN)10.1186/s12014-018-9184-2 (DOI)000426761800001 ()29507545 (PubMedID)2-s2.0-85042706257 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg Foundation
Note

QC 20180322

Available from: 2018-03-22 Created: 2018-03-22 Last updated: 2018-03-22Bibliographically approved
Djureinovic, D., Dodig-Crnkovic, T., Hellström, C., Holgersson, G., Bergqvist, M., Mattsson, J. S., . . . Micke, P. (2018). Detection of autoantibodies against cancer-testis antigens in non-small cell lung cancer. Lung Cancer, 125, 157-163
Open this publication in new window or tab >>Detection of autoantibodies against cancer-testis antigens in non-small cell lung cancer
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2018 (English)In: Lung Cancer, ISSN 0169-5002, E-ISSN 1872-8332, Vol. 125, p. 157-163Article in journal (Refereed) Published
Abstract [en]

Objectives: Cancer-testis antigens (CTAs) are defined as proteins that are specifically expressed in testis or placenta and their expression is frequently activated in cancer. Due to their ability to induce an immune response, CTAs may serve as suitable targets for immunotherapy. The aim of this study was to evaluate if there is reactivity against CTAs in the plasma of non-small cell lung cancer (NSCLC) patients through the detection of circulating antibodies. Materials and methods: To comprehensively analyze autoantibodies against CTAs the multiplexing capacities of suspension bead array technology was used. Bead arrays were created with 120 protein fragments, representing 112 CTAs. Reactivity profiles were measured in plasma samples from 133 NSCLC patients and 57 cases with benign lung diseases. Results: Altogether reactivity against 69 antigens, representing 81 CTAs, was demonstrated in at least one of the analyzed samples. Twenty-nine of the antigens (45 CTAs) demonstrated exclusive reactivity in NSCLC samples. Reactivity against cancer-testis antigen family 47; member A (CT47A) genes, P antigen family member 3 (PAGE3), variable charge X-linked (VCX), melanoma antigen family B1 (MAGEB1), lin-28 homolog B (LIN28B) and chromosome 12 open reading frame 54 (C12orf54) were only found in NSCLC patients at a frequency of 1%–4%. The presence of autoantibodies towards these six antigens was confirmed in an independent group of 34 NSCLC patients. Conclusion: We identified autoantibodies against CTAs in the plasma of lung cancer patients. The reactivity pattern of autoantibodies was higher in cancer patients compared to the benign group, stable over time, but low in frequency of occurrence. The findings suggest that some CTAs are immunogenic and that these properties can be utilized as immune targets. 

Place, publisher, year, edition, pages
Elsevier Ireland Ltd, 2018
Keywords
Adenocarcinoma, Cancer immunity, Lung cancer, MAGE, Squamous cell cancer, Tumor markers
National Category
Clinical Medicine
Identifiers
urn:nbn:se:kth:diva-236616 (URN)10.1016/j.lungcan.2018.09.012 (DOI)000450378500023 ()2-s2.0-85054035205 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg FoundationSwedish Cancer Society, 2012/738
Note

Export Date: 22 October 2018; Article; CODEN: LUCAE; Correspondence Address: Djureinovic, D.; Department of Immunology, Genetics and Pathology, Uppsala UniversitySweden; email: dijana.djureinovic@igp.uu.se; Funding details: SciLifeLab, Science for Life Laboratory; Funding details: Knut och Alice Wallenbergs Stiftelse; Funding details: 2012/738, Cancerfonden; Funding text: This study was supported by the Swedish Cancer Society Cancerfonden (2012/738), Lions Cancer Foundation Uppsala, Sweden, Erik, Karin and Gösta Selanders Foundation, Sweden as well as grants for SciLifeLab and the Human Protein Atlas funded by the Knut and Alice Wallenberg foundation.; Funding text: We thank the Human Protein Atlas team, the Uppsala Biobank and everyone in the Division of Affinity Proteomics and the Autoimmunity Profiling facility at SciLifeLab for their support. Appendix A. QC 20181119

Available from: 2018-11-19 Created: 2018-11-19 Last updated: 2018-12-10Bibliographically approved
Sjöberg, R., Andersson, E., Hellström, C., Mattsson, C., Schwenk, J. M., Nilsson, P. & Ayoglu, B. (2018). High-density antigen microarrays for the assessment of antibody selectivity and off-target binding. In: Epitope Mapping Protocols: (pp. 231-238). Humana Press Inc.
Open this publication in new window or tab >>High-density antigen microarrays for the assessment of antibody selectivity and off-target binding
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2018 (English)In: Epitope Mapping Protocols, Humana Press Inc. , 2018, p. 231-238Chapter in book (Refereed)
Abstract [en]

With the increasing availability of collections of antibodies, their evaluation in terms of binding selectivity becomes an important but challenging task. Planar antigen microarrays are very suitable tools to address this task and provide a powerful proteomics platform for the characterization of the binding selectivity of antibodies toward thousands of antigens in parallel. In this chapter, we describe our in-house developed procedures for the generation of high-density planar antigen microarrays with over 21,000 features. We also provide the details of the assay protocol, which we routinely use for the assessment of binding selectivity of the polyclonal antibodies generated within the Human Protein Atlas. © Springer Science+Business Media, LLC, part of Springer Nature 2018.

Place, publisher, year, edition, pages
Humana Press Inc., 2018
Series
Methods in Molecular Biology, ISSN 1064-3745 ; 1785
Keywords
Affinity proteomics, Antibody selectivity, Antigen microarrays, Protein microarrays
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-238338 (URN)10.1007/978-1-4939-7841-0_15 (DOI)2-s2.0-85046366884 (Scopus ID)978-1-4939-7839-7 (ISBN)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20181115

Available from: 2018-11-15 Created: 2018-11-15 Last updated: 2018-11-15Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-8141-8449

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